Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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TITLE
DETERGENT-FREE SIMULTANEOUS MULTIOMICS SAMPLE PREPARATION
METHOD USING NOVEL NEW VESICLE DESIGN
RELATED APPLICATIONS
[0001] This application claims priority from U.S. Provisional Application
Serial Number
62/894,201, filed August 30, 2019. The entirety of the aforementioned
applications is
incorporated herein by reference.
FIELD
[0002] The present application relates to methods and devices for the
preparation of
samples containing proteins and/or small molecules and/or DNA/RNA.
BACKGROUND
[0003] The combination of omic technology and techniques is gaining in
popularity and
advancing our understanding of biological systems and human pathologies.
However,
integrating analyses across comics platforms has introduced new technical
challenges. Parallel
sample handling in which a sample is split and portions are processed for
different molecular
classes, e.g. proteins and metabolites, is one potential solution. However,
when the sample
amounts are limited, as is often the case with clinical material, or
heterogeneity exists, for
example across different tissue sections, using a simultaneous extraction
methodology for
several molecular classes is essential however such methods have been lacking.
The only
methods available have been based on phase separation, e.g. chloroform-
methanol extraction,
and are limited by their complexity and laboriousness. They are not practical
for either
implementation with small sample amounts or high-throughput analyses.
[0004] In chemistry, biochemistry and clinical and research settings, there is
often need
for samples to be treated, for example including, and not limited to, steps of
chemical or
enzymatic reactions, or precipitations or coagulations, before said sample is
subject to
subsequent steps which might include filtration, capture, clarification,
chromatography or
myriad other processes, all of which require the sample to flow through some
kind of a matrix
adapted to the process of interest such as and not limited to filtration,
capture, clarification,
enrichment or chromatography. Simultaneous Trapping (SiTrap) facilitates
direct measurement
of, at a minimum, the proteome and metabolome in the same sample extract.
SiTrap represents a
method and system of sample preparation and separation on a depth filter to
fractionate biology
into two or many classes of biological moieties. SiTrap can be detergent-free
and is extensible to
nucleic acid polymers (DNA and RNA) as well as lipids, glycans and other
molecular classes. A
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new novel vesicle allows for maximum SiTrap function and increased processing
speed and
convenience.
100051 Each treatment step often requires time, i.e. incubation, and such
steps are often
serial, such as a precipitation or depletion or enzymatic or chemical
reaction, followed by some
kind of chromatography or enrichment or affinity or enzymatic treatment. Such
treatments are
most typically achieved by running a reaction in one tube or vesicle or
container, transferring the
contents of that reaction, perhaps including any precipitant or solid material
(or excluding it,
depending), to some matrix such as, and not limited to, a filter or porous
material or a
chromatography column of many formats (cartridges, tips, sheets, membranes,
spin columns or
filters, gravity flow columns, solid-phase extraction [SPE] columns, etc.),
which then flows into
a second tube or vesicle or container. It is noted that depending on the needs
of the system at
hand, such matrices might be in serial, for example a filter might be placed
before a
chromatography cartridge to prevent clogging.
100061 After processing on or through the matrix, some combination of (1) the
flow
through that passes through the matrix, (2) the retentate which did not enter
into the matrix, or
(3) the material bound on or to or within the matrix are taken for further
work or analysis; the
fraction(s) which are desirable and not desirable are completely a function of
the processing at
hand.
100071 After whatever portion of the sample has passed through the matrix, the
matrix
and/or retentate is often further processed such as by washing, chemical or
enzymatic treatment,
affinities, elution, etc. Depending on the workflow, a treatment that requires
time to work, i.e.
incubation(s), might be applied to the matrix, or the retentate, or both. Such
incubation might be
at lower or higher temperature than is ambient. Depending on the task at hand,
such incubations
might also involve the introduction of electromagnetic radiation in the form
of light or
microwaves or radiowaves, or of ultrasonic energy.
100081 Because treatment steps including and not limited to chemical or
enzymatic
reactions or precipitations or other reactions which cause a phase change
require incubation
times, the output side of the matrix must be somehow plugged or stopped to
prevent liquid from
flowing through the matrix so that the process happening on or in or on top of
the matrix can be
afforded the requisite amount of time. In the above examples, if the protease
K solution were to
drip through, it would not act on the tissue; if the HRP solution were to flow
through, no ELISA
reaction would occur; and if the biotin elution solution were to flow through,
no elution would
occur. Similarly, if the solution containing salts or other dissolved moieties
flowed through, no
desired concentration reduction would occur, and for biopolymers, should
incubation be needed
to effect a phase change, the biopolymers would then pass through the matrix
into the sample
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which was supposed to be cleared of biopolymers to later cause clogging. In
the potential second
(or more than second) incubation, if the listed enzymes were to flow through,
they would not
process any biomolecules atop or in or within or on the matrix, causing
failure. Thus, plugging
of the output side of the matrix is essential in such treatments to afford the
treatment sufficient
time,
100091 This requirement to plug or stop flow through the matrix causes
multiple negative
issues. First, not only is plugging a hassle, but it adds significant
additional experimental time
especially when handling large numbers of samples. The use of a matrix with
plugging often
happens in the following sequence: 1) apply sample to a matrix, perhaps in a
spin column (but
other formats are of course possible); 2) make the flow through pass through
the matrix with
positive pressure on the input side of the matrix or negative pressure on the
output side or
alternatively centrifugation; 3) lift the spin column containing the matrix;
4) plug the column or
other vesicle holding the matrix; 5) close the former container which holds
the flow through; 6)
place the now plugged column in a new tube or container; 7) open the spin
column such that
other solutions of the treatment can be added, perhaps (and not limited to) an
enzyme solution
which works on material on and in and atop the matrix; 8) cap the column
again; 9) incubate the
column with the matrix at the necessary temperature for the necessary amount
of time; 10)
remove the spin column from the new tube; 11) remove the plug, probably very
carefully,
directly above the new tube; 12) return the spin column to the new tube or
container; 13) apply
positive or negative pressure or centrifugal force to remove the contents of
the matrix and any
contents held by the spin column; 14) potentially elute from the matrix or
wash the matrix, as
determined by the needs of the system and properties of the matrix; and 15)
repeat this process,
each time using a fresh tube, if additional incubations are to be performed,
such as recovery first
of nucleic acids, then enzymatic treatment with a glycosidase, then chemical
treatment with
reduction and allcylation reagents, among many other possibilities (such as
citraconic anhydride
or hydroxylamine or NHS or isothiocyanate or many other chemistries and
chemical treatments),
then washing such reagents away, then processing with a protease the proteins
bound in and on
and within and atop the matrix. Needless to say, for large numbers of sample,
this becomes
simply intractable.
100101 Beyond the tedious nature of plugs, they can leak, losing sample and
causing
failure. Indeed because matrices and their vesicles are inside tubes and thus
not directly visible,
the presence of a leak is often not detected until a treatment or experiment
has run its course_
Plugs can also break off during removal, also losing sample from an inability
to recover sample
from the matrix,
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10011] Plugging introduces additional experimental error because the timing of
the
plugging may be variable and can affect results: some samples might drip
through more, or
samples might have longer Or shorter incubation times depending on when they
were plugged
and unplugged. Indeed, by the very nature of the process, the first sample to
be plugged in a
series will be plugged for longer than the last sample to receive a plug,
subjecting the samples to
additional and undesirable experimental variability.
[0012] Plugging also causes problems when it is desired to capture all of the
material
which flows from or out of or off or through the matrix. Indeed especially for
small volumes, the
plug itself may retain a significant amount of material one wishes to work to
obtain. This is
particularly the case if the plug is of a female variety and caps the end of a
nozzle or flow
director or connector or lure lock: the act of removing the plug creates a
vacuum, filling the plug
with material which, depending on the process, may be desirable and precious_
In such a case
one must pipette out the sample back into the vesicle atop the matrix, if
possible. Such sample
loss can also occur if plugs are a male variety: when the plug inserted in to
the inside of the
connection on the post-flow side of the matrix, the act of unplugging creates
the same suction
which can lead to the sample flowing out and potentially being lost. To
address such situations,
one may place a capture tube under the connector which comes off the matrix,
carefully remove
the plug and attempt to capture any sample which drips out. In summary,
plugging is an error-
prone process necessitating significant amounts of time. Finally, the use of
plugs necessitates
significant manual manipulation of the vesicles holding the matrix. This
manipulation is poorly
translatable to automation.
[0013] Thus, there exists a need for a device and method and process which:
affords
support to a matrix or matrices, which might be serially employed or stacked;
affords a space
before the matrix into which samples can be added, and a space after the
matrix which can hold
the portion of the sample which has passed through the matrix; affords easy
start and stop of
flow through a matrix with minimal handling and importantly no loose plug;
allows for the easy
use of multiple sequential treatments including on- or in-matrix treatments;
allows for the easy
introduction of heat or electromagnetic radiation like light or sonic energies
such as
ultrasonication; limits the potential for treatment failure due to dripping or
lack of sealing.
SUMMARY
[0014] In one aspect, the present application provides a method of preparing a
sample
comprising one or more fractions of molecule of interest, the method
comprising: Exposing the
sample to an extraction solvent, wherein the extraction solvent can be around
neutral, or basic or
acidic, and wherein the extraction solvent can be detergent-free or
alternatively contain
detergents or surfactants; Exposing said sample combined with said extraction
solvent to
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physical disruption such as bead beating, sonication or ultrasonication.
Preferably, but not
obligatorily, sonication and ultrasonication is used; In the case that a
sample was extracted with
a basic extraction solvent, neutralizing the basic extraction solvent with an
acid so that the pH is
around neutral.
100151 In certain embodiments, in the case that a sample was extracted with an
acidic
extraction solvent, neutralizing the acidic extraction solvent with a base so
that the pH is around
neutral; Exposing said sample combined with said extraction solvent to a
molecule coagulant
which facilitates binding of especially larger molecules upon a matrix,
preferably a porous
matrix, or a collection of small particles which can be manipulated and/or
retained, wherein said
coagulant may consist of a single- or multi-phase solution; During the (large)
molecule
coagulation step, bringing said sample combined with said large molecule
coagulant into contact
with a matrix adapted to capture said large molecules in the presence of the
coagulant and most
preferably a matrix which prevents excessive aggregation of coagulated large
molecules such
that flow through the matrix is not impeded; Collection into a removable
vesicle of the smaller
non-coagulated and unbound molecules, where the classes of smaller non-
coagulated molecules
collected are dependent on the choice and use of large molecule coagulant;
Typically, though
not obligatorily, washing the matrix and the captured large molecules to clean
them; such a step
is not optional if the extraction solvent contained surfactants or detergents,
and such a step is
typically always performed after a chemical manipulation like reduction and
alkylation; Most
preferably, elution of classes of separate classes of coagulated captured
molecules from the
capture matrix into a removable vesicle with extraction solvents chosen to
match the solubilities
of the captured molecules.
100161 In certain embodiments, nucleic acids and polynucleic acids such as DNA
and
RNA, or alternatively free glycans as well as other kinds of molecules, are
water-soluble and can
be eluted by flowing an aqueous buffer through said capture matrix into a new
removable
vesicle. Similarly some lipids and some hydrophobic peptides are soluble in
organic solvents
like alcohols, which serves only as an illustrative example. Optionally during
this step physical
andVor thermal energy may be added such as via shaking or sonication or
ultrasonication or
heating or rnicrowaving or other techniques apparent to one skilled in the
art. It is explicitly
noted that elutions may be serial using different elution solvents. By
example, captured DNA
and RNA might be eluted with an aqueous buffer and then captured lipids might
be eluted with
an organic extraction solvent, the choice of which is determined by the
solubility properties of
the classes of molecules of interest; Most preferably, either before or after
the above mentioned
elution of classes of captured coagulated large molecules upon the capture
matrix, processing of
the captured molecules with enzymes or chemistries, such as nucleases,
proteases, glycosidases,
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lipases, or cyanogen bromide cleavage of proteins, and other enzymes and
chemistries which
alter the state of the coagulated large molecules to facilitate further down-
stream processing.
[0017] In certain embodiments, specific classes of molecules may be liberated
and/or
processed from larger molecules into smaller molecules which often have
different solubility
properties. Such a step may be performed prior to or after steps of elution,
and it is apparent to
one skilled in the art that there is great flexibility in sample processing
which can yield similar
results.
[0018] In embodiments of the application, example classes of molecule which
might be
fractionated and/or prepared include amino acids, nucleosides, nucleotides,
oligonucleotides,
nucleic acids, sugars, carbohydrates, oligosaccharides, polysaccharides, fatty
acids, lipids,
hormones, metabolites, heterocyclic aromatic compounds, carcinogens, mutagens,
compounds
of the exposome such as plasticizers, pesticides, mold release agents, and/or
fire retardants
among many others, peptides, metabolites, cofactors, inhibitors, drugs,
agents, nutrients,
vitamins, polypeptides, proteins, glycoproteins, lipoproteins, antibodies,
growth factors,
cytokines, chemokines, receptors, neurotransmitters, antigens, prions,
allergens, antibodies,
substrates, biological hazardous materials, infectious substances including
viruses, protozoan,
bacteria and fungi, and wastes.
[0019] In certain embodiments, a sample can be first captured on the matrix,
then
optionally treated with a nuclease to form smaller molecules of DNA and RNA,
which can be
eluted and fractionated with the addition of an aqueous buffer, then captured
lipids can be
extracted with an organic solvent such as ethanol, hexane, methanol, ether, or
chloroform, either
separately or in combination, then captured proteins can be processed in or on
the matrix, by
example by glycosidases and the liberated glycosidases can be eluted with
another aqueous
elution, then the proteins can be reduced and alkylated in-situ within the
trapping matrix, then
subsequently they can be processed with a protease such as try psin, or
alternatively via chemical
means such as cyanogen bromide or acidic degradation or alternatively sheeting
from strong
sonic forces, and the peptides resulting from the captured proteins can be
captured in a separate
fraction.
[0020] In certain embodiments, one would obtain small molecules such as
metabolites in
the first flow through fraction, a lipid fraction, a nucleic acid fraction, a
glycan fraction, and a
peptide fraction, all of which are ready for analysis by mass spectrometry or
other detection
technique. Such enzymatic or chemical reactions or elutions may be accelerated
with the
addition of physical and/or thermal energy may be added such as via shaking or
sonication or
ultrasonication or heating or microwaving or other techniques apparent to one
skilled in the art.
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[0021] In certain embodiments, the trap may allow molecules or molecule
fragments
suitably pass to one or more secondary matrix or matrices, where the matrix or
matrices might
provide chromatographic separation or enrichment of various classes or
subclasses of molecules.
[0022] In certain aspects, the application is a method, system and device for
preparing a
samples containing many classes of biomolecules such as (and not limited to)
DNA, RNA,
protein, glycans, small molecules, lipids and other metabolites and small
molecules solubilized
without a surfactant for analysis by mass spectrometry, e.g. LC-MS/MS.
[0023] In certain aspects, the application is a method, system and device for
preparing a
sample containing multiple molecule classes for multi-omics analyses. Such
attempts to date
have typically involved lysis of cells and extraction of proteins, and have
failed to generate
multiple molecule classes from one sample. A suitable lysis medium comprises
30 mM
ammonium acetate. Another suitable lysis medium is 1.8% ammonium hydroxide.
Another is 1
MHO.
[0024] In certain embodiments, the method comprises the step of reducing and
simultaneously alkylating the disulphide bonds of proteins in situ on the
trapping matrix. This is
achieved by heating the sample at 80 C in 60 iriM triethylammonitun
bicarbonate (TEAB), 10
mM tris(2-carboxyethyl)phosphine (TCEP), 25 mM chloroacetamide (CAA). Other
suitable
reagents may be used. The use of such reagents prevents formation of
disulphide bonds between
cysteine residues, especially of different peptides.
[0025] In certain embodiments, centrifugation is performed to drive the
various media,
reagents, buffers and the like through the matrix (matrices) as required.
[0026] In certain embodiments, pumps or the like can be used to move the
various
media, reagents, buffers and the like through the matrix (matrices) of the
present application.
[0027] In certain aspects, the present application provides a sample
preparation device
for molecules extracted in a liquid medium, the device comprising a vessel
having an inlet and
an outlet, a matrix disposed between the inlet and the outlet, the matrix
being adapted to capture
and retain particles of molecules of interest from a medium as is flows from
the inlet to the
outlet.
[0028] In certain embodiments, the matrix is formed from a depth filter
material.
[0029] In certain embodiments, the matrix extends across the entire lumen of
the vessel
such that anything flowing from the inlet to the outlet must pass through at
least a portion of the
matrix.
[0030] In one aspect, this application provides a new multi-part vesicle which
speeds
digestion or solubilization of intact proteins, which minimizes the number of
transfer steps and
which affords quick use. This new vesicle provides first for the ability to do
the flow through
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described herein. It also provides for the ability to seal an inner vial
within an outer vial, and that
sonic energy and heat can be transferred from the outside to the inside. It
further provides that
flow through the matrix is most preferably uniform and uni-directional. The
inner vial can be
sealed against the outer vial so that solutions can be added to the inner vial
which can act on the
matrix and such solutions can be permeated into the matrix by capillary action
as well as
centrifugation. The inner vial can then be raised such that there is space
between the inner and
outer vials, so that the solution which was initially added can be centrifuged
into the outer vial.
The outer vial then becomes the container holding the oinks sample which will
be analyzed.
[0031] In an aspect of the application, a two-piece assembly for sequential
through-
matrix processing of solutions and/or solids is provided, the assembly having
an inner vial which
maintains and holds the matrix and an outer vial which is configured to
receive the inner vial at
the upper or lower parked positions, to respectively allow or impede passage
of the solution
through the matrix of the upper vial. The capability of the outer vial to
reversibly seal the inner
vial obviates the need for plugs and eliminates loss of sample to the plug.
The inner vial has an
inner chamber to receive a sample which may contain solids and liquids and via
treatments
solids may form from liquids. The outer vial has an inner chamber which can
alternately seal the
inner vial in the lower parked position or receive sample which flows from the
inner vial
through the matrix into the receiving space of the outer vial in the upper
parked position. The
inner vial and outer vial have opening on the top and are both afforded caps
or lids to protect the
sample and seal the space for samples. The inner vial lid additionally has a
vent to allow for
gases to escape in the case of heating, and the inner vial has an opening on
the bottom to allow
flow through the matrix it supports. In one preferred embodiment, the inner
and outer vials are
substantially cylindrical. The inner and outer vials have a locking or parking
or support system
such that the inner vial can be supported in the lower or upper parked
positions. In one preferred
embodiment, the support system consists of ridges and U-shaped stops; one
skilled in the art will
recognize that many other embodiments are possible, so long as the upper and
lower positions
are able to be maintained. The inner vial supports the matrix at its lower
portion.
[0032] In certain embodiments, in the upper parked position, the outer vial is
configured
to receive sample placed into the sample holding space of the upper vial which
flows from the
upper vial through the matrix supported by the inner vial through the opening
in the bottom of
the lower vial when the upper vial is in the upper parked position.
[0033] In certain embodiments, in the lower parked position the outer vial is
configured
to seal the inner vial and impeded flow through the matrix, allowing
incubation of the contents
of the inner vial, while also reducing the dead volume of solution in the
inner vial. In the lower
parked position, the inner and outer vials can be centrifuged to drive out any
air in the matrix
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and put a solution in full contact with the matrix, such as and not limited to
solutions containing
enzymes or chemicals to allow them to work on the material and molecules held
within or atop
the matrix.
100341 In an aspect of the application, a kit is provided that includes the
inner and outer
vials, the inner vial being charged with a matrix to meet the needs of the
required sample
treatment, and optionally any reagents or solutions or materials to implement
the steps of the kit.
100351 In an aspect of the application, a method of sample processing is
provided
including passing a solution having solvent, soluble contaminants, and
insoluble solid
components, all of which may be of interest, through the matrix of the present
application, such
that soluble materials without affinity for the matrix pass through into the
outer vial, materials
which bind to the matrix are retained and any insoluble materials are retained
in or on the
matrix.
100361 In an aspect of the application, a method of sample processing is
provided
including solubilizing some desired component of a sample, such as biological
molecules
including metabolites, lipids, proteins, nucleic acids, glycans and proteins,
capturing or trapping
or separating some fraction of molecules of interest, such as biopolymers,
often and not only by
the addition of coagulation agents including mild precipitants such as and not
limited to a wide
variety of organic solvents, potentially by inducing a phase change or
coagulation or binding of
said molecules and in all cases resulting in retention of the molecules of
interest, which can then
be trapped in or on or atop or within the matrix, or by affinities afforded to
the matrix for the
molecules of interest. With the sample fraction which is not trapped or
retained by the matrix
separated, the retained molecules can be subject to a wide range of treatments
including
chemical and/or enzymatic and/or chromatographic treatments which cause the
release of
desired components of the retained material, which can then be eluted. Sample
can be driven
through the matrix by positive pressure on the input side of the matrix or
negative pressure on
the output side or centrifugation.
BRIEF DESCRIPTION OF THE DRAWINGS
100371 FIG. 1 shows protein capture and digest from non-ionic detergent
lysates. Fig.
IA Protein capture in cellulose depth filter tips. 3% Octyl Glucoside (OG) and
3% Poloxamer
407 (P407) lysates were prepared in 30 tnivl Ammonium Acetate from IvIDA-MB-
231 cells by
sonication on ice. The lysates were loaded into the tips immediately or
diluted with equal
volumes of methanol in 30 mM Ammonium Acetate (final methanol concentration ¨
50%). The
captured proteins were eluted with 2X Laemmli buffer. Fig. 1B SiTrap-type in-
tip digestion of
MDA-MB-231 cellular lysate prepared with 3% Octyl Glucoside. The capture tips
were
constructed either with quartz or cellulose materials. Digestion was performed
according to
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SiTrap protocols. The digest products were eluted with 2X Laemmli buffer.
Samples were
analyzed on NuPAGE 4-12% Bis-Tris Protein Gels. A block flow diagram of a
general approach
for denaturing a biochemical agent using an activated cleaning fluid mist.
[0038] FIG. 2 shows MDA MB 231 cells were lysed by probe sonication on ice
using 30
mM ammonium acetate (AA), 1.8% ammonium hydroxide (AH) or 3% SDS in 30 mM
ammonium acetate (SDS). The lysates were centrifuged at 11,000 x g for 2 min
to remove
debris. For AA and SDS lysates 4 volumes of methanol in 30 mM acetate were
added to the
samples; for AH lysates equal volume of 1 M acetic acid was added to the
sample followed by
addition of 2 volumes of methanol. The proteins were then captured in
cellulose depth filters
and consequently eluted with 2X Laemmli buffer and run on NuPAGE 4-12% Bis-
Tris Protein
Gels.
[0039] FIG. 3 shows SiTrap processing of cellular material. Fig. 3A Basic
scheme. A
cell pellet is sonicated or otherwise physically disrupted and/or heated in
excess of either 30 mM
ammonium acetate (AA) or 1.8 % ammonium hydroxide (AEI). For AA extraction
four volumes
of methanol in 30 mM AA are added to the lysate. For AH extraction an equal
volume of 1M
acetic acid is added to the lysate followed by two volumes of methanol. The
resultant mix is
loaded into the SiTrap unit (1), the proteins are captured in the depth filter
trap and the flow-
through is collected (2, 3). Following a wash with 50% methanol, the proteins
are denatured,
reduced and allcylated in situ by heating at 80 C in 60 mM triethylammonitun
bicarbonate
(TEAR), 10 mM tris(2-carboxyethyl)phosphine (TCEP), 25 mM chloroacetamide
(CAA)
solution (4). After the wash (5) an enzyme is introduced to the trapped
proteins (6). After the
digestion, the peptides are eluted from the SiTrap tips (7). The peptides are
concentrated by
Stage tips for downstream analysis by mass spectrometry. Fig. 3B-Fig. 3D
Proteomics
comparison of SiTrap ammonium hydroxide (AH), SiTrap ammonium acetate (AA) and
standard SDS-based digests of MDA-MB-231 cells. Fig. 38 Box-plot diagram of
identified
protein numbers (at least two peptides were required for protein
identification). Fig. 3C Protein
distributions in the main GO cellular component categories. Fig. 3D Venn
diagram showing
distributions of the number of proteins identified with at least two peptides
for each of the three
sample preparation methods.
[0040] FIG. 4 shows digestion of a cellular lysate by SiTrap using cellulose
tips. MDA-
MB-231 cells were lysed by probe sonication on ice either with 30 mM ammonium
acetate (AA)
Fig. 4A or 1.8 % ammonium hydroxide (AH) Fig. 4B. The 30 pig of lysate was
loaded into
SiTrap tip according to the described protocol and the flow-through (FT1) was
collected.
Trapped proteins were reduced and allcylated in-situ for 30 min with 10 mM
TCEP and 25 mM
chloroacetamide in 60 mM TEAB at 80 C (FT2), digested with trypsin at 47 C
for 45 min and
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eluted with 2X Laemmli buffer. Samples were run on NuPAGE 4-12% Bis-Tris
Protein Gels.
[0041] FIG. 5 shows volcano plot significance analysis of the metabolomics and
proteomics profiling data for normal vs tumor renal sections. The significance
cut-offs were set
to 0.05 for false discovery rates (FDR). Fig. 5A The results of the
metabolomics analysis
indicate a decrease in both short chain acylcarnitines (C5, C5:1 and C3) and
in polyunsaturated
free fatty acids (C20:5, C20:4, C22:6) in the tumor samples. Fig. 5B The
results of the
proteomics analysis indicate downregulation of enzymes in the camitine
pathway, Camitine 0-
acetyltransferase (CRAT), Camitine 0-pahnitoyltransferase 2 (CPT2) and
Camitine 0-
palmitoyltransferase 1 (CPT1A) in the tumor samples. Downregulation of enzymes
in the
polyunsaturated fatty acid pathway, Acyl-CoA Thioesterase 1 (ACOT1) and long
chain Fatty
acid-CoA ligase (ACSL1), is also observed in the tumor samples.
[0042] FIG. 6 shows SiTrap proteomic and metabolomic analysis of renal tumors
identifies dysfunctional acylcarnitine (AC) metabolism. Fig. 6A Metabolomics
analysis
identifies decreased short chain acyl carnitines (C5, C5:1 and C3) in the
tumor samples. The Y
axes represent mean-centered relative concentrations. Fig. 6B Proteomics
analysis indicates
downregulation of Camitine 0-acetyltransferase (CRAT), Carnitine 0-
palmitoyltransferase 2
(CPT2) and Camitine 0-palmitoyltransferase 1 (CPT1A) in the tumor samples. The
Y axes
represent label-free quantitation (LFQ) intensity values.
[0043] FIG. 7 shows 0.5 pl of human serum from a healthy volunteer was either
digested
directly by SiTrap technology (6 replicate samples in total) or diluted with
20 mIvI TEAB buffer
and processed by fractionation using SiTrap quartz tips. SiTrap processing
produced two
fractions, captured and flow-through (3 replicates each for each fraction, 6
samples in total). The
MS results from tryptic digests of the 6 samples in each approach were merged.
[0044] FIG. 8 shows human renal FFPE tissue was deparaffinized by standard
xylene/ethanol treatment and then lysed in 30 mM ammonium acetate by probe
sonication. ¨50
pg of the resultant protein lysate was processed either by SiTrap or SDS
methods. The sample
was cleared of SDS by the standard protocol and the flow-through was collected
(FT). Similarly
to SiTrap, the proteins were digested at 48C by two consecutive 1-hour
digestions with L25 pg
of trypsin (Promega) in 100 mivI ammonium bicarbonate (trypsin concentration
0.07 pg/p1).
Digest products were eluted consecutively by 500 mM ammonium bicarbonate and
50%
acetonitrile in 0.2% formic acid. The leftover material was eluted by 2X
Laemmli buffer.
[0045] FIG. 9 shows a schematic of the use of the assembly. In Fig. 9A, the
matrix 117
is in contact with the solution 120 first applied to the inner sample holding
space of the inner
vial, potentially for treatments that require incubations, with the inner and
outer vials are in their
lower parked positions. In Fig. 9B, the inner vial has been moved to the upper
parked position
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and the solution 124 has passed through the matrix 117. Depending on the
matrix, it may have
molecules bound in it or potentially material 123 that cannot pass through the
matrix. In Fig. 9C,
treatment solution 127 has been applied to work on materials bound or retained
in or on or by
matrix 117 and any materials potentially not passing into the matrix 123. It
is in Fig. 9C that the
nested assembly is exposed in its lower portion 273 is exposed to heat or
sonic energy such as
ultrasonication or light or electromagnetic radiation such as microwaves, to
speed or facilitate
reactions, the details of which depend entirely on the experimental system.
After this processing
has been complete, as shown in Fig. 913 the vial is moved to its upper parked
position; in this
view the inner vial stops 153 and outer vial support mechanism 174 are not
visible. The solution
which has worked on the matrix and its retentate 125 is propelled through the
matrix 117 by
positive or negative pressure or by centrifugation to transfer the now
processed sample 126 to
the bottom of the outer vial in its sample collection area. After this process
is complete, the
sample is ready to store or analyze further as shown in Fig. 9E.
[0046] FIG. 10 shows the complete assembly of the inner vial rotated to engage
the
locking mechanisms of the inner and outer vials, in this embodiment three of
them, to hold the
inner vial in the upper parked position within the outer vial, and how sample
from the inner
space of the inner vial can flow through the matrix supported by the inner
vial to the receiving
space of the outer vial.
[0047] FIG. Ills another view of the inner and outer vial assembly showing the
other
side of the assembly where two locking mechanisms are engaged to hold the
upper parked
position.
[0048] FIG. 12 shows the inner and outer vial assembled in the lower parked
position in
which the inner vial is sealed against the outer vial to transmit externally
applied treatments and
seal the inner vial while removing dead space of the output of the inner vial
with a pin.
[0049] FIG. 13 shows both sides of the inner vial.
[0050] FIG. 14 is a cutaway of the lower parked position of the nested inner
and outer
vials illustrating the position of the matrix, pin which serves to remove dead
volume, and sample
collection region, as well as the tight interface between the inner and outer
vials through the
whole lower region of the inner and outer vials.
[0051] FIG. 15 shows an embodiment of the application arrayed in a 96-well
plate
format in which the inner plate bearing a matrix of inner vials is in the
lower parked position
with the outer plate and which is held in place by moveable hinged tab stops;
the array is
unmodified in the sealing and treatment transmission capabilities between the
inner and outer
vials/plates.
[0052] FIG. 16 shows an embodiment of the application arrayed in a 96-well
plate
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format in which the inner plate bearing a matrix of inner vials is supported
in the upper parked
position by moveable hinged tab stops to allow the contents of the inner plate
to flow through
the matrix of the wells of the inner plate to the sample collection region of
the outer plate.
[0053] FIG. 17 shows an embodiment of a locking mechanism to establish a lower
and
upper park position consisting of posts with corresponding notches which
afford the two
positions.
[0054] FIG. 18 shows an embodiment of a locking mechanism to establish two
positions
will allow or prohibit flow thorough the matrix via a side release design in
which the outer vial
either seals or does not seal against the inner vial depending on the position
of rotation. A snap
fit locking mechanism can afford sealing
[0055] FIG. 19 shows an embodiment of a locking mechanism to establish a lower
and
upper park position consisting of snaps which hold the inner vial at various
vertical heights
within the outer vial.
[0056] FIG. 20 shows an embodiment of a locking mechanism to establish a lower
and
upper park position consisting of multiple ridges, fine or coarse, which hold
the inner vial at
various vertical heights within the outer vial.
[0057] FIG. 21 is like Fig. 20 in demonstrating a potential embodiment of a
locking
mechanism to establish a lower and upper park position consisting of multiple
ridges to hold the
inner vial at various vertical heights within the outer vial, but has the
added advantage of having
a release where the ridges are disengaged by rotation to gaps that lack
interlocking ridges.
[0058] FIG. 22 shows a potential embodiment of a locking mechanism to
establish a
lower and upper park position consisting of a coarse thread where the inner
vial is screwed down
to seal or unscrewed to allow flow from the inner to outer vial.
[0059] FIG. 23 shows the various transitions of SARS-CoV-2 nucleoprotein
peptide
WYFYYLGTGPEAGLPYGANK
[0060] FIG. 24 shows the various transitions of SARS-CoV-2 nucleoprotein
peptide
DGIIWVATEGALNTPK.
[0061] FIG. 25 illustrates reversible SiTrap capture and release of RNA from
detergent-
containing and detergent-free conditions.
100621 Parts legend
101 Inner vial
109 Outer vial
111 Inner and outer vial assembled in the lower parked position to hold and
treat solutions
within the space of the inner vial 122 and/or materials on or atop or in or
within matrix 117
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113 Inner and outer vial assembled in the upper parked position to pass
solutions from the space
of the inner vial 122 through matrix 117 to the sample holding space of the
outer vial 222
115 Inner and outer plate assembly including means to support the upper and
lower parked
positions.
117 Matrix held in place by the inner vial
120 Sample first added to the inner vial in the parked position prior to
processing.
122 Space within the inner vial to hold sample including solid and/or liquid
sample and/or
treatment reagents in the upper or lower parked positions
123 Material potentially retained by the matrix
124 Initial flow through fraction which might be devoid of coagulated
material, have been
depleted of something via affinity in the matrix 117, might be free of
insoluble matter, etc.
125 Solution post processing as it is passing through the matrix of the inner
vial having worked
on material retained or bound in or on or by the matrix
126 Solution post processing that has passed through the matrix of the inner
vial
127 Treatment solution applied to work on material retained by or in the
matrix 117 and/or
anything on it like 123
129 Opening of the inner vial with surface to interface with the rib sealing
mechanism 248
137 Vent of the lid of the inner vial
145 D-pin which is configured to plug the inner vial and remove the dead space
of the output of
the inner vial up to the bottom of the matrix
153 Locking/stop/support mechanisms of the inner vial which engage with the
support
mechanism of the outer vial 174 to form a supported upper parked position to
allow the contents
held on the inside of the inner vial to flow through the matrix into the
receiving space 222 of the
outer vial
168 Living hinge of the outer vial connecting outer vial lid 217 with the main
body of the outer
vial
174 Support mechanism of the outer vial which interfaces with the locking
mechanisms or stops
of the inner vial 153
185 Hinge of the inner vial
195 Sample collection region of the outer vial at the lowest depth
203 Output of the outer vial; as depicted the outside dimensions fit lure lock
receptacles
217 Lid of outer the vial with tab 299 for opening and closing
222 Space within the outer vial which can receive and hold flow through of
sample that passes
through the matrix
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226 Space within the outer plate which can receive and hold flow through of
sample that passes
through the matrix supported by the inner plate
237 Lid of the inner vial with tab 281 for opening and closing
248 Rib sealing mechanism of the inner vial lid which seals the inner vial at
its top
256 Rib sealing mechanism of the outer vial lid which seals the inner vial at
its top
269 Inner vial bottom opening for receiving flow from the matrix and
transmitting it out of the
inner vial
273 Region of the outer vial which can receive the inner vial with a tight fit
and transmit from
the exterior of the outer vial heat and sonic energy to the inner vial, the
matrix of the inner vial
and any sample held in the inner vial, when the inner vial is in the lower
parked position
276 Tight interface between the inner and outer vials which facilitates flow
from the exterior of
the outer vial to the interior of the inner vial, its contents and matrix of
treatments such as heat
or light or electromagnetic radiation or sonic energy like ultrasonication
281 Tab of inner vial lid for opening and closing by hand or automation
299 Tab of outer vial lid for opening and closing by hand or automation
303 Plates bearing embodiment bearing the equivalent to 96 inner vials
308 Plates bearing embodiment bearing the equivalent to 96 outer vials
311 Hinge region of the outer plate which allows the inner plate to be held
down and sealed in
the lower parked position or held up in the upper parked position to
facilitate flow through the
matrices to the outer plate.
326 Support of the outer plate which holds the inner plate down to seal it
against the outer plate
in the lower parked position
331 Support of the outer plate which holds the inner plate up in the upper
parked position to
allow solution to flow
100631 Throughout the drawings, the same reference numerals and characters,
unless
otherwise stated are used to denote like features, elements, components or
portions of the
illustrated embodiments. Moreover, while the present disclosure will now be
described in detail
with reference to the figures, it is done so in connection with the
illustrative embodiments and is
not limited by the particular embodiments illustrated in the figures and
appended claims.
DETAILED DESCRIPTION
100641 Reference will be made in detail to certain aspects and exemplary
embodiments
of the application, illustrating examples in the accompanying structures and
figures. The aspects
of the application will be described in conjunction with the exemplary
embodiments, including
methods, materials and examples, such description is non-limiting and the
scope of the
application is intended to encompass all equivalents, alternatives, and
modifications, either
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generally known, or incorporated here. Unless otherwise defined, all technical
and scientific
terms used herein have the same meaning as commonly understood by one of
ordinary skill in
the art to which this application belongs. One of skill in the art will
recognize many techniques
and materials similar or equivalent to those described here, which could be
used in the practice
of the aspects and embodiments of the present application. The described
aspects and
embodiments of the application are not limited to the methods and materials
described.
100651 As used in this specification and the appended numbered paragraphs, the
singular
forms "a," "an" and "the" include plural referents unless the content clearly
dictates otherwise.
100661 Ranges may be expressed herein as from "about" one particular value,
and/or to
"about" another particular value. When such a range is expressed, another
embodiment includes
from the one particular value and/or to the other particular value. Similarly,
when values are
expressed as approximations, by use of the antecedent "about," it will be
understood that the
particular value forms another embodiment. It will be further understood that
the endpoints of
each of the ranges are significant both in relation to the other endpoint, and
independently of the
other endpoint. It is also understood that there are a number of values
disclosed herein, and that
each value is also herein disclosed as "about" that particular value in
addition to the value itself.
For example, if the value "10" is disclosed, then "about 10" is also
disclosed. It is also
understood that when a value is disclosed that "less than or equal to "the
value," greater than or
equal to the value" and possible ranges between values are also disclosed, as
appropriately
understood by the skilled artisan. For example, if the value "10" is disclosed
the "less than or
equal to 10" as well as "greater than or equal to 10" is also disclosed.
100671 The present application concerns, at least in part, a two-piece
"processing and
containment" assembly which includes an inner vial that maintains and supports
a matrix, which
may be multiple matrices in series such as a porous capture surface followed
by a
chromatographic media as is common in SPE such as C4, C8, C18, or ion exchange
resins such
as SCX, SAX, or metal binding surfaces such as IMAC, and which facilitates
processing a
sample into multiple fractions, and an outer vial which seals the inner vial
during incubation
andVor reaction steps in the lower parked position, which then in the upper
parked position
serves as a containment vesicle. The upper and lower parked positions are
afforded by locking
mechanisms between the inner and outer vial which in the lower parked
position, allow the inner
vial to seal against the very bottom of the outer vial, and which in the upper
parked position,
effected by minimal rotation, allow the inner vial to be supported at the
upper parked position so
that its contents can be passed through the matrix into the sample holding
space of the outer vial.
The combination of a containment vesicle and a sealing ability in the outer
vial, complete with a
pin to remove dead space of the output of the inner vial, minimizes sample
losses, minimizes
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elution volumes and maximizes throughput. The inner vial serves as a reaction
vessel in
capturing and processing steps. Reactions can occur within the inner vial
including atop or
within or on the matrix held by the inner vial. It the lower parked position,
the inner and outer
vial assembly can be centrifuged to ensure the surfaces and pores of the
matrix held by the inner
vial have all been cleared of air and are exposed to treatment reagents. The
assembly can be
disposable.
100681 The assembly can be manufactured in multiplexed formats such as 96-well
plates;
many other multiplexed samples can be contemplated. Some workflows may utilize
multiple
outer vials to capture first the initial flow through, then the results of
other treatment steps
received by the material retained insider the inner vial and on, atop, within
or in its matrix. In
preferred embodiments, molecules are forced by the addition of reagents to
bind to or within the
matrix, or to coagulate to themselves or onto the matrix or the molecules
themselves, allowing
all non-coagulated molecules to be passed through the matrix. After the inner
vial, now
containing the coagulated material retained via the matrix, has been placed
into a new outer vial
in the lower parked position, reagents are added to the sealed interior of the
lower vial to process
the material held atop or in or within or on the matrix.
100691 Treatments might include various elutions from chromatographic media
such as
salt cuts from ion exchange or organic solvent cuts from reverse phase, as
well as the above -
referenced chemical, enzymatic, heat, sonic or other kinds of treatments. This
application
simplifies sample treatments and eliminates the need to manually process
plugs. By integrating
steps of coagulation or precipitation or chemical treatment steps with matrix
processing that can
include filtration as well as binding including binding to chromatographic
surfaces (which can
be serial simply by stacking matrices), into a two-part assembly, this
application facilitates high
throughput including in via automation, robustness and reproducibility, as
well as cost-
effectiveness which will be essential as treatments are applied to large
scales such as in
personalized or precision medicine.
100701 Protein and DNA and glycan and other molecules and biopolymers are
captured
through a combination of at least two capture mechanisms. Any precipitant
particles such as
protein or biopolymer precipitant are physically trapped in the filter pores
and in-solution
protein material in the presence of the coagulant is adsorbed on the matrix
via non-covalent
interactions with the matrix surface by intentional modulation of the
chaotropicity of the solvent
containing the analyte molecules. Importantly, the flow-through after this
capture contains the
extracted physiological small molecules and does not include contaminants,
save the volatile or
non-interfering buffer components. Thus, this application provides that the
flow-through is a
suitable medium for profiling of metabolites and other unbound molecules.
Significantly and
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surprisingly, the captured biomolecules like proteins can still be reduced and
alky fated while in
the trap, consequently facilitating downstream in situ protein digestion and
proteomics analysis.
Other treatments, chemical and enzymatic, to proteins and other trapped
molecules can
surprisingly be performed in situ. It is a significant unexpected advantage of
the present
application that captured molecules can be treated with enzymes and/or
chemistries in situ in the
matrix, and without the need for the use of strong chaotropic agents such as
urea or detergents
like SDS.
100711 The present methods and systems can involve the use of extraction
solvents
which, despite having no detergent, are strongly solubilizing. For example, a
preferred buffer
for the present application is 1.8% ammonium hydroxide, which by proteomics
results
surprisingly showed similar ability to retrieve proteins as SDS. Unexpectedly,
the capture of
molecules from a neutral (or neutralized) extraction solution, without the
presence of a detergent
or chaotrope, supplemented with a mildly chaotropic coagulant, for example the
aqueous
methanolic compositions described herein, provides very favorable conditions
for the method of
the present application, namely capture of molecules in native or near-native
state, and capture
with a high surface area-to-volume ratio which makes the molecules
particularly sensitive to
enzymatic or chemical treatments and/or manipulations within the trapping
matrix, while also
allowing for the selective recovery of various classes of molecules. For
example, proteins so
captured are highly protease-sensitive. Additionally, capture in the native
state allows the use of
enzymes which require native tertiary structure of biomolecules. By non-
limiting example, the
enzyme FabRICATOR digests IgG at a specific site below the hinge region,
generating a
homogenous pool of F(ab4)2 and Fc/2 fragments. FabRICATOR can be used to
enzymatically
process antibodies in the workflow of this application, by contrast,
FabRICATOR cannot be
used after other techniques of sample preparation such as protein
precipitation.
[0072] Another specific and preferred embodiment of the present application is
the
addition of two volumes of methanol to sample first extracted with 1.8%
ammonium hydroxide
sonication (by probe or otherwise), which was then neutralized by addition of
an equal volume
of 1 M acetic acid. To this neutralized solution can be additionally added
coagulant, specifically
such as two volumes of methanol, although other ratios may be advantageous.
This particular
approach is unique and totally surprising in that upon neutralization,
biomolecules instantly form
enzyme-sensitive aggregates which can be captured, separated from smaller non-
aggregated
molecules, washed, and further extracted and/or processed by chemical or
enzymatic means.
Definitions
100731 As used herein, the term "virus" can include, but is not limited to,
influenza
viruses, herpesviruses, polioviruses, noroviruses, and retroviruses. Examples
of viruses include,
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but are not limited to, human immunodeficiency virus type 1 and type 2 (HIV-1
and HIV-2),
human T-cell lymphotropic virus type I and type II (HTLV-I and HTLV-II),
hepatitis A virus,
hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis delta virus (HDV),
hepatitis E virus
(HEV), hepatitis G virus (HGV), parvovirus B19 virus, hepatitis A virus,
hepatitis G virus,
hepatitis E virus, transfusion transmitted virus (TTV), Epstein-Barr virus,
human
cytomegalovirus type 1 (ICMV-1), human herpesvirus type 6 (HI-[V-6), human
herpesvirus
type 7 (11:11V-7), human herpesvirus type 8 (HUY-8), influenza type A viruses,
including
subtypes H1N1 and H5N1, human metapneumovirus, severe acute respiratory
syndrome
(SAPS) coronavirus, SARS-CoV-2, Middle East respiratory syndrome (MERS),
hantavints, and
RNA viruses from Arenaviridae (e.g., Lassa fever virus (LFV)), Pneumoviridae
(e.g., human
metapneumovirus), Filoviridae (e.g., Ebola virus (EBOV), Marburg virus (MBGV)
and Zika
virus); Bunyaviridae (e.g., Rift Valley fever virus (RVFV), Crimean-Congo
hemorrhagic fever
virus (CCHFV), and hantavirus); Flaviviridae (West Nile virus (WNV), Dengue
fever virus
(DENV), yellow fever virus (YFV), GB virus C (GBV-C; formerly known as
hepatitis G virus
(HGV)); Rotaviridae (e.g. rotavirus), and combinations thereof In one
embodiment, the subject
is infected with HIV-1 or HIV-2.
[0074] The genetically diverse Orthocoronavirinae family is divided into four
genera
(alpha, beta, gamma, and delta coronaviruses). Human CoVs are limited to the
alpha and beta
subgroups. Exemplary human CoVs include severe acute respiratory syndrome
coronavirus-2
(SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle
East
respiratory syndrome coronavirus (MERS-CoV), HCoV-229E, HCoV-0C43, HCoV-NL63,
and
HCoV-HKUl.
[0075] Nonlimiting examples of subgroup la alphacoronaviruses and their
GenBank
Accession Nos. include FCov.FIPV.79.1146.VR.2202 (NV_007025), transmissible
gastroenteritis virus (TGEV) (NC 002306; Q811789.2; DQ811786.2; DQ811788.1;
DQ811785.1; X52157.1; AJ011482.1; KC962433.1; AJ271965.2; JQ693060.1;
KC609371.1;
JQ693060.1; JQ693059.1; JQ693058.1; JQ693057.1; JQ693052.1; JQ693051.1;
JQ693050.1);
porcine reproductive and respiratory syndrome virus (PRRSV) (NC_001961.1;
DQ811787), as
well as any subtype, clade or sub-clade thereof, including any other subgroup
la coronavirus
now known (e.g., as can be found in the GenBank Database) or later identified
in the
GenBank Database.
[0076] Nonlimiting examples of a subgroup lb alphaeoronaviruses and their
GenBank
Accession Nos. include HCoV.NL63.Amsterdam.I (NC 005831),
BtCoV.HKU2.HK.298.2006
(EF203066), BtCoV.HKUIHK.33.2006 (EF203067), BtCoV.HKU2.HK.46.2006 (EF203065),
BtCoV.HKU2.613.430.2006 (EF203064), BtCoV.1A.AFCD62 (NC 010437),
19
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BtCoV.1B.AFCD307 (NC_010436), BtCov.HKU8.AFCD77 (NC_010438), BtCoV.512.2005
(DQ648858); porcine epidemic diarrhea viruses (NC_003436, DQ355224.1,
DQ355223.1,
DQ355221.1, JN601062.1, JN601061.1, JN601060.1, JN601059.1, JN601058.1,
JN601057.1,
JN601056.1, JN601055.1, JN601054.1, JN601053.1, JN601052.1, JN400902.1,
JN547395.1,
FJ687473.1, FJ687472J, FJ687471.1, FJ687470.1, FJ687469,1, FJ687468.1,
FJ687467J,
FJ687466.1, FJ687465.1, FJ687464.1, FJ687463.1, FJ687462.1, FJ687461.1,
FJ687460.1,
FJ687459.1, FJ687458.1, FJ687457.1, FJ687456.1, FJ687455.1, FJ687454.1,
FJ687453
FJ687452.1, FJ687451.1, FJ687450.1, FJ687449.1, AF500215.1, ICF476061.1,
1CF476060.1,
KF476059.1, KF476058.1, KF476057.1, ICF476056.1, KF476055.1, KF476054.1,
1CF476053.1,
KF476052.1, KF476051.1, ICF476050.1, ICF476049.1, KI476048.1, KF177258.1,
KF177257.1,
KF177256.1, KF177255.1), HCoV.229E (NC 002645), as well as any subtype, clade
or sub-
dade thereof, including any other subgroup lb coronavirus now known (e.g., as
can be found in
the GenBank Database) or later identified in the GenBank Database.
100771 Nonlimiting examples of subgroup 2a belacoronaviruses and their GenBank
Accession Nos. include HCoV.HKUl.C.N5 (DQ339101), NIFIV.A59 (NC_001846),
PHEV.VW572 (NC 007732), HCoV.0C43.ATCC.VR.759 (NC 005147), bovine enteric
coronavirus (BCoV.ENT) (NC_003045), as well as any subtype, Glade or sub-
cladle thereof;
including any other subgroup 2a coronavirus now known (e.g, as can be found in
the
GenBank Database) or later identified in the GenBank Database.
MOM] Nonlirniting examples of subgroup 2b betacoronavirases and their GenBank
Accession Nos. include human SARS CoV-2 isolates, such as Wuhan-Hu-1
(NC_045512.2) and
any CoV-2 isolates comprising a genomic sequence set forth in GenBank
Accession Nos., such
as MT079851.1, MT470137.1, MT121215.1, MT438728.1, MT470115.1,MT358641.1,
MT449678.1, MT438742.1, LC529905.1, MT438756.1, MT438751.1, MT460090.1,
MT449643.1, MT385425.1, MT019529.1, MT449638.1, MT374105.1, MT449644.1,
MT385421.1, MT365031.1, MT385424.1, MT334529.1, MT466071.1, MT461669.1,
MT449639.1, MT415321.1, MT385430.1, MT135041.1, MT470179.1, MT470167.1,
MT470143.1, MT365029.1, MT114413.1, MT192772.1, MT135043.1, MT049951.1; human
SARS CoV-1 isolates, such as SARS CoV.A022 (AY686863), SARSCoV.CUHK-W1
(AY278554), SARSCoV.GDO1 (AY278489), SA1RSCoV.HC.SZ.61.03 (AY515512),
SARSCoV.SZ16 (AY304488), SARSCoV.Urbani (AY278741), SARSCoV.civet010
(AY572035), SARSCoV.MA.15 (DQ497008); bat SARS CoV isolates, such as
BtSARS.HKU3.1 (DQ022305), BtSARS.HKU3.2 (DQ084199), BtSARS.HKU3.3 (DQ084200),
BtSARS.Ftml (DQ412043), BtCoV.279.2005 (DQ648857), BtSARS.Rfl (DQ412042),
BtCoV.273.2005 (DQ648856), BtSARS.Rp3 (DQ071615), ), as well as any subtype,
clade or
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sub-clade thereof, including any other subgroup 2b coronavirus now known
(e.g., as can be
found in the GenBank Database) or later identified in the GenBank Database.
[0079] Nonlimiting examples of subgroup 2c betacoronavintses and their GenBank
Accession Nos. include Middle Fast respiratory syndrome coronavirus (MERS)
isolates, such as
Riyadh 22012 (1CF600652.1), Al-Hasa_18_2013 (1CF600651.1), Al-Hasa 17_2013
(KF600647.1), Al-Hasa 152013 (ICF600645.1), Al-Hasa 16 2013 (KF600644.1), Al-
Hasa 21 2013 (1CF600634), Al-Hasa 19_2013 (CF600632), Buraidah 1 2013
(KF600630.1),
Hafr-A1-Batin_1_2013 (1C_F600628.1), Al-Hasa 122013 (1CF600627.1),
Bisha.ltoreq.1_2012
(KF600620.1), Riyadlc3 2013 (KF600613.1), Riyadh_l 2012 (1CF600612.1), Al-Hasa
3 2013
(KF186565.1), Al-Hasa 1_2013 (CF186567.1), Al-Hasa_2_2013 (KF186566.1), Al-
Hasa 4 2013 (K.F186564.1); Betacoronayirus England 1-N1 (NC 019843), SA-N1
(KC667074); human betacoronayirus 2c Jordan-N3/2012 (KC776174.1); human
betacoronavirus 2c EMC/2012, (JX869059.2); any bat coronavirus subgroup 2c
isolate, such as
bat coronavirus Taper/CII_KSA_287/Bisha/Saudi Arabia (TU493885,1), bat
coronavirus
Rhhar/CII KSA 003/Bisha/Saudi Arabia/2013 (1CF493888.1), bat coronavirus
Pikuh/CII KSA 001/Riyadh/Saudi Arabia/2013 (CF493887.1), bat coronavirus
Rhhar/CII_KSA 002/Bisha/Saudi Arabia/2013 (1CF493886.1), bat coronavirus
Rhhar/CII KSA 004/Bisha/Saudi Arabia/2013 (KF493884.1), bat coronavirus
BtCoV.HKU4.2
(EF065506), bat coronavirus BtCoV.HKU4.1 (NC_009019), bat coronavirus
BtCoV.HKU4.3
(EF065507), bat coronavirus BtCoV.FIKU4.4 (EF065508), bat coronavirus
BtCoV133.2005
(NC_008315), bat coronavirus BtCoV.HKU5.5 (EF065512), bat coronavirus
BtCoV.HKU5.1
(NC_009020), bat coronavirus BtCoV.HKU5.2 (EF065510), bat coronavirus
BtCoV.HKU5.3
(EF065511), and bat coronavirus HKU5 isolate (KC522089.1); any additional
subgroup 2c, such
as KF192507.1, KF600656.1, ICF600655.1, ICF600654.1, KF600649.1, ICF600648.1,
1CF600646.1, KF600643.1, ICF600642.1, ICF600640.1, KF600639.1,
KF600638.1,1CF600637.1,
KF600636,1, KF600635.1, ICF600631. 1, KF600626.1, 'U600625.1, 1CF600624,1,
1CF600623.1,
KF600622.1, KF600621.1, KF600619.1, KF600618.1, KF600616.1, 1CF600615.1,
1CF600614.1,
ICF600641.1, ICF600633.1, KF600629.1, KF600617.1, KC8696782; KC522088.1,
KC522087.1, KC522086.1, KC52208.5.1, KC522084.1, KC522083.1, KC522082.1,
KC522081.1, KC522080.1, KC522079.1, KC522078.1, KC522077.1, KC522076.1,
KC522075.1,KC522104.1, KC522104.1, KC522103.1, KC522102.1, KC522101.1,
KC522100.1, KC522099.1, KC522098.1, KC522097.1, KC522096.1, KC522095.1,
KC522094.1, KC522093.1, KC522092.1, KC522091.1, KC522090.1, KC522119.1,
KC522118.1,KC522117.1, KC522 n 6.1, KC52211_ 5.1, KC522114.1, KC522113.1,
KC522112.1,KC522111,1, KC522110.1,KC522109.1, KC522108.1õKC522107,1,
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KC522106.1, KC522105.1); Pipistrellus bat coronavirus HKU4 isolates
(KC522048.1,
KC522047.1, KC522046, 1, KC522045.1, KC522044.1, KC522043.1, KC522042.1,
KC 522041.1, KC522040.1, KC522039.1õ KC 522038.1,, KC522037.1õ KC 522036.1,,
KC522048.1, KC522047.1, KC522046.1, KC522045.1, KC522044.1, KC522043.1,
KC522042.1, KC522041.1, KC522040, 1, KC522039.1, KC522038.1, KC522037.1,
KC522036.1, KC522061.1, KC522060.1, KC5220591, KC522058.1, KC522057.1,
KC522056.1, KC522055.1, KC522054.1, KC522053.1, KC522052.1, KC522051.1,
KC522050.1, KC522049.1, KC522074.1, KC522073.1, KC522072.1, KC522071.1,
KC522070.1, KC522069.1, KC522068.1, KC522067.1, KC522066.1, KC522065.1,
KC522064.1, KC522063.1, KC522062.1 ), as well as any subtype, clade or sub-
clade thereof,
including any other subgroup 2c coronavirus now known (e.g., as can be found
in the
(lenBank Database) or later identified in the GenBank Database.
[0080] Nonlimiting examples of subgroup 2d betacoronaviruses and their GenBank
Accession Nos. include BtCoV.HKU9.2 (EF065514), BtCoV.HKU9.1 (NC_009021),
BtCoV.HkU9.3 (EF065515), BtCoV.HKU9.4 (EF065516), as well as any subtype,
clade or sub-
dade thereof, including any other subgroup 2d coronavirus now known (e.g., as
can be found in
the GenBank Database) or later identified in the GenBank Database.
[0081] Nonlimiting examples of subgroup 3 gammacoronaviruses include
IBV.Beaudette.IBV.p65 (DQ001339) or any other subgroup 3 coronavirus now known
(e.g., as
can be found in the GenBank Database) or later identified in the GenBank
Database.
[0082] A coronavirus defined by any of the isolates or genomic sequences in
the
aforementioned subgroups la, lb, 2a, 2b, 2c, 2d and 3 can be targeted.
[0083] As used herein, the term "bacteria" shall mean members of a large group
of
unicellular microorganisms that have cell walls but lack organelles and an
organized nucleus.
Synonyms for bacteria may include the terms "microorganisms", "microbes",
"germs", "bacilli",
and "prokaryotes." Exemplary bacteria include, but are not limited to
Mycobacterium species,
including M. tuberculosis; Staphylococcus species, including S. epiderinidis,
S. aureus, and
methicillin-resistant S. aureus; Streptococcus species, including S.
pneumoniae, S. pyogenes, S.
mutans, S. agalactiae, S. equi, S. canis, S. bovis, S. equinus, S. anginosus,
S. sanguis, S.
salivarius, S. mitis; other pathogenic Streptococcal species, including
Enterococcus species,
such as E. faecalis and E. faecium; Haemophilus influenzae, Pseudomonas
species, including P.
aeruginosa, P. pseudomallei, and P. mallei; Salmonella species, including S.
enterocolitis, S.
typhimurium, S. enteritidis, S. bongori, and S. choleraesuis; Shigella
species, including S.
flexneri, S. sonnei, S. dysenteriae, and S. boydii; Brucella species,
including B. melitensis, B.
suis, B. abortus, and B. pertussis; Neisseria species, including N.
meningitidis and N.
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gonorrhoeae; Escherichia coil, including enterotoxigenic E. coli (ETEC);
Vibrio cholerae,
Helicobacter pylori, Geobacillus stearothermophilus, Chlamydia trachomatis,
Clostridium
difficile, Cryptococcus neoformans, Moraxella species, including M.
catarrhalis, Campylobacter
species, including C. jejuni; Corynebacteritun species, including C.
diphtheriae, C. ulcerans, C.
pseudotuberculosis, C. pseudodiphtheriticum, C. urealyticum, C. hemolytictun,
C. equi; Listeria
monocytogenes, Nocardia asteroides, Bacteroides species, Actinomycetes
species, Treponema
pallidum, Leptospirosa species, Klebsiella pneumoniae; Prateus sp., including
Proteus vulgaris;
Serratia species, Acinetobacter, Yersinia species, including Y. pestis and Y.
pseudotuberculosis;
Francisella tularensis, Enterobacter species, Bacteriodes species, Legionella
species, Borielia
burgdorferi, and the like. As used herein, the term "targeted bioterror
agents" includes, but is
not limited to, anthrax (Bacillus antracis), plague (Yersinia pestis), and
tularemia (Franciscella
tularensis).
100841 As used herein, the term "fungi" shall mean any member of the group of
saprophytic and parasitic spore-producing eukaryotic typically filamentous
organisms formerly
classified as plants that lack chlorophyll and include molds, rusts, mildews,
smuts, mushrooms,
and yeasts. Exemplary fungi include, but are not limited to, Aspergillus
species,
Dermatophytes, Blastomyces derinatitidis, Candida species, including C.
albicans and C.krusei;
Malassezia furfur, Exophiala wemeckii, Piedraia hortai, Trichosporon beigelii,
Pseudallescheria
boydii, Madurella grisea, Histoplasma capsulatum, Sporothrix schenckii,
Histoplasma
capsulatum, Tinea species, including T. versicolor, T. pedis T. unguium, T.
cruris, T. capitus, T.
corporis, T. barbae; Trichophyton species, including T. rubrum, T.
interdigitale, T. tonsurans, T.
violaceum, T. yaotuidei, T. schoenleinii, T. megninii, T. soudanense, T.
equinum, T. erinacei,
and T. verrucosum; Mycoplasma genitalia; Microsporutn species, including M.
audouini, M.
ferrugineum, M. canis, M. nanum, M. distortum, M. gypseum, M. fulvurn, and the
like.
100851 As used herein, the term "protozoan" shall mean any member of a diverse
group
of eukaryotes that are primarily unicellular, existing singly or aggregating
into colonies, are
usually nonphotosynthetic, and are often classified further into phyla
according to their capacity
for and means of motility, as by pseudopods, flagella, or cilia. Exemplary
protozoans include,
but are not limited to Plasmodium species, including P. falciparum, P. vivax,
P. ovate, and P.
malariae; Leishmania species, including L. major, L. tropica, L. donovani, L.
infantum, L.
chagasi, L. mexicana, L. panamensis, L. braziliensis and L. guyanensi;
Cryptosporidium,
Isospora belli, Toxoplasma gondii, Trichomonas vaginalis, and Cyclospora
species.
100861 By 'capture', 'retain' and related terms in the context of the matrix
and biological
molecules including large molecules and fragments of large molecules, it is
meant that the
matrix and molecules interact such that the molecules especially large
molecules are retained on
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and/or in the matrix after the molecules are exposed to the coagulant. The
interaction is typically
non-covalent, and may be an intermolecular interaction or simple retention on
the basis of size.
The specific nature of the interaction is not critical. However, the matrix
can retain molecules
especially large molecules such as (and not limited to) DNA, RNA, protein and
glycans
following addition of the coagulation medium, allows for washes as needed,
prevents excessive
aggregation, allow for smaller molecules to be captured in the flow through
thus separating the
small molecules from the large molecules, that the matrix allows for chemical
and/or enzymatic
treatment such as with (and not limited to) protease, nucleases and
glycosidases, and the matrix
allows the molecules or fractions of molecules to be eluted afterwards, most
preferably in
separate elution steps. If needed, bound molecules can be washed with a
solvent which does not
dissolve the captured molecules; correspondingly, different classes of
molecule are eluatable and
thus fractionateable with different solvents.
[0087] In the following, "matrix" shall mean "one or a combination of
matrices."
[0088] The term herein "analyte" means the molecule or molecules desired to
analyze
which could include proteins, DNA, RNA, gjycans, lipids, small molecules such
as metabolites,
drugs and vitamins, etc. Analytical techniques which can be used to analyze
analytes are well
known to one skilled in the art and include mass spectrometry, NMR, antibody
assays,
nanopores, nucleic acid tagging technologies, and many others.
[0089] The term herein "contaminant," mean moieties which interfere with
downstream
processing and/or analysis. Contaminants may include salts, buffers,
chaotropes, detergents, or
components which are naturally found in the sample such as phospholipids or
components
which are added to the sample by the user during other sample treatment steps
such as reduction
and alkylation reagents.
[0090] The term herein "robustness," means that use of the system, its
assembly or
components in the methods of the present application produce results which are
highly
reproducible.
[0091] The term herein "throughput" means the speed at which a single sample
can be
processed, or the speed and ability to process multiple samples in parallel,
often by automation.
[0092] The term herein "simple-to-use," means the ability to maintain all
desired aspects
of sample treatment and manipulation including recovery and separation of
analytes, robustness
and throughput with minimal former training with minimal chance for the
perturbations in the
sample processing to result in failed treatment.
100931 As referred to herein, "a strong chaotropic agent" is a reagent that
causes total
denaturation of biological molecules and typically prevents capture and
binding to the capture
matrix. Chaotropes are compounds of many kinds that induce disorder in
biological
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macromolecules and supramolecular assemblies, disrupting especially hydrogen
bonds. They
tend to disrupt phospholipid membranes and weaken or unfold the three-
dimensional structures
of proteins and nucleic acids. The exact mechanism in which chaotropes work is
complex and
depends on the specific substance; some are more disordering than others at
the same
concentration and we thus recognize stronger and weaker chaotropes. Urea and
guanidinium
salts are commonly recognized as strong chaotropes that, in high enough
concentrations, cause
total denaturation and typically dissolution of biological samples and their
molecules at large
concentrations such as 8 M or 6 M. Strong chaotropes are to be avoided in the
choice of
extraction solvents of the present invention as they inhibit coagulation and
thus prevent binding.
10094] As referred to herein, "a mild chaotropic agent" is a chaotropic agent
that does
not fully denature biomolecules and that facilitates binding to the trapping
matrix. Mild
chaotropes give molecules structural freedom and encouraging protein extension
and
denaturation while not completely linearizing biopolymers and denaturing
biomolecules. Such
mild chaotropes reduce the amount of order in the structure of a protein
formed by water
molecules, both in the bulk and the hydration shells around hydrophobic amino
acids, which
allows molecules to present typically hydrophobic interior regions to each
other as well as the
binding surfaces of the matrix, resulting in binding. Many kinds of molecules
are chaotropes that
can effect various levels of disorder in biological molecules including, and
are not limited to,
alcohols and other organic solvents such as benzene, sugars, glycerol,
zwitterions, even vanillin
among many, many other compounds (see Timson, D. J. (2020). The roles and
applications of
chaotropes and kosmotropes in industrial fermentation processes. World Journal
of
Microbiology and Biotechnology, 36(6). doi:10.1007/s11274-020-02865-8). Like
the science of
chaotrops in general, with regard to the present invention, the determination
of the strength
chaotropicity of a given agent is an empirical one which hinges on the
chaotrope's ability to
facilitate coagulation of the analyte molecules of interest to the binding
matrix at hand. Mild
chaotropes function only in the context of the combination of the extraction
solvent and
molecule coagulant.
10095] As referred to herein, a "molecule coagulant" shall mean a reagent or
combination of reagents which when mixed with the extraction solvent, which
may have been
pH adjusted, promotes the retention and binding of analytes of interest in the
protein trap via
especially non-covalent mechanisms such as hydrophobic or hydrophilic
interactions or ionic
interactions. Molecule coagulants are so chosen that they do not cause
excessive aggregation of
coagulated analyte molecules, which would impede flow through the binding or
capture matrix,
though they may promote intermolecular interactions to make analyte molecules
more apt to
bind to the capture matrix. In many embodiments, while coagulants promote
binding, they do so
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in a mostly native state of biomolecules. A potential coagulant can be easily
tested by first
determining if it hinders sample processing, which would indicate that it
causes excessive
aggregation, second if it promotes binding (e.g. analyze flow through for, by
example, the
presence or absence of protein, if that molecular class is of interest), and
third if it hinders
subsequent processing steps (such as treatment with reduction and alkylation
reagents followed
by trypsin). Effective molecule coagulants must not hinder sample processing,
must promote
binding, and must not hinder subsequent processing done on- or in-matrix.
100961 As referred to herein, an "extraction solvent" shall mean a solvent
with the ability
to dissolve or substantially dissolve, perhaps under conditions of agitation
such as physical
agitation or thermal agitation or sonic or ultrasonic agitation, one or more
classes of desired
analyte molecule. While in principle extraction solvents might have components
such as urea or
detergents, the presence of such non-volatile compounds interferes with
downstream analysis.
Examples of extraction solvents include hydrophobic organics chosen to
dissolve hydrophobic
components of a sample like lipids and hydrophobic proteins, which might then
be made less
hydrophobic by the addition of a more polar molecule coagulant, causing the
hydrophobic
components to bind to the matrix, volatile acids and bases such as
hydrochloric acid or formic or
acetic acid or other mostly volatile acids, or ammonium hydroxide or
tetramethylamrnonium
hydroxide, all of which can be neutralized, which are mass-spec compatible and
which can be
mixed with molecule coagulants to cause the aggregation of analytes on the
matrix.
100971 Extraction solvents and molecule coagulants should preferably be
volatile
mixtures, or mixtures which do not interfere with down-stream analysis, or
alternatively
mixtures from which interfering components can be easily and quickly removed
to non-
interfering levels. Extraction solvents and molecule coagulants must be so
chosen they, once
combined, yield conditions that promote analyte binding to the matrix_
Extraction solvents and
molecule coagulants can be mixtures and dissolve substances of interest, can
be easily handled
in or do not interfere with downstream analysis and processing, and must have
conditions (of
temperature, time, pH, concentration, etc. all of which might be different
depending on the kind
of matrix) that foster the binding or coagulation or capture of one or more
class of molecule of
interest to the matrix,
Capture Matrix
100981 Herein disclosed is a two-piece sample processing assembly with
integrated
matrix which enhances the speed and simplicity of sample processing that
requires incubation
steps, which formerly necessitated the repeated application and removal of
plugs, causing delay,
irreproducibility, inability to automate and sample loss. The assembly can be
used in any case
where some fraction of a sample must be passed through a matrix, and the
material which is
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retained on or in or by the matrix will be further treated with a reagent that
requires time to
operate i.e. requires an incubation under some conditions of time,
temperature, etc. The
assembly is consequently useful in many analytical fields from analysis of
environmental to
clinical samples. While the exact protocols of use depend fully on the
composition of the matrix
and treatments received by the samples, disclosed herein are the steps to
produce samples of
metabolites, lipids, nucleic acids, glycans and proteins. In many embodiments,
the assembly is
to be fabricated by injection molding of plastics and is disposable to prevent
sample crossover
and contamination. The assembled system especially in the lower parked
position is specifically
contemplated to be exposed to conditions conducive to treatments afforded to
the samples bound
or retained on or in or by the matrix. Such treatments might include
especially temperature and
sonic energy, but other treatments are possible.
[0099] It is preferred that the matrix is a porous or fibrous material which
is able to be
penetrated by the medium comprising the large molecules. Such a porous or
fibrous material
may also be formed from a powder or pieces or beads. Furthermore, the matrix
should be a
suitable material to permit the large molecules to be reversibly captured by
the matrix. The
matrix, through its pores and rough surface, affords in ultrasonication
nucleation promoting
features which lower cavitation thresholds (ultra)sonication bubble
nucleation, growth and
collapse, and in so doing, promote the action of (ultra)sonication in an on
and within the matrix.
The methods herein, thus may speed sonic or ultrasonic processing steps.
[0100] The presence of such a matrix allows for aggregation of the large
molecules to be
moderated from the medium to which the coagulant has been added. If no such
matrix were
present, the large molecules would tend to aggregate together in an
uncontrolled manner. This is
undesirable as it makes further processing of the large molecules more
difficult or impossible.
For example, digestion of captured proteins with a protease is impeded without
aggregates first
being disrupted by a chaotropic agent such as concentrated urea, or a
detergent, either and all of
which can then interfere in downstream analysis. Similarly, a nuclease may be
unable to access
DNA which is aggregated together with proteins and other co-aggregated
molecules, or a
glycosidase unable to access gly cans including those attached to proteins.
[0101] Essentially, capture of large molecules in the matrix also allows for
their
sequential elution, which is essential to the generation of multiple classes
of analyte for multi-
omics analysis. Furthermore, having the large molecules captured in the matrix
allows for
washing (rinsing) of the matrix and large molecules to be performed to remove
any
contaminants and/or separate different molecular classes whilst ensuring the
captured molecules
are not lost or diluted excessively, which would make further processing
problematic.
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[0102] There are many materials which are potentially suitable for use as a
matrix in the
present application, and therefore the choice of a specific set of materials
is not limiting.
Various exemplary suitable materials, and general properties of such
materials, will be described
below, but it will be apparent to the skilled person that other materials can
be used, including
small beads used in chromatography, or surfaces otherwise derivatized for
sample processing
such as a C18 surface (on beads or on a membrane) or a mixed bed containing,
by example,
reverse phase and ion exchange media comingled, or any other material which
fulfils the below
criteria, and which may have additional properties.
[0103] While many other matrices are possible, particularly preferred matrixes
comprise
depth filter materials.
[0104] The key consideration in the context of the present application is that
the matrix
(typically a depth filter) is able to bind and retain the (typically large(r))
molecules
supplemented with a coagulation media and retain those molecules during
subsequent washing
and processing steps, maintain them in a form such that enzymes can be used to
alter the
physical state of the retained molecules, especially to reduce the size of
larger molecules such as
proteins, DNA and RNA, or glycans, or to free from captured molecules moieties
of interest
such as glycans or lipids or ubiquitination or other molecular feature which
can be accessed with
chemical and/or enzymatic treatments. The suitability of any putative matrix
can be assessed by
testing it in a protocol as described in the examples below. One of ordinary
skill will be able to
identify alternative suitable matrix materials.
[0105] By way of general guidance, the matrix typically:
¨ is adapted to capture and retain fine and very fine particles, e.g. from
several
micrometers (e.g. 20 pm or less, 10 pm or less, 5 pm or less, or 2 pm or less)
to sub-
micrometer size range (e.g. down to 0.2 inn or even 0.1 pm in size);
¨ is substantially inert with respect to the molecules of the sample;
¨ is able to reversibly capture (La retain) molecules such as proteins and
DNA or RNA or
lipids or glycans, from the sample when the sample is exposed to a coagulation
medium;
¨ allows for chemical and/or enzymatic processing of the captured
molecules, for example
a protease can be used to digest the proteins in situ, or a nuclease can be
used to produce
smaller sizes of DNA or RNA, or a glycosidase can release glycans from protein
or does
not bind to, and therefore retain, the surfactant to any significant extent.
[0106] The capture matrix, which can be a depth filter, but which simply must
be porous,
may in some embodiments be derivatized with enzymes for processing of the
sample, for
example with proteases or nucleases or lipases or glyc,osidases.
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[0107] The matrix is held within the inner vial by a variety of techniques
known to one
skilled in the art including hermetically sealing or plastic welding or heat
welding or ultrasonic
welding or alternatively by the physical size of the matrix and friction as it
is forced into the
narrowing bottom of the inner vial or the use of adhesives or fits or support
systems such as
screens or plastic scaffolding, which could include support or retention rings
or screens among
other possibilities.
[0108] The matrix or matrices can be anything porous such as filtration
material,
chromatographic material, material with affinities, membranes, frits, SPE
material, filters, depth
filters, etc. In the case of lose chromatographic beads, frits can be afforded
at the bottom and
top, or only the bottom Suitable materials for the matrix of the present
application include
porous matrices such as sintered materials or porous plastics or membranes of
defined or
approximately defined porosity. Exemplary materials include porous
polyethylene (PE),
polypropylene (PP), polytetrafluoroethylene (PTFE) and sintered
polytetrafluorethylene.
Suitable materials might also include porous materials made by sintering glass
or other
materials, various filters including paper or glass or depth filters, glass
membrane filters,
membranes with specific molecular weight cutoffs, or membranes with specific
pore sizes such
as 0.2 micron, 2 micron, 20 micron, among many others. In addition to
membranes and sheets,
matrices can include lose beads or powders, depending on the use case. If lose
powder, the
dimensions of the particles (particle diameters) may be of a size similar to
liquid
chromatography, or may be slightly larger or smaller to afford control over
the force required to
move solvent through said matrix material. Similarly, pore size of porous
matrices including
membranes can be modified to alter the rate of flow of solution through the
matrix. The matrix
may be hydrophilic or hydrophobic and may be chosen to be water or organic-
solvent wettable.
[0109] One skilled in the art will recognize that a huge variety of surfaces
or medias can
be employed for the matrix or matrices including materials useful for SPE
materials, reverse
phase materials such as bonded phase silica and including by example C4, C8 or
C18 packing
materials, or chelating surfaces to capture materials like metals, or
polymeric polymer particles
which present a hydrophobic surfaces, or ion exchange resins such as SCX, SAX
which present
negatively or positively charged surfaces, or weak cation or ion exchange, or
gel filtration
material of particles with pores of a given size to facilitate retention of
analytes of some given
radius or molecular weight, or affinity based support, including surfaces such
as IMAC for metal
affinity chromatography, or other affinities such as and not limited to
antibodies against antigens
or haptens or PTMs of peptides such as phosphorylations of YST or ubiquitin or
acetylations or
methylations or lipidations or antibodies against particular motifs or
titanium dioxide to capture
phosphorylated residues or silicon carbide for nucleic acid (DNA and RNA)
affinity or
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streptavidin for biotinylated moieties or fluorinated surfaces to capture
halogenated compounds,
or antibody based capture materials such as protein A or G, or chelators, or
any similar matrix
material used in chromatography such as high performance liquid
chromatography. The matrix
may be partly or fully comprised of a monolithic material with any of the
above affinities, or
other affinities.
[0110] The device may comprise a secondary matrix, likely a hydrophobic matrix
disposed between the primary matrix and the outlet, Le. downstream of the
primary matrix. One
skilled in the art will recognize that many other matrices are possible.
[0111] Suitably the secondary matrix extends across the entire lumen of the
vessel such
that anything flowing from the inlet to the outlet must pass through at least
a portion of the
secondary matrix.
[0112] The outlet may lead to a sump or reservoir adapted to collect various
media,
reagents, buffers and the like which pass through the matrix, and in
particular different fractions
of different molecules with different solubilities.
[0113] The eluted molecules or fragments can suitably pass to a secondary
matrix.
Suitably the secondary matrix might be a hydrophobic matrix, e.g. a stationary
hydrophobic
phase suitable for reverse phase chromatography (RPC). The most column FtPC
matrices are
based upon silica substrates, for example, silica with alkyl chains bonded
thereto, but any inert
hydrophobic solid phase could, in theory, be used. A particularly preferred
hydrophobic matrix
comprises octadecyl carbon chain (C18)-bonded silica, C8-bonded silica, or a
combination of
the two, but other suitable matrices include cyano-bonded silica and phenyl-
bonded silica.
Alternative secondary matrices might ion exchange, or hydrophobic interaction
chromatographies or alternatively affinity chromatography based either on
larger molecule
affinity reagents such as aptameres or antibodies or alternatively chemistries
like IMAC or
titanium dioxide for phosphorylation. Similarly RNA can be enriched with oligo-
thymidine, or
glycans with boron affinity chromatography or lectins. One skilled in the art
will understand that
could be coupled with this application in many different embodiments such as
with loose beads
held with a frit, or derivatized membranes such as Empore C18. The secondary
matrix can have
several roles, e.g.: it functions as a mechanical support for the primary
matrix, it acts as a guard
filter to capturing stray particles and shed fiber material from the primary
matrix, and it assists in
the final clean-up of the molecules and fragments of molecules which were
captured and
processed; and it can allow for chromatographic resolution of the molecules
and fragments of
molecules which were captured and processed.
[0114] In some preferred embodiments of the application which include a
reverse phase
secondary matrix, the application comprises the step of eluting the molecules
and fragments of
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molecules which were captured and processed from a hydrophobic, secondary
matrix using a
series of eluents or gradient of eluent of increasing hydrophobicity. Thus the
method can
provide a degree of chromatographic separation of the molecules and fragments
of molecules
which were captured and processed based on their hydrophobicity. This allows
the population
of captured molecules or fragments thereof to be resolved on the basis of
hydrophobicity which
can aid in later analysis. For this purpose a secondary matrix comprising C8-
bonded silica is
very useful. A suitable series of eluents comprises, consecutively, 5% ACN in
water, 10% ACN
in water, 15% ACN in water and then 60% acetonitrile in 0.5% formic acid (FA);
such a series
allows for four fractions to be obtained from the captured molecules.
[0115] The device may be a modified pipette tip. Other types of vessels are
contemplated, e.g. vessels adapted for automated and/or high throughput sample
preparation
and/or spin columns
Capture
[0116] Especially large molecule capture, after addition of coagulation
medium, is
achieved through a combination of two capture mechanisms. Any precipitated
particles are
physically trapped in the filter pores and other in-solution material is
adsorbed on the filter via
non-covalent interactions with the filter surface. One skilled in the art will
recognize that there
are many different solutions which can specifically cause the coagulation of
specific classes or
combinations of classes of kinds of biomolecules, such as lipids, glycans,
proteins, peptides,
nucleic acids. For example, the coagulation and capture of proteins is
facilitated by the addition
of an organic solvent such as methanol, other alcohols or many other organic
solvents.
[0117] Altematively, the capture of lipids is facilitated by the use of an
aqueous solvent,
and lipids and other small molecules can be separated in the flow through by
the use of a
biphasic organic solution such as mixtures of methanol, water and methyl-tert-
butyl-ether
(MTBE), solutions which also cause protein and DNA and RNA and glycans to
precipitate
and/or be bound within the trapping matrix.
[0118] In the case of a size-based retention, i.e. where particles are trapped
in pores
because of their size or size of aggregated particles or particulate, elution
may be achieved after
chemical and/or enzymatic treatment to reduce large molecules to smaller
sizes. By non-limiting
example, captured protein can be broken into protein fragments of smaller size
(peptides) by
proteases or chemical treatment or sheering by sonic energy. Similarly glycans
can be liberated
from captured proteins by treatment with glycosidases, or larger glycans can
be processed to
smaller pieces by glycosidases, lipids can be freed from trapped material
including protein (for
lipidated proteins) or larger lipids broken into smaller pieces, and nucleases
or sonic sheering
can produce shorter lengths of DNA and RNA, by non-limiting example for the
generation of
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libraries. This method relies on the capture of one or more class of molecule
while another or
others is/are left soluble, and thus pass(es) through the pores for subsequent
analysis.
[0119] The capacity (and hence volume) of the matrix should generally be
sufficient to
trap substantially all of the (large) molecules in the sample without becoming
clogged regardless
by which mechanism the (large) molecules are retained. However, it will be
apparent that the
required matrix capacity depends, inter alia, on the concentration of the
molecules in the sample.
Suitable matrix volumes can be determined by trial and error, and typically
there will be no
problem encountered if a higher volume of matrix is provided than is strictly
required, other than
it may require more reagents to wet, wash, and enzymatically or chemically
process the sample
and to elute the resultant processed molecules.
[0120] The coagulant causes some part or fraction of the biomolecules to
adhere to or be
captured or retained upon or within the matrix in a reversible manner.
Typically, though not
exclusively, this will include proteins, DNA, RNA and glycans. Most typically
small molecules
such as (and not limited to) metabolites will pass through. However by varying
the extraction
solvent(s) used, it is possible to capture and retain other classes of
molecule such as (and not
limited to) lipids. With enough time, it is possible that especially protein
and DNA/RNA will
precipitate and form a suspension of fine particles; this precipitation is not
obligatory. What is
obligatory is that the coagulant does not cause severe precipitation which
renders the precipitant
insensitive to enzymatic treatment (e.g. with digestion try psin or LysC or
PNGase F or
nucleases), especially under aqueous conditions. It is noted that while a
sample can be clarified
by example by centrifugation after exposer to the extraction solvent, the
entire sample can also
be loaded including debris; this will then be subjected to whatever further
extractions and/or
chemical and/or enzymatic processing steps.
Depth Filters
[0121] Depth filters are a type of filters that use a porous filtration medium
to retain
particles throughout the medium, rather than just on the surface of the medium
(as is the case
with membrane/surface filters). Depth filters are commonly used when the fluid
to be filtered
contains a high load of particles because, relative to other types of filters,
they can retain a large
mass of particles before becoming clogged (for more information on depth
filters, and other
filters, see Derek B Purchas and Ken Sutherland, Handbook of Filter Media (2nd
Edition),
Elsevier Advanced Technology (2002)).
[0122] Depth filters typically have a random network of pore channels that
vary in size
and geometry. They are manufactured from a variety of solid materials.
Materials of
construction include various forms of quartz, polymers, cellulose, and glass,
either singly or in
combination. The processes used to manufacture depth filters do not result in
a regular
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arrangement of the solid matrix. Instead, there is a range of pore sizes
within a given structure
that includes pores significantly larger and significantly smaller than the
nominal pore rating.
[0123] Depth filters are typically made out of one or more of the following
materials:
= Quartz;
= Glass fiber;
= Polymers;
= Cellulose; and
= Cellulose with other additions such as diatomaceous earth.
[0124] Preferred depth filters for use in the present application are formed
from
cellulose, filled cellulose, quartz, glass fiber or polymers. The filter
material should typically be
inert with respect to the molecules which are being processed and reagents
used in the method,
so that undesirable reactions are avoided.
[0125] Depth filters are not typically characterized by a defined pore size in
the same
way as membrane filters (surface filters), and the pore size is typically
highly variable. Thus it
is imprecise to define a specific pore size for a depth filter-based matrix.
Depth filters are often
referred to in terms of target particle size retention, e.g. 5 p.m, 1 p.m or
the like. The format of
depth filters is highly diverse from sheets to cartridges to pleated filters
in a huge variety of
physical formats.
[0126] Particularly preferred depth filters for the present application
include quartz,
borosilicate depth filters, cellulose and/or cellulose plus diatomaceous earth
or minerals or
carbon or other materials, many forms of which are available from many
suppliers such as
Ahlstrom, Eaton, EMD Millipore, EitelAlsop, Filtrox, HOBRA-Skolnik, Pall,
Sartorius,
Whatman, or alternatively of a proprietary composition and construction, so
long as the material
substantially corresponds to the properties of a depth filter.
[0127] Depth filters have a random network of pore channels that vary in size
and
geometry. They are manufactured from a variety of solid materials. Materials
of construction
include various forms of plastics, cellulose, and glass, either singly or in
combination. The
processes used to manufacture depth filters do not result in a regular
arrangement of the solid
matrix. Instead, there is a range of pore sizes within a given structure that
includes pores
significantly larger and significantly smaller than the pore rating.
101281 The randomness of the structure does not allow the assignment of a
definitive
upper limit on the size of particles that may pass through the filter. A
portion of the particles in
the filtrate will exceed the pore rating. Depth filters also can entrap a
large percentage of
particles smaller than the pore rating. Because depth filters trap particles
throughout the
structure, they typically exhibit a high particle-handling capacity. This
makes them particularly
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useful in applications where the solution being filtered has a high particle
load. Depth filters are
not considered sterilizing-grade.
[0129] Various grades of depth filters may have different pore sizes, La Grade
4 (20-25
pm pores), Grade 598 (8-10 pm pores) and Grade 3 (6 gm pores) can be used and
will achieved
some degree of retention, but the finer or coarser filters may provide
improved performance,
depending on the properties of the molecules to be allowed to pass through and
those desired to
be retained on the filter. Thus the indication is that depth filters in the
trapping range of from 15
pm down to 0.1 pm (or even smaller) are preferred, e.g. about 15 pm or finer,
about 5 pm or
finer, about 1 gm or finer, about 0.5 gm or finer being suitable.
[0130] Depth filters are typically used as pre-filters because they are an
economical way
to remove > 98% of suspended solids and protect elements downstream from
fouling or
clogging. They owe their high capacity to the fact that contaminants are
trapped and retained
within the whole filter depth.
[0131] Conventional depth filters can be made out of the following materials:
= Quartz
= Glass Fibre
= Polymers
= Cellulose
= Cellulose with fillers such as diatomaceous earth
[0132] Quartz. Filter media made of pure micro-quartz fibres. Such media can
be
produced with or without glass fibres and binder. Media without glass fibres
and binder are
particularly appropriate for emission control at high temperatures of 900-950
C and wherever
absolute purity of the filter medium is required. Excellent filtration
properties, minimal metal
contents, outstanding weight and dimension stability.
[0133] Glass Fibre. As implied by the name, glass fibre depth filters are made
from
glass fibres. In sheet form the fibres are initially held together only as a
consequence of
mechanical interaction. To improve the handling characteristics, the filter is
sometimes treated
with a polymeric binder, such as polyvinyl alcohol, which serves to hold the
matrix together.
Glass fibre filters are also prone to fibre shedding. If required, a membrane
filter can be placed
downstream to retain any fibres. Examples include GF/D (Whatman), a filter
material which is
utilised in the above-mentioned examples.
[0134] Polymers. Polymeric depth filters are manufactured from plastic fibres
of various
lengths, morphologies, and diameters. To improve the strength of these filters
and reduce the
level of fibre shedding, the filter can be calendared, the process of running
the material between
cylindrical rollers to apply pressure and/or heat. Most polymeric depth
filters are inherently
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hydrophobic. For low pressure aqueous filtration, the filter may require a
surface treatment to
render it wettable. Polymeric depth filters are normally very strong and easy
to handle.
[0135] Cellulose. As implied by the name, cellulosic depth filters are made
from
cellulose fibres. The fibres can be derived from a relatively crude source,
such as wood pulp, or
a highly purified source, such as cotton. The filters are manufactured by
techniques very similar
to paper manufacture and are very economical. Although they are generally very
easy to handle
when dry, they are mechanically very weak when wet. Cellulosic filters are
prone to fibre
shedding during fabrication into a device and when used in filtration. If
required, a membrane
filter can be placed downstream to retain any fibres. Cellulose fibres may
also be a source of
contaminants, however the ability of cellulose filters to be embedded with
other materials such
as diatomaceous earth presents unique opportunities. Various such forms,
highly purified, may
well be useful.
Buffers
[0136] Conventional methods for precipitating in preparation for mass
spectrometry are
harsh and cause dramatic precipitation and aggregation which render them
rather insensitive to
enzymatic activity. By example with proteins, exemplary precipitants in prior
art methods
include trichloroacetic acid (TCA), typically a 100% w/v solution (500g TCA
into 350 ml
dH20). See, for example Cuff Protoc Protein Sci. 2010 February; CHAPTER: Unit-
16.12.
Such precipitated proteins must be treated with strong chaotropic agents to
render them
susceptible to protease action. Exemplary chaotropic agents for such purposes
include urea (e.g.
at 8M concentration) and the like, or with detergents or the like.
101371 The present application can involve the use of buffers and the like
which could be
considered to be mildly chaotropic. For example, a preferred buffer for the
present application
is based upon methanol and ammonium acetate. One specific embodiment is 50%
methanol as a
coagulant with 50% 30 mM ammonium acetate containing 3% nonionic detergent
Another
specific and preferred embodiment is four volumes of methanol as a coagulant
containing 30
mM ammonium acetate (made in anhydrous methanol from an aqueous 1 M ammonium
acetate
stock solution) added to sample extracted in 30 mM ammonium acetate with probe
sonication.
Another specific embodiment is 50% methanol with 30 mM ammonium acetate as a
wash
solution. These compositions have far less chaotropic effects on biomolecules,
including those
which are precipitated and aggregated, including (and not limited to) DNA and
protein, quite
unlike urea or guanidinitum hydrochloride.
[0138] Suitably the coagulant comprises a mixture of aqueous and organic
solvents,
most typically in the range of two parts methanol to one part aqueous
extraction solution to ten
parts methanol to one part aqueous extraction solution. It is apparent to one
skilled in the art that
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methanol is only a representative organic solvent, and that many others might
be used to the
same ends.
[0139] The aqueous extraction solution can be neutral, such as in the specific
embodiment of 30 mM ammonium acetate, which is near pH 7, or basic, such as
1.8%
ammonium hydroxide, or acidic, such as 1 M HO or formic acid.
[0140] Aqueous extraction solvents around neutral are the preferred embodiment
when
proteins are to be captured in their native states, for example for later
enzymatic processing The
extraction solution can contain detergents, and detergents are typically not
ideal because they
interfere with downstream analysis of the classes of molecule which did not
bind to the capture
matrix. The concentrations of buffer such as ammonium acetate in a neutral
aqueous extraction
can be varied from 1 mM to as high as multiple molar, depending on the kind
and class of
molecules desired. Similarly, the concentration of base can vary from less
than 1% to the
maximum of solubility, for example for ammonium hydroxide a maximum of 35.6%
w/w;
typically 1% ¨5% base is most suitable, though other embodiments are possible.
[0141] In basic extractions ammonium hydroxide is preferred due to it volatile
nature.
The concentration of acid in an aqueous acidic extraction solution falls
between 10 mM and
multiple molar, again optimized depending on the desired class of molecules.
It should be noted
that in preferred embodiments, volatile acids, bases and buffers are desired
as they can be
removed by speed-vacing. It should also be noted that capture is best around
neutral pH, and
also noted that while the extraction solution is typically aqueous, there is
no reason it must be
aqueous so long as it enables the capture and fraction mechanism(s) described
below. It is
apparent to one skilled in the art that there exist many buffers, acids and
bases, and coagulants,
and that the substances described in this paragraph serve only as illustrative
examples and are
not limiting.
[0142] Typically acidic or basic extractions are favored because at non-
physiological pH
values, enzymes that could degrade a sample such as and not limited to
proteases, phosphatases,
lipases, glycosidases, nucleases and other are inactive or poorly active.
[0143] To determine the needed concentration of coagulant, typically a
solution of the
sample extracted with extraction solvent is exposed to varying concentrations
of coagulant,
allowed to flow through the trapping matrix (typically a depth filter) and the
flow through is first
concentrated, then analyzed for the class of molecule which were intended to
be captured. For
example, in the embodiment where the flow through contains small molecules
such as
metabolites and protein and DNA and RNA and glycans are retained on the trap,
one would
analyse for proteins by example by SDS PAGE and for DNA by e.g. polyacrylamide
gels, each
kind of gel being visualized by their respective stains (e.g. colloidal
Coomassie, lectins and
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ethidium bromide, among many other stains for both proteins, nucleic acids and
glycans). If the
desired class of protein has not been captured, a different coagulant or
different concentration
must be tried, until reversible capture of the molecular class is achieved. In
one test, it was found
for an aqueous solution containing antibodies that a six-to-one volume excess
of methanol
afforded good capture.
[0144] Other methods of coagulating molecules onto the matrix may also be
suitable for
the present application. For example, salts can be used to drive 'salting out'
precipitation. In
such embodiments, the downstream implications of such coagulation methods must
be
considered. For example, PEG can be used to drive things out of solution,
however PEG would
make downstream analysis near impossible.
[0145] The suitability of any coagulant for use in the present application can
be tested
using the methodology described below. In particular, any coagulant should be
able to cause
biomolecules or molecules which have been solubilized with an extraction
solution, which if it
was not new neutral, will be brought to around neutral before capture, to be
captured on the
trapping matrix, and the molecules so retained should be capable of being
treated in the device
and/or on the capture matrix with enzymes such as (and not limited to) a
nuclease, protease
(typically trypsin), glycosidase, among many other enzymes or alternatively
various chemistries,
without the need for solubilization with a strong agent such as a chaotropic
like urea or a
surfactant or detergent. As mentioned above, given too much time molecules can
aggregate and
precipitate which might potentially lead to enzymes such as protease no longer
being effective.
Accordingly, sensitivity to enzymatic and/or chemical treatments should be
assessed
immediately after capture of molecules on the trapping matrix, or, ideally,
following capture of
the molecules in the depth filter trapping matrix as described above, Time
courses for the period
of exposure of the sample to coagulant may also be performed. Furthermore, the
coagulant
should not prevent downstream analysis of extracted and potentially processed
molecules using
mass spectrometry.
[0146] The sample comprising a sample extraction solvent and coagulant is
typically
brought into contact with the trapping matrix, although the matrix can also
deal with any
precipitation or debris from the sample, and the vessel holding the trapping
matrix may hold the
coagulant and the extraction solvent may be directly added to the coagulant.
[0147] Where the extraction solvent/coagulant mixture is added to the matrix,
the matrix
may already be permeated with a fluid medium (phase), i.e. a solution and
typically a fluid
which matches the composition of the extraction solvent/coagulant mixture.
Preferably the fluid
medium which permeates the matrix is mildly chaotropic. For example it can
comprise an
aqueous solution of a short chain alcohol, e.g., methanol, ethanol or
propanol, or other organic
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solvents. Most preferred is an aqueous methanolic solution, e.g. typically
comprising 60% or
higher.
[0148] An exemplary, and generally preferred, extraction solvent/coagulant
mixture is
sample extraction with one volume of 30 mNI ammonium acetate mixed with four
volumes of a
coagulant in particular for this example methanol containing 30 'TIM ammonium
acetate,
whereby anhydrous methanol is supplemented to 30 mNI ammonium acetate from an
aqueous 1
M ammonium acetate stock solution.
101491 The step of washing the trapping matrix with captured molecules is not
obligatory for captured endogenous molecules however it is obligatory if
proteins have been
reduced and alkylated or otherwise chemically manipulated on the trapping
matrix; this will
typically take place after recovery of the smaller molecules such as lipids
and metabolites.
Washing also removes contaminants, and any suitable washing liquid which
solubilizes the
contaminants or reduction/alkylating reagents (or reagents of any other
chemical treatment such
as cleavage or deamidation or oxidation) which does not solubilize molecules
of interest can be
used. A suitable liquid is the various aqueous methanolic solutions containing
ammonium
acetate described above. However, other liquids would be suitable, and the
suitability of any
putative washing liquid could be readily tested. Typically mild chaotropes are
useful for this
purpose. They should ideally be mass spec compatible.
[0150] In some situations it may be desirable to remove the washing liquid,
e.g. where
presence of that liquid might have an adverse effect on the activity of
subsequently administered
processing enzymes such as proteases or nucleases or glycosidases, and to
replace it with
another buffer. This is easily accomplished with a first wash step using an
aqueous methanolic
solution to remove reduction and alkylation reagents (other organic solvent
compositions could
be used), and a second rinse to remove residual methanolic solution, e.g.
using water or an
aqueous ammonium bicarbonate solution. Aqueous buffers containing reagents
such as
ammonium bicarbonate or acetate, and many other known to a person skilled in
the art, plus any
necessary cofactors, can then be used for the purpose of downstream
processing.
Processing
[0151] In this application, the enzymes such as proteases or nucleases or
glycosidases
used to treat the captured molecules are administered after the small
molecules have been
separated and captured in the flow through of the solvent extraction solution
combined with the
coagulation solution. Digestion of the proteins with a protease is a
conventional step in the
preparation of proteins for analysis by mass spectrometry. Typical proteases
include trypsin or
LysC, but it can be any other suitable protease, e.g. chymotrypsin and many
others. For
example, 0.07iig/p1 of trypsin (03708985001, Roche or V5111, Promega) in 50
tnM ammonium
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bicarbonate can be used in embodiments of the application. Glycans can be cut
or processed or
liberated by enzymatic means. N-linked glycans can be released with peptide-N-
glucosidase F
(PNGaseF). PNGaseF releases most glycans except those that contain 1-3 linked
fucose to the
reducing terminal GlcNAc. In that case, the enzyme peptide-N-glucosidase A
(PNGaseA) is
used. There are fewer enzymes comparable to PNGAseF for 0-linked glycan
release and
subsequent analysis. Typically the release of 0-linked glycans is achieved
through chemical
methods such as 13-elimination. However, Genovis offers an o-protease for 0-
glycan-specific
digestion of glycoproteins (OpeRATOR), an endoglycosidase (0-glycosidase) for
0-glycans of
core 1 and core 3 (OglyZOR) and an exoglycosidase which acts on sialic acids
(Sia1EX0); all
these enzymes can be used in embodiments of this application, most preferably
applied to the
trapping matrix.
[0152] The enzymes used to process captured molecules such as proteases or
nucleases
or glycosidases or lipases or other enzymes are typically added to the medium
permeating the
matrix. Multiple enzymes can be used either serially or in parallel. For
example, large molecules
can be separated and captured in the trapping matrix as described above with
capture of the
small molecules. Glycans can be freed or cut with PNGase F, which can be
recovered with an
aqueous wash as they are highly water-soluble. Proteins are left behind, which
can then be
reduced and alkylated in situ, followed by digestion with proteases as
described above. In
another example, in the case that the nucleic acids of samples are not of
interest, nucleases and
proteases can be added simultaneously, so long as the protease does not
instantly digest the
nuclease.
101531 Suitably the method comprises the step of desalting the captured
molecules
and/or their fragments. Desalting can be achieved by rinsing the molecules
with salt free buffer
and/or water and/or water mixed with organic solvents such as methanol.
Desalting can be either
within the trapping matrix, in which case molecules are simply washed, or
alternatively in some
embodiments the application has other affinities such as C8 or C18 (see
below).
Elution
101541 The molecules and their fragments can be eluted using any suitable
agent. Water
is useful for dissolving glycans, and can solubilize DNA and RNA. DNA and RNA
can be
solubilized in TE buffer (1 mM EDTA 10 mM tris pH 8.0). Basic solutions (e.g
ammonium
bicarbonate), or acidic solutions (e.g trifluoroaceric acid) or salt solutions
(e.g. sodium chloride)
and solutions of water supplemented with an organic such as 10% acetonitrile
are suitable for
eluting proteins/fragments from the matrix. Notably, independent of any other
processing for
other molecular classes, captured proteins can be eluted from the depth filter
using high
concentrations of formic acid (60% or 80% or more, keeping the solution cold
to avoid
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formylation), or 8 M urea or 6 M GuHC1 or base such as 1.8% ammonium
hydroxide. It should
be noted that sonication assists for any of these reagents, and that
carbamylation by urea can be
limited by using amine containing buffers.
[0155] The method may further comprise eluting the molecules and fragments of
molecules which were captured and processed from the secondary matrix using a
suitable
elution solution, e.g. 70% acetonitrile, 0.5% formic acid in H20 for
embodiments which use
reverse phase capture.
[0156] Preferably the application uses at least in part a coagulation medium
substantially
comprising methanol or another alcohol or organic solvent. This medium is
useful not only for
capture in the trapping matrix but also for washing. A particularly preferred
medium is a buffer
at an approximately neutral pH (e.g. from 6.5 to 7.5) comprising methanol or
another alcohol
(typically 60% or higher v/v methanol) and ammonium acetate or other buffer
with a pKa
around neutral at, e.g. specifically 80% methanol containing 30 mM ammonium
acetate. This
formula and composition may be reached only after combination of the
extraction solvent with
the coagulation medium. Other suitable media for the present application will
be apparent to the
skilled person.
[0157] The presented method is suitable for processing samples which comprise
many
conventional surfactants. SDS is commonly used as a surfactant for
solubilizing and extracting
membrane bound proteins from cells, but other surfactants are also used,
including sodium
cholate, sodium deoxycholate, n-dodecyl-beta-D-maltoside, Triton X-114, NP-40
(Thermo
Scientific), and Brij 35 (Thermo Scientific). However, surfactants hamper down-
strcam analysis.
101581 Where the device is a pipette tip or a spin column, it preferably
comprises a layer
of primary matrix and a layer of secondary matrix, the layers being arranged
such that the
primary matrix is upstream of the secondary matrix relative to the net
direction of flow through
the device. Typically the primary and secondary matrices are provided in the
tapered portion of
the device, with the secondary matrix being located nearer to the narrow tip
end (nozzle), and
the primary matrix being located nearer to the wide end.
101591 The primary andVor secondary matrix may each comprise one or more flat
layers
(e.g. disks for a vessel which is circular in cross section) of the relevant
material (e.g depth filter
or hydrophobic silica). Two or more layers of the relevant material can be
stacked to provide
the desired total depth, and hence volume and capacity, of matrix.
Alternatively, a thicker and
thus larger capacity material can be used.
101601 The matrices can be retained in the device in any suitable manner, e.g.
mechanically (e.g. by friction with the wall of the device, or using a clip,
frame or other support
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means) or by an adhesive or the like (provided such an adhesive or the like is
compatible with
the method).
[0161] The device is suitably adapted to be mounted in a centrifuge to
facilitate driving
of the various media, reagents, buffers and the like through the matrix.
[0162] Alternatively the device is adapted to connect to one or more pumps to
drive of
the various media, reagents, buffers and the like through the matrix.
[0163] The device can suitably be a microfluidic device.
[0164] The device can be provided in association with a holder, e.g a support
which
allows the device to be mounted in a centrifuge or other piece of laboratory
equipment.
[0165] The present application provides a system comprising a device and
associated
sample handling apparatus.
[0166] In certain embodiments, the trap may be combined with a computer
control
system, or with microfluidics and/or other provisions described below which
allow for
automated sample processing. In an exemplary embodiment, the computer system
includes a
memory, a processor, and, optionally, a secondary storage device. In some
embodiments, the
computer system includes a plurality of processors and is configured as a
plurality of, e.g.,
bladed servers, or other known server configurations. In particular
embodiments, the computer
system also includes an input device, a display device, and an output device.
In some
embodiments, the memory includes RAM or similar types of memory. In particular
embodiments, the memory stores one or more applications for execution by the
processor. In
some embodiments, the secondary storage device includes a hard disk drive,
floppy disk drive,
CD-ROM or DVD drive, or other types of non-volatile data storage. In
particular embodiments,
the processor executes the application(s) that are stored in the memory or the
secondary storage,
or received from the intemet or other network. In some embodiments, processing
by the
processor may be implemented in software, such as software modules, for
execution by
computers or other machines. These applications preferably include
instructions executable to
perform the functions and methods described above and illustrated in the
Figures herein. The
applications preferably provide GUIs through which users may view and interact
with the
application(s). In other embodiments, the system comprises remote access to
control and/or
view the system.
[0167] The system may be adapted to perform several steps of the method of the
present
application, e.g. at least the steps of molecule capture, transfer of
molecules to the matrix,
fractionation and washing as needed, subsequent treatment with enzymes and
subsequent
fractionation.
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[0168] Furthermore the system may additionally be adapted to perform one or
more of
cell lysis, extraction of biomolecules and elution of biomolecule fragments
from the matrix.
[0169] The present application provides a kit comprising a device and one or
more
containers comprising at least one of: a buffer medium for use in the device;
reagents for cell
lysis and solubilization of membrane bound proteins; enzymes including (by
example)
proteases, nucleases, gylcosidases, lipidases, et.; washing/rinsing agents as
needed; and multiple
elution reagents, which are chosen per biomolecular class property as
described herein. Various
suitable media, reagents and the like are discussed herein.
Spin Column Assembly
[0170] There remains a need for a simple, efficient and reproducible sample
preparation
tool, compatible with small sample amounts, which produces from the same
biological sample
separate fractions of different classes of molecules from a sample, typically
(but not necessarily)
a biological sample such as a biopsy or blood sample or other piece of
biology. The present
application provides an elegant and simple-to-use two-part nesting system of
an inner and outer
vial to satisfy these experimental needs. The inner vial has a space to retain
and support one or
more matrices and other components well known to one familiar in the art such
as flits and
membranes. The inner and outer vials interface in two positions, the "lower"
and "upper" parked
positions. In the lower parked position, the outer column seals the inner
vial, preventing flow out
of the inner vial and allowing reactions to happen in whatever incubation
conditions are required
within and atop the matrix and within volume of the inner vial. The inner vial
can then be raised
to the upper parked position in which solution can flow from the interior of
the inner vial
through the matrix to the interior of the outer vial. The outer vial then
becomes a receptacle for
the flow though of whatever incubation step, and there are no issues with
losses to plugs because
the outer vial replaces plugs.
[0171] The two-piece system does not require any plugging to effect
incubations or
reactions, minimizing handling and maximizing sample processing speed as well
as sample
recovery. The system has upper and lower parked positions, effected by
engaging or disengaging
locking mechanisms between the inner and outer vials, which blocks flow out of
the matrix or
allows it. In a preferred embodiment, the locking mechanism is comprised of
upside down U-
shaped stops on the exterior of the inner vial and support pillars on the
inside of the outer vial
which can engage the middle of the U to support it in the upper parked
position or be disengaged
from the support pillars to allow the inner vial to seat into the very bottom
of the outer vial. In
the upper parked position, the contents of the inner vial and matrix can be
transported to the
outer vial through the porous matrix, for example by positive pressure or
centrifugation, or by
appropriate application of negative pressure. In the lower parked position,
the inner vial is sealed
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by the outer vial, allowing for treatments that require incubation such as
coagulation steps. The
inner vial is tightly interfaced against and sealed to the outer vial,
allowing the inner vial and its
matrix and contents to receive heat, light, electromagnetic radiation like
microwaves, sonic
energy like ultrasonication, pressure or any other of a variety of treatments
applied to the outside
of the outer vial. Using such external treatments, especially sonication and
heat and pressure,
can dramatically reduce reaction times of chemical and enzymatic reactions, or
the time it takes
to solubilize materials.
101721 This application conveniently provides a method of affording such
treatments to
samples by submersion into solutions (including and not limited to e.g.
ultrasonication and
incubation) while preventing contamination; devices with plugs cannot be
relied upon to keep
seals during such treatments. In the lower parked position, dead volume is
minimized with a pin
that fills most of the void space in the output beneath the matrix. This
allows for minimal
reaction volumes and maximal elution concentration of reactions such as and
not limited to
enzymatic reactions, on, in, within or atop the matrix, or elutions from
chromatographic media
like C18 or SCX among many others. Once the outer vial has received either
flow though of the
matrix or sample after subsequent processing steps, the outer vial can be
closed and sealed with
its integrated cap to become a storage container for the eluted or processed
material; multiple
outer vials may be used for multiple processing steps resulting in multiple
fractions, and the
outer vial obviates sample transfers and plugging of the matrix. Adsorptive
losses, which
become exasperated during frequent sample transfers, are thus minimized. The
outer vial has a
sloped lower surface facing the flat surface of a D-shaped pin to form a
region first into which
sample flows due to gravity and/or centrifugal force and second a region
afforded enough space
such that the sample can be removed or withdrawn or sampled by standard means
such as
pipette tips or aspiration needles, among many other techniques of sample
transport and
handling. The design of the inner and outer vials can remain the same yet be
easily changed with
many different matrices, allowing application of this system to many treatment
workflows.
Finally, the lure lock output of the inner vial is standard and allows easy
attachment of the inner
vial to many other constnnables and devices, such as Sep-Pak C18 disposable
SPE units, or
vacuum manifolds, affording the system flexibility to be adapted to the
largest number of
workflows.
101731 A representative embodiment of spin-column assembly 113 is detailed in
Figs. 10
¨ 14, and a representative embodiment in a 96-well plate format 115 in Figs.
15 and 16. It will
be apparent to one skilled in the art that these are merely representative
embodiments, and that
they are not limiting.
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[0174] The inner vial 101 consists of an opening 129 into which samples of
liquids
and/or solids can be deposited, a space 122 to hold samples, a hinge 185
connected to the lid
237, said lid having a tab 281 to open and close it, as well as a ribbed
sealing mechanism 248 to
seal the inner vial during incubations, and said sealing mechanism is afforded
a vent 127 which
allows air pressure to equalize between the inside and outside of the inner
vial. Sample holding
space 122 is connected and exposed to a porous matrix 117 which sits at the
bottom of the inner
vial in a conical section, conical to afford better sealing during
manufacturing and operation via
centrifugation or positive or negative pressure, though conical simply a
representative shape and
is not limiting, and said matrix 117 opens on the bottom to the inner vial
bottom opening 269
which in the specific embodiment as presented has the outer dimensions of a
lure lock 203, but
which can have other dimensions. The inner vial has, in the presented
embodiment, three stops
153 in the shape of upside down U-s (see Fig. 13) which, if rotated to the
correct angle offset
between the caps of the inner and outer, can interface with supports on the
interior of the outer
vial 174 to support the inner vial 101 in an upper parked position, as is
shown in Figs, 10 and 11.
In this upper parked position, the sample can pass from space 122 through
matrix 117 through
the bottom opening 269 of the inner vial and into the sample holding space of
the outer vial 222.
However if the inner vial is positioned such that the inner vial lid 237 and
outer vial lids 217 are
directly above each other as shown in Figs. 12 and 14, the inner vial passes
to the bottom of the
outer vial where it is sealed by a tight interface 276 with the outer vial and
D-pin 145 of the
outer vial; this is the lower parked position (see Figs. 12 and 14). In this
way the combination of
the inner and outer vials obviate the need for plugs in processing through a
matrix, especially
when incubations are required. The output channel 269 of the inner vial 10115
so fashioned to
exactly fit the space at the bottom of the outer vial 109 and especially the
slope of the outer vial
which affords a sample collection space 195.
[0175] The outer vial 109 consists of a vial with an opening configured to
receive the
inner vial 101 in either an upper parked position (Figs. 10 and 11) with
support bars 174
engaged in the stops 153 of the inner vial, or alternatively in a lower parked
position (Figs 12
and 14) in which the outer vial seals the inner vial through the tight
interface 276 and D-pin 145.
In the most preferred embodiment, D-pin 145 nearly touches the matrix 117 and
thus occupies
much or all of the dead space of inner vial output channel or opening 269. The
outer vial has a
lid 217 attached via a hinge 168, said lid having a tab 299 to open and close
it as well as a rib
sealing mechanism 256 that interfaces with the lid 217. When the lids of the
inner vials are
aligned, the support bars 174 do not engage the inner vial and the inner vial
passes to the lower
parked position shown in Figs. 12 and 14. The tight interface 276 is
intentionally tight to most
efficiently communicate treatments such as and not limited to heat, light,
ultrasonic energy or
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electromagnetic energy applied to the exterior of the outer vial at the bottom
region of the
assembly 273 to the interior of the inner vial including to the matrix 117 and
any material bound
on, in or on top of it, as well as any solution held within the inner vial
sample holding space 122.
The bottom of the outer vial is sloped to create a sample collection region
195 which is directly
opposed to the flat side of the D-pin; the D-pin shape affords sufficient room
for pipette tips of
normal size to pass to the bottom of the sample collection region 1195. The
low nature of this
sample collection region 195 allows solution held in the sample holding region
222 of the outer
vial to flow down via gravity or centrifugation and be recovered by standard
means such as
pipetting nearly quantitatively. The D-pin 145 and form-fitted, tight
interface 176 between the
inner and outer vials removes the need for a plug in processing through a
matrix.
[0176] Sample processing begins in the inner vial 101. Samples that do not
require an
initial incubation, or which have already been incubated with the necessary
reagents in a
separate vesicle, can be immediately applied to the inner vial's interior
sample holding space of
112 through the upper opening 129 when the inner vial 101 and outer vial 109
are in the upper
parked position held by use of the locking/stop/support mechanism afforded to
the inner vial 153
and the outer vial 174 (see Figs. 10 and 11). Samples may contain solids or
coagulated or
precipitated or flocculent materials, or beads or other insoluble components,
all of which may or
may not be loaded along with any liquid, as required by the experiment. In the
case of no initial
incubation, the inner and outer vial assembly 113 is then typically closed
with the lid 237 which
interfaces with the opening of the inner vial 1129 with a rib sealing
mechanism 248.
[0177] Alternatively and depending on the case, the inner vial might be placed
in the
lower parked position (Figs. 12 and 14) and the interior space 122 can be
preloaded with a
reagent, such as a coagulation reagent or a precipitation or a solubilization
reagent. Sample can
then be directly introduced into the liquid reagent to be incubated with the
needed conditions for
the needed length of time in the inner vial.
[0178] During incubations, the inner vial 101 can be sealed for incubation
with its lid
237 which bends at its hinge 185 which is manipulated to be open or closed by
hand or
automation with handling tab 281. Incubations that require heat cause the gas
also contained in
the interior space 122 to expand; thus the inner vial lid 237 is afforded a
vent 137 to allow the
internal pressure to equalize with the atmosphere external to the inner vial
101. This same vent
137 provides that a vacuum does not build up during passage of the sample from
the inner space
122 of the inner vial to inner space of the outer vial 222; this step occurs
when the two pieces
are engaged in the upper parked position.
101791 In the lower parked position (Figs. 12 and 14), the bottom portion 273
of inner
vial 101 and outer vial 109 nest extremely tightly because the inner
dimensions of the outer vial
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is sized to exactly fit the outer dimensions of the inner at an interface 276.
This tight interface
facilitates flow from the exterior of the outer vial to the interior of the
inner vial, its contents and
matrix treatments such as heat or light or electromagnetic radiation or sonic
energy like
ultrasonication; such treatments are afforded to the inner vial 101 and its
contents by treatment
of the lower portion 273 of the engaged nested assembly 113 in the lower
parked position, for
example by placing at least portion 273 in an ultrasonication bath, or by
placing it in an
incubator, or by exposing it to light or microwaves, or other treatments.
Exposure to such
treatments can speed and facilitate chemical and enzymatic reactions, as well
as solubilize or
physically disrupt materials held within the assembly.
10180] In the lower parked position, outer vial pin 145 mostly occupies the
dead space
interior of the output of the inner vial 203. This is important to minimize
reaction or elution
volumes, to maintain maximal concentration of analytes and to minimize waste
of reagents that
might be expensive, such as mass spec grade enzymes like proteases.
10181] The tight fit seals the inner vial 101 against the outer vial 109 in
region 273 when
in the lower parked position seals the inner vial and prevents flow from its
interior 122 or matrix
117, obviating need for a plug. The tightness of interface 276 is important as
well because its
volume is negligible, typically < 10 L and usually <2 IA, depending on the
consistency of
manufacturing, that any leak from the internal space 122 of the inner vial 101
is limited to that
small space as the solution level in interface 276 equalizes with the solution
level on the interior
space of the inner column 122; leaks of this volume, should they occur, are
negligible and
acceptable. Indeed it is advantageous to fill any space remaining at interface
276 with solution
via centrifugation to facilitate flow of external treatments like heat or
ultrasonication from the
exterior of the outer vial to the interior of the inner vial including its
matrix and contents. One
skilled in the art will recognize that this application can be scaled to much
larger or much
smaller than the presented examples, and that such examples as are presented
here are not
limiting, and that the exemplary dead volumes listed herein will change as a
function of the scale
at which the particular embodiment is made. As presented, the application is
scaled to fit in a
standard benchtop centrifuge. Specifically contemplated forms include standard
sizes used in
laboratory and analytical settings such as tubes from 02 mL to 2 inL and
including 0.5 and 1.7
mL sizes, as well as conical tubes such as a 15 and 50 nal, conical tube
(Falcon tube).
101821 Regardless if the sample did or did not require an initial incubation,
after the
incubation complete with whatever potential treatment such as time or heat or
ultrasonication
applied in the lower parked position as shown in Fig. 12 and 14, the inner
vial 101 can be
returned to the upper parked position within the outer vial 109 as shown in
Figs. 10 and 11. The
sample within the sample holding space 1122 of the inner vial can then be
passed through the
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matrix 117 by centrifugation, gravity flow, or positive or negative pressure.
The application of
positive pressure necessitates that the lid 237 remain open. A typical force
at which the
assembly might be centrifuged is 4,000 g; it can be higher or lower depending
on the use case
and the strength of matrix 117.
101831 If the matrix is afforded an affmity or chromatography or filtration
function, the
first fraction will be a flow through fraction of the moieties which passed
through matrix 117;
this fraction will pass into the sample holding space of the outer vial 222.
Washes, should they
be necessary, can subsequently be performed by moving the inner vial 101 to
fresh outer vials
109, which can capture the washes if desired. Alternatively washes can be
directly into the flow
though, as directed by the needs of the experiment.
[0184] Either with or without washes, material bound or retained in, on or by
the matrix
117 is ready for further processing. The inner column 101 is lifted to
disengage the
locking/support mechanisms 153 and the inner vial, perhaps in a fresh outer
vial, is placed in the
lower parked position to seal the inner vial against the outer vial. Treatment
reagents are then
added to the interior of the inner vial via opening 129 into sample holding
and processing space
122. Most typically, the assembled inner and outer vial will be subject to
centrifugation to
displace all air from the matrix 117 and fill any dead space with treatment
reagents. Treatment
reagents most commonly include: elution solutions, such as aqueous buffers to
elute nucleic
acids or hydrophobic solvents to dissolve lipids or other hydrophobic
compounds, in both cases
and others heat and or sonication may be applied to help dissolve and elute
the fractions of
interest; enzymes, such as proteases like trypsin, pepsin, chymotrypsin, Lys-
C, Lys-N, Asp-N,
Arg-C and Tryp-N, nucleases of which hundreds to thousands of enzymes exist,
glycosidases like PNGase F, and other enzymes or proteins like HRP conjugated
enzymes or
antibodies or proteins; chemical treatments such as derivatizations with
reactive chemistries like
isothiocyanates like FITC or NHS esters or isobaric labels or cysteine labels,
among many other
reactions, or reductions and alkylations; or other treatments. These
treatments are then afforded
their requisite time, temperature, energy addition be it from heat or
sonication etc. to complete
the treatment. After the treatment is complete, the inner vial 101 is again
moved to the upper
parked position as show in Figs. 10 and 11 and again the portion of the sample
released from the
matrix is propelled through the matrix 117 by pressure or centrifugation.
[0185] Such treatments can be sequential, each likely occurring in a fresh
outer vial, and
each releasing a new fraction of the sample retained or bound on, in or by the
matrix. For
example, in the case a coagulant such as an organic solvent is first added to
the inner vial in the
lower parked position, and then a sample, for example serum containing
ammonium acetate, is
added to the coagulant, and the sample is passed through a matrix 117 adapted
to capture the
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biopolymers of the sample after the inner vial is raised to the upper parked
position, the first
resulting fraction will be of small molecules. If then the inner vial is then
returned to the lower
parked position in a new outer vial, and an aqueous solution is applied,
especially with the aid of
heat and/or sonicatiort, nucleic acids can be dissolved and eluted once the
inner vial is again in
the upper parked position. Returning the inner vial to a new outer vial in the
lower parked
position, the retained and bound biopolymiers can be treated with PNGse F to
release glycans.
The inner vial is again placed in the upper parked position and the gly cans
are eluted. Putting the
inner vial in a new outer vial, the proteins can be exposed to reduction with,
e.g., TCEP, and
alkylation with, e.g., MMTS. After reduction and alkylation the proteins can
be washed free of
the reagents, likely into a waste tube, and as a final step a protease such as
try psin can be applied
to process proteins bound or retained on or in or by the matrix to peptides.
This reaction can be
accelerated with heat and sonication applied to the lower region of the
assembled inner and outer
vials 273 afforded by the tight interface between the inner and outer vials
276.
101861 The embodiment shown in Figs. 10¨ 14 requires rotation to mesh the
upside
down U-shaped stops 153 with the corresponding support mechanism 174 of the
outer vial. One
skilled in the art will appreciate that many such locking mechanisms are
possible including
support posts which hold the inner vial or, when placed in recesses, allow the
inner vial to seal
against the outer vial (see Fig. 18), push-on bumps or snaps or ridges to
support the inner vial at
various vertical heights within the outer vial (Figs. 21 ¨ 22), which may have
a break radially
within the outer vial and on the outside of the outer vial to allow the bumps
or snaps or ridges to
be disengaged by rotation (Fig. 21) or threads (Fig. 22). Alternatively,
embodiments exist in
which a locking mechanism that allow or prohibit flow thorough the matrix via
a side release
design in which the outer vial either seals or does not seal against the inner
vial depending on
the position of rotation and in which a snap fit locking mechanism can afford
sealing is
contemplated (Fig. 19).
101871 It is noted that the lure lock 203 allows the reversible attachment of
cartridges
such as SPE cartridges like C18 Sep-Palcs directly to the inner vial. Such
cartridges may have
any of the affinities listed for the matrix 117, and make the assembly
particularly flexible and
able to use currently existent sample preparation and chromatography products.
101881 The above described steps and method and assembly are easily
parallelized by
making arrays of individual columns, as is illustrated by the example 96-well
plate embodiment
of Figs. 15 and 16. Such embodiments maintain exactly the same tight sealing
and interfacing
mechanism 276, D-pin 145, matrix 117, sample collection region 195, and sample
holding
spaces of the inner (122) and outer (222) vials. The difference lies in the
mechanism used to
support the upper and lower parked positions. In Figs. 15 and 16, two supports
326 and 331 exist
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on side tabs supported by hinges 311 which allows the side tabs to be engaged
or disengaged in
the upper and lower parked positions by swinging them out. In Fig 15, the
lower parked position
in which the inner plate 303 is sealed against the outer plate 308 is
maintained by support tab
326 on both sides which holds the inner plate 303 down against the outer plate
308_ In Fig 16,
support tab 331 on both sides holds the inner place 303 up such that contents
of the inner plate in
sample holding space 122 can flow though the matrices 117 through the bottom
openings of the
inner plate 269 into the sample holding space of the outer plate 226. There is
no difference in
use or treatment steps between individual spin columns and plates with the
sole exception that
plates or other arrays must be supported in slightly different fashions. One
skilled in the art can
contemplate many mechanisms to support the inner plate within the sample-
receiving space 226
of the outer plate including, by example, clips or support rings or collars or
tabs, which might be
integrated into either the inner or outer plates, Or both, or which might be a
third piece, or pillars
or supports which swing up or collapse to afford the upper and lower parked
positions.
101891 The present application is further illustrated by the following
examples that
should not be construed as limiting. The contents of all references, patents,
and published patent
applications cited throughout this application, as well as the Figures and
Tables, are incorporated
herein by reference.
EXAMPLES
101901 This work outlines and demonstrates the concept of the Simultaneous
Trapping
technology, SiTrap, for detergent-free proteomics and metabolomics sample
preparation, which
is extensible to many other classes of molecules such as DNA, RNA and glycans
including
glycans covalently attached to proteins. SiTrap methodology provides the
opportunity for simple
and robust multiomics profiling performed on the same sample which has
significant impact for
comparative biological inference in nines data, high-throughput ornics
analysis, and is of key
importance when working with limited sample amounts in translational medical
research.
Methods
SiTrap tips
101911 SiTrap tips were constructed using either cellulose or quartz depth
filter
materials. 1.6-mm-diameter plugs were inserted into a pipette tip (D200,
Gilson). SiTrap
cellulose tips were used for cellular and tissue analyses. For the sample
processing steps
involving centrifugation (load_ wash and elution), the tips were placed in 2.0
or 1.5-nil sample
tubes with the aid of tube adapters.
Sample processing
Cell pellets
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10192] MDA-MB-231 cell pellets (1,000,000 cells per pellet) were lysed by
probe
sonication on ice in 250 pl of lysis solutions (30 mM ammonium acetate, 1.8%
ammonium
hydroxide (prepared by dilution of the stock 28% ammonium hydroxide solution
(Sigma)), 3%
SDS in 30 mM ammonium acetate, 3% P407 in 30 DIM ammonium acetate) for SiTrap,
and in
3% SDS in 50 mM Tris-HC1, pH 7.6 for SDS based approaches. The extracts were
clarified by
centrifugation at 11,000 g for 2 min at 18 'C. Protein concentration was
measured by Pierce
RCA Protein Assay Kit (Thermo). 30 pg of protein was processed in six
replicates in each case.
For ammonium acetate (AA) extraction SiTrap processing, four volumes of
methanol containing
30 InNI AA were added to the lysate whereby anhydrous methanol was
supplemented to 30 mM
AA from an aqueous 1 M ammonium acetate stock solution. For ammonium hydroxide
(All)
extraction SiTrap, an equal volume of 1 M acetic acid was added to the lysate
followed by the
addition of two volumes of methanol. The samples were loaded into SiTrap
cellulose tips. The
tips were inserted into 2.0-ml sample tubes and were centrifuged at 2000 g to
capture the
proteins. Captured proteins were washed by adding 80 gl of 50% methanol in 30
mM AA to the
tips followed by centrifugation at 2500 g for 30 sec. The tips were removed
and placed into 1.5-
ml sample tubes. The captured proteins were further denatured, reduced and
allcylated in situ by
adding 60 mM triethylarrunonium bicarbonate (TEAB), 10 mM tris(2-
carboxyethyl)phosphine
(TCEP), 25 inAil chloroacetamide (CAA) solution to the tips followed by
heating at 80 C for 30
min (the reduction/allcylation solution should be prepared prior to the start
of experiment and
thoroughly vortexed right before use). After a wash with 80 pl of 20 mlyi TEAR
at 2500 g for 30
sec, the tips were removed and placed into new 1.5-ml sample tubes. 20 p.1 of
Sequencing Grade
trypsin (Promega) in 100 mM ammonium bicarbonate at a concentration of 0. 07
Lig,41 was
added to the tips. The ttypsin solution was pushed down using a syringe with a
customized tip
adapter described previously till the solution meniscus was positioned ¨ 3 mm
above the top of
the cellulose plug. Tryptic digestion was done by incubation at 47 C for 1
hour. Post-digest
elution was performed consecutively with 70 IA 300 rtiM ammonium bicarbonate
and 70 pi of
3% formic acid. The peptides were concentrated using Cs Stage tips for the
downstream analysis
by mass spectrometry. For SDS processing 30 pg of protein was processed with
trypsin after
SDS removal. The digestion, peptide elution and concentration were the same as
for SiTrap.
10193] One skilled in the art will recognize that a tip format is only one
embodiment of
many different physical forms such as plates or spin columns or cartridges in
many different
formats. Specifically, and not exclusively, the application might be embodied
in a 96-well or
384-well plate or many other formats including cartridges and units integrated
with
chromatographic media or chromatographic separation systems, among many
others, and that
such an embodiment might also be coupled to sample collection and/or storage.
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Renal tissues
101941 Frozen renal tissue from three matched clear cell renal carcinoma (62
pT3a, G2
pT1b, 61 pT2) /adjacent normal sample pairs were obtained from The Leeds
Multidisciplinary
Research Tissue Bank. Approximately 1 cm' sections with a thickness of 10 pm
were cut for
each sample and placed into 1.5 ml sample tube. 80 gl of 1.8% ammonium
hydroxide was added
to the tube and the tissue was lysed by probe sonication. The tube was
centrifuged at 11,000 g
for 2 min at 18 I- C to remove the debris. The supematant was removed for
further processing.
The SiTrap load was normalized by protein concentration. 50 jig of protein was
loaded into the
SiTrap cellulose tips as described above for ammonium hydroxide lysates. The
collected flow-
through fraction, devoid of proteins, was dried down using a Speed-Vac for
targeted
metabolomics analysis. The captured protein fraction, in turn, was digested as
described above,
the resulting peptides were concentrated for proteomics analysis_
SiTrap serum processing method
101951 SiTrap quartz tips were constructed as described in this document.
Serum from a
healthy volunteer was obtained from The Leeds Multidisciplinary Research
Tissue Bank. 0.5 pl
of the serum was processed either directly by protein solubilization with 25
pl of 5% SDS in 50
mM Tris-HCl, pH 7.6 and hyptic digestion in OQ STrap tips (6 replicate samples
in total), or by
SiTrap, diluted with 30 gl of 20 inNI TEAB buffer and processed by
fractionation on SiTrap
quartz tips resulting in two fractions, 'captured' and 'flow-through' (3
replicates each for each
fraction, 6 samples in total). The diluted in TEAB serum was loaded into
SiTrap quartz tip and
gently pushed through with the aid of a syringe with a tip adapter, the 'flow-
through' fraction
was collected. The tip with the 'captured' fraction was inserted into 2.0-ml
sample tube and
washed consecutively with 100 gl and 40 gl of 20 inM TEAB using centrifugation
at 2500xg.
Reduction/alkylation and digestion of the trapped proteins were performed in
the same way as
described for cellular lysates in the Methods. The 'flow-through' fraction was
diluted with six
volumes of 30 mM ammonium acetate in methanol and consequently trapped and
digested in
another SiTrap tip in the same way as the 'captured' fraction. The resulting
peptides were
analyzed by LC-MS/MS using 100 min acquisition time as described in the
Methods. The
obtained data were processed as described below.
Proteomics
101961 Peptides were separated online by reversed-phase capillary liquid
chromatography using an EASY-nLC 1000 system (Proxeon) connected to a custom-
made 30-
cm capillary emitter column (inner diameter 75 pin, packed with 3 pin Reprosil-
Pur 120 C18
media, Dr. Maisch). The chromatography system was hyphenated with a linear
quadrupole ion
trap - orbitrap (LTQ-Orbitrap) Velos mass spectrometer (Thermo). The total
acquisition time
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was 100 min for cellular and 140 min for tissue analyses; the major part of
the chromatographic
gradient was 3% - 22% acetonitrile in 0.1% formic acid. Survey MS scans (scan
range of 305-
1350 amu) were acquired in the orbitrap with the resolution set to 60,000. Up
to 20 most intense
ions per scan were fragmented and analyzed in the linear trap. Data were
processed against a
Uniprot human protein sequence database (October, 2018) with MaxQuant 1522
software
package (www.maxquant.org) (Cox, J., Mann, M., MaxQuant enables high peptide
identification rates, individualized p.p.b.-range mass accuracies and proteome-
wide protein
quantification. Nature biotechnology 2008, 26, 1367-1372).
Carbamidomethylation of cysteine
was set as a fixed modification, with protein N-terminal acetylation and
oxidation of methionine
as variable modifications. Up to three missed cleavages and at least one
unique peptide for valid
protein identification were chosen. The maximum protein and peptide false
discovery rates were
set to 0.01. Analysis of Gene Ontology (GO) features was undertaken with
Panther 14.0
(www.pantherdb.org) (Thomas, P. D., Campbell, M. J., Kejariwal, A., Mi, H., et
al.,
PANTHER: a library of protein families and subfamilies indexed by function.
Genome Res
2003, 13, 2129-2141.). Perseus software package 1.6.2.3
(https://maxquant.net/perseus/)
(Tyanova, S., Temu, T., Sinitcyn, P., Carlson, A., et at., The Perseus
computational platform for
comprehensive analysis of (prote)omics data. Nat Methods 2016, 13, 731-740.)
was used for
volcano plot significance analysis ¨ the mean LFQ intensities of proteins were
1og2-transformed
and their differences plotted against the corresponding p values from 1-lest,
the significance cut-
offs were set to 0.05 for FDR and 0.01 for SO. For data comparison only
proteins identified with
at least two peptides and one unique peptide were used.
Metabolomics
Targeted metabolomic LC-MS analysis of acylcamitines, free fatty acids and
bile acids
101971 A solution of 10 gM palmitoyl-L-camitine-(N-methyl-d3) (Sigma), 10 pM
palmitic acid-d31 (Sigma) and 10 p,M deoxycholic acid-d6 (Sigma) in LC-MS
grade methanol
was prepared as internal standard spiking solution (1555). Samples were
reconstituted in 100 gl
LC-MS grade water and 100 p1 ISSS, vortex mixed and sonicated for 30 min
before being
transferred to LC vials. Chromatography was performed using an ACQUITY UPLC
system
(Waters) equipped with a CORTFCS T3 2.7 p.m (2.1 X 30 mm) column, which was
kept at
60 C. The ACQUITY UPLC system was coupled to a Xevo TQ-XS mass spectrometer
(Waters
Corporation). The binary solvent system used was solvent A comprising LC-MS
grade water,
0.2 mM ammonium formate and 0.01% formic acid, and solvent B comprising
analytical grade
acetonitrile /isopropanol 1:1, 0.2 mM ammonium formate, and 0.01% formic acid.
For all
analyses a 10 pi injection was used and mobile phase was set at a flow rate of
1.3 ml/min. For
acylcamitine analysis, the column mobile phase was held at 2% solvent B for
0.1 min followed
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by an increase from 2% to 98% solvent B over 1.2 min. The mobile phase was
then held at 98%
solvent B for 0.9 min. The mobile phase was then returned to 2% solvent B held
for 0.1 mins to
re-equilibrate the column. For free fatty acid analysis, the column mobile
phase was increased
from 50% to 98% solvent B over 0.7 min. The mobile phase was then held at 98%
solvent B for
0.5 min. The mobile phase was then returned to 50% solvent B held for 0.1 min
to re-equilibrate
the column. For bile acid analysis, the column mobile phase was held at 20%
solvent B for 0.1
min followed by an increase from 20% to 55% solvent B over 0.7 min. The mobile
phase was
increased to 98% solvent B and held for 0.9 min. The mobile phase was then
returned to 20%
solvent B held for 0.1 mins to re-equilibrate the column. Analyses were
performed using
multiple reaction monitoring (MRM). Transitions and ionization conditions are
given in tables 1,
2 and 3. For acylcamitine analyses the Xevo TQ-XS was operated in positive
electro-spray
ionization (ESI) mode. For free fatty acid and bile acid analyses the Xevo TQ-
XS was operated
in negative ES! mode. A cone gas flow rate of 50 ml/hr and desolvation
temperature of 650 C
was used.
Metabolomics data analysis
[0198] Data were processed and peak integration performed using the Waters
Targetlynx
application (Waters Corporation). Integrated acyl-carnitine, free fatty acid,
and bile acid peak
areas were normalized to the palinitoyl-L-carnitine4N-methyl-d3), palmitic
acid-d31 or
deoxycholic acid-d6 internal standard respectively.
[0199] Multivariate data analysis was performed using Metaboanalyst version
4.0
(Chong, J., Soufan, O., Li, C., Caraus, I., et al., MetaboAnalyst 4.0: towards
more transparent
and integrative metabolomics analysis. Nucleic Acids Res 2018, 46, W486-
W494.). Data sets
were mean-centered and analyzed using principal components analysis (PCA) and
partial least
squares-discriminant analysis (PLS-DA). Metabolite changes responsible for
clustering or
regression trends within the pattern recognition models were identified by
interrogating the
corresponding loadings plot. Metabolites identified in the variable importance
in
projections/coefficients plots were deemed to have changed globally if they
contributed to
separation in the models with a confidence limit of 95%. These were verified
using univariate
volcano plots with a fold change cut off of 1.2 and P-value cut off of 0.05.
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10200] Table 1 Multiple Reaction Monitoring Parameters for acyl-carnitines
species. Acyl-camitines are designated by acyl chain length in carbons and
degree of
unsaturated double bonds. Internal standard (IS).
Acyl-Carnitine Parent Ion Fragment Ion Cone
Voltage Collision
WO (neZ)
(v) Energy (ev)
C18:2 424.3 85
50 28
C18:1 426.4 85
50 28
C180 428.4 85
50 28
C16:1 398.3 85
50 26
C16:0 400.3 85
50 26
C14:2 368.3 85
46 26
C14:1 370.3 85
46 26
C14:0 372.3 85
46 26
C12:1 343.3 85
46 24
C12:0 344.3 85
46 24
C10:1 314.2 85
42 24
C10:0 316.2 85
42 24
C8:1 286.2 85
42 22
C8 288.2 85
42 22
C6 260.2 85
54 20
C5:1 244.2 85
38 22
C5 246.1 85
38 22
C4 232.1 85
34 20
C3 218.1 85
32 18
C2 204.1 85
32 18
C16:0-d3 IS 403.4 341.26
8 18
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10201] Table 2 Multiple Reaction Monitoring Parameters for free fatty acid
species.
Free fatty acids are designated by acyl chain length in carbons and degree of
unsaturated double
bonds. Internal standard (IS).
Free Fatty Parent Ion Fragment Ion Cone
Voltage Collision
Acid WO (nez)
(v) Energy (ev)
C22:6 327.25 327.25
45 7
C22:5 329.25 329.25
45 7
C22:4 331.25 331.25
45 7
C22:1 337.25 337.25
45 7
C22:0 339.25 339.25
45 7
C20:5 301.25 301.25
45 7
C20:4 303.25 303.25
45 7
C20:3 305.25 305.25
45 7
C20:2 307.25 307.25
45 7
C20:1 309.25 309.25
45 7
C20:0 31L25 311.25
45 7
C18:3 277.25 277.25
45 7
C18:2 279.25 279.25
45 7
C18:1 281.25 281.25
45 7
C18:0 283.25 283.25
45 7
C17:1 267.25 267.25
45 7
C17:0 269.25 269.25
45 7
C16:2 251.25 251.25
45 7
C16:1 253.25 253.25
45 7
C16:0 255.25 255.25
45 7
C15:1 239.25 239.25
45 7
C15:0 241.25 241.25
45 7
C14:1 225.25 225.25
45 7
C14:0 227.25 227.25
45 7
C12:1 197.25 197.25
45 7
C12:0 199.25 199.25
45 7
C16:0-d31 IS 286.62 286.62
45 7
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10202] Table 3 Multiple Reaction Monitoring Parameters for bile acid species.
Internal standard (IS).
Bile Acid Parent Ion
Fragment Cone Collision
(nut) Ion
(m/z) Voltage (v) Energy (ev)
Glycoursodeoxycholic acid 448.25
74 60 35
Tauroursodeoxycholic acid 498.25
80 60 60
Taurohyodeoxycholic acid 498.25
80 60 60
Taurocholic acid 514.25
80 60 64
Glycocholic acid 464.25
74 60 34
Taurochenodeoxycholic acid 498.25
80 60 60
Taruodeoxycholic acid 498.25
80 60 60
Ursodeoxycholic acid 391.25
391.25 60 16
Cholic acid 407.25
343.25 60 34
Glycochenodeoxycholic acid 448.25
74 60 35
Glycodeoxycholic acid 448.25
74 60 35
Taurolithocholic acid 482.25
80 60 60
Chenodeoxycholic acid 391.25
391.25 60 16
Glycolithocholic acid 432.25
74 60 35
Deoxycholic acid 391.25
391.25 60 16
Lithocholic acid 375.2
373.25 60 32
Deoxycholic acid-d6 IS 397.23
331.32 80 36
10203] A tip format SiTrap is made according to the disclosure of this
application and a
portion of sample is added according to the disclosure such that 50 jug of
protein are captured in
the trapping matrix in a total volume of ammonium acetate and methanol
totaling 150 FtL; there
will be more than 50 p.g of total captured molecules because DNA, RNA and
glycans among
other molecules will be trapped. The flow through is kept which contains small
molecules and
lipids, which will be taken to lipidomics and metabolomics analysis. Some
lipids and small
molecules may be retained. DNA and RNA is first eluted by the addition of 3x
of 50 pL TE
buffer commonly used in molecular biology; this fraction may be analyzed by
transcriptomics,
RNAseq and genomics techniques. The sample is next treated with 2 p.g of
PNGase F in 50 1.11,
of a phosphate buffer for at least one hour and up to overnight at 37 C. The
glycans are spun out
and recovered for glycomics analysis. The proteins are reduced and allcylated
as disclosed
below. 2 lug of trypsin is added in 40 laL of 50 inlvi TEAR at pH 8.5 and the
proteins are
digested 1 hr at 47 C; other incubation times and temperatures can be added.
The capture matrix
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may be sonicated or ultrasonicated to speed digestion. Resulting peptides are
taken into
proteomics analyses. Alternatively, proteins can be eluted for top-down
proteomics by
sonication in, by example 40 pL 60% formic acid, 8 M urea or 6 M GuHCI; these
reagents work
best with sonication.
102041 To solve the need for multi-omics analysis, where detergent or
chaotrope or other
solubilization agents prevent downstream analysis of molecules not bound on
the trapping
matrix, the system, device, process and method herein were devised to match
the protein
processing power and simplicity of detergent based methods but with the use of
a detergent-free
composition for lysis which allows for post-capture in situ
reduction/allcylation of the trapped
proteins, providing a contaminant-free flow-through fraction for complementary
`oinics'
analysis. The process can be automated to make a sample processing machine.
Example 1
102051 While working with cell lysates and non-ionic detergents such as octyl
glucoside
and Poloxamer 407, unexpectedly proteins in their native state can be captured
by cellulose or
quartz depth filters at near-neutral pH (Fig. 1) with or without detergents.
This is surprising and
represents a significant new advance which enables this method. The capture
was robust. One
skilled in the art will recognize that there are many other surfactants and
detergents.
Unexpectedly, further denaturation, reduction, alkylation and wash steps in
situ was possible
with subsequent digestion performed in the device and in the trapping matrix.
One skilled in the
art will recognize that there are many reduction and allcylation reagents can
be used. This
application provides the optimal composition and means of lysing cells and
samples and
biological without detergents, capturing extracted proteins and other molecule
classes such as
DNA and gly cans in situ, while the metabolites and lipids and small molecules
are separated and
kept in the flow through, and further allowing the retained molecules to be
processed by
example by enzymatic digestion for example with a protease or nuclease or
glycosidase. This
application shows that sonication of a cell pellet either at near-neutral or
basic pH efficiently
releases proteins into solution with a similar extraction efficiency to SDS
(Fig. 2); this
application shows that proteins can be captured by either cellulose or quartz
depth filter trap.
One skilled in the art will recognize that, as described above, many other
filter materials or
porous materials could be employed, so long as the proteins are captured.
102061 To outline SiTrap cellular processing, firstly a cell pellet is
sonicated in excess of
either 30 m1VI ammonium acetate (AA) or 1.8 % ammonium hydroxide (AH) with
subsequent
centrifugation to remove debris. Using AH for lysis, and analyzing UV
absorbance at 280 nm in
a microvolume spectrophotometer, may provide a coarse direct estimation of
protein
concentration in cell lysates I . If AA extraction is used, four volumes of
methanol containing 30
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niM AA are added to the lysate. The sample is loaded into a SiTrap tip
containing a depth filter
compartment where proteins are instantly trapped. If AH extraction is used,
then first an equal
volume of 1M acetic acid is added to the lysate to bring the pH close to
neutral, then two
volumes of methanol are added before loading into the SiTrap tip. The
resulting flow-through is
collected for additional comics' processing. The captured proteins are
denatured, reduced and
alkylated in situ by heating at 80 C in 60 inM thethylammonium bicarbonate
(TEAB), 10 inlY1
tris(2-carboxyethyl)phosphine (TCEP), 25 mM chloroacetamide (CAA) solution.
After a wash,
trypsin is added and the sample incubated at 47 C for one hour to provide
digestion of the
proteins. The peptides are eluted and then concentrated using CS or Cis Stage
tips for analysis by
mass spectrometry (MS) (Fig. 3A). It is noted that DNA is also co-captured,
and that other
enzymes can be used such as gjycosidases to extend this application to other
molecule classes.
102071 To test the proteomics performance of the new SiTrap methodology, it
was
compared with SDS based sample preparation. MDA-MB-231 cells were extracted
using either
cell lysis and probe sonication on ice with AA or AH followed by SiTrap try
ptic processing in
cellulose SiTrap tips (Fig. 4) or cell lysis and probe sonication with SDS
followed by digestion.
30 mg of protein was processed in six replicates in each case. Tryptic
digestion was performed at
47-C for one hour both for the samples. The test identified 1293 (+12 SD) and
using SDS, 1278
(+44 SD) proteins on average with at least two peptides using AA or AH SiTrap
lysis,
respectively. This was comparable with the 1230 ( 27 SD) average number of
proteins
identified for SDS lysis (Fig. 3B). The protein distributions in the main GO
cellular component
categories were very similar in all cases (Fig. 3C) and the majority of
proteins were identified by
all three approaches indicating an absence of bias (Fig. 3D).
Example 2
102081 The ability of the SiTrap method and device to provide a simultaneous
multiomics analysis platform was probed using a comparative proof-of-principle
proteomics/
metabolomics profiling study of clear cell renal carcinoma and corresponding
adjacent
noncancerous tissue sections. One skilled in the art will realize that this is
simply an exemplary
example, and is not limiting. The tissue sections (three normal/tumor pairs)
were lysed by
sonication with AH, the lysates were loaded into the SiTrap cellulose tips,
the flow-through
fractions were collected for targeted metabolomics profiling whereas the
captured proteins were
digested for proteomics analysis. Proteomics analysis resulted in a proteome
dataset of 2655
proteins. The targeted metabolomics screen included 62 species across three
metabolite classes ¨
26 free fatty acids, 20 acyl carnitines, and 16 bile acids. Of these 59
metabolites were observed
and quantified ¨ 25 free fatty acids, 19 acylcarnitines, and 15 bile acids,
The metabolomics
analysis indicated a decrease both in short-chain acylcarnitines (CS, CS:1 and
C3) and in
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polyunsaturated free fatty acids (C20:5, C20:4, C22:6) in the tumor samples
(Fig. 5A, Fig. 6A).
Camitine 0-acetyltransferase (CRAT), Carnitine 0-palmitoyltransferase 2 (CPT2)
and Camitine
0-palmitoyltransferase 1 (CPT1A), the enzymes with crucial roles in
acylcarnitine metabolism,
were identified, quantified and found to be significantly decreased in the
tumor samples, in
concordance with the metabolomics results (Fig. 58, Fig. 68), A significant
decrease in the
tumor samples of other enzymes relevant to the polyunsaturated fatty acid
metabolism was
detected, in concordance with the metabolomics results: Acyl-CoA Thioesterase
1 (ACOT1)
which releases C20:4, C20:5 and C22:6 from their CoA equivalents, and long
chain Fatty acid-
CoA ligase (ACSL1) which activates long-chain fatty acids to form acyl-CoAs
(Figure 5B).
Exantple 3
[0209] While working with serum samples it was observed that at basic pH, e.g.
diluted
in 20 mM TEAR, serum albumin is not trapped by a depth filter. However, many
other serum
proteins are captured (Fig. 7). This simple serum fractionation results in two
fractions - the
captured fraction, devoid of albumin, which can be directly processed in-tip
by SiTrap. The
alternative flow-through 'albumin' fraction can then be diluted with six
volumes of 30 mM
ammonium acetate in methanol and consequently captured and digested in another
SiTrap unit.
To test this approach 0.5 1 of human serum from a healthy volunteer was
either digested
directly by STrap technology (6 replicate samples in total) or diluted with 20
mM TEAB buffer
and processed by fractionation using SiTrap quartz tips. SiTrap processing
produced two
fractions, captured and flow-through (3 replicates each for each fraction, 6
samples in total). The
MS results from tryptic digests of the 6 samples in each approach were merged.
The SiTrap
fractionation lead to -30% increase in protein identifications compared with
the direct approach
(Fig. 78, C). This example demonstrates fractionating serum into two or more
fractions.
Example 4
102101 SiTrap sample processing also works on FFPE samples, due to their
ubiquitousness in pathology, stability at room temperature and the sheer
number of FFPE
samples. Such samples, while representing a rich resource, are however very
difficult due to
their formalin-crosslinked nature and being embedded in wax. Surprisingly,
SiTrap functioned
well. Human renal FFPE tissue was deparaffinized by standard xylene/ethanol
treatment and
then lysed in 30 mlY1 ammonium acetate by probe sonication. -50 pg of the
resultant protein
lysate was processed either by SiTrap or SDS methods. For SiTrap -4 volumes of
methanol in
30 mM ammonium acetate was added to the lysate followed by protein capture in
SiTrap
cellulose tip, the tip was further washed with 60% methanol in 30 mM ammonium
acetate, the
flow-through was collected (FT1). Then 10 mM TCEP/30 mM chloroacetamide/60 mM
TEAB
solution was added and the tip was heated at 95C for 1 hour. The tip was then
washed with 20
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mM TEAB, the flow-through was collected (FT2). The trapped proteins were
digested at 48C by
two consecutive 1-hour digestions with 1.25 pg of trypsin (Promega) in 100 mM
ammonium
bicarbonate (ftypsin concentration 0.07 mg/pi). Digest products were eluted
consecutively by
500 mM ammonium bicarbonate and 50% acetonitrile in 0.2% formic acid. The
leftover material
was eluted by 2X Laenrunli buffer. For SDS processing, the lysate was mixed
with equal volume
of 5% SDS in Tris-HCI pH 7.6, DYE was added to the final concentration of 20
mM, the sample
was heated at 95C for 1 hour. Chloroacetamide was added to the final
concentration of 120 mM
with consequent incubation for 30 min. The sample was cleared of SDS by the
standard protocol
and the flow-through was collected (FT). Similarly to SiTrap, the proteins
were digested at 48C
by two consecutive 1-hour digestions with 1.25 pg of trypsin (Promega) in 100
mM ammonium
bicarbonate (trypsin concentration 0.07 pg/p1). Digest products were eluted
consecutively by
500 inIVI ammonium bicarbonate and 50% acetonitrile in 0.2% formic acid. The
leftover material
was eluted by 2X Laemmli buffer. This example demonstrates use of SiTrap
technology for
FFPE tissue (Fig. 8).
Example 5
102111 One exemplary embodiment of this application comprises capture of
proteins and
small molecules as described herein from one volume of a sample, wherein the
sample was
sonicated in the 30 mM ammonium acetate extraction solvent to physically
disrupt the sample,
mixed with four volumes of methanol containing 30 inNI ammonium acetate,
whereby
anhydrous methanol was supplemented to 30 triM ammonium acetate from an
aqueous 1 M
ammonium acetate stock solution. The mixture was then applied to a cellulose
depth filter
employed as a trapping matrix, and the flow through, which contains small
molecules of the cell
in particular and not limited to metabolites and lipids, was kept for
metabolomics and lipidomics
analysis. The proteins were then reduced and alkylated in situ by heating at
80 C in 60 mM
triethylammonium bicarbonate (TEAB), 10 mM tris(2-carboxyethyl)phosphine
(TCEP), 25 mM
chloroacetamide (CAA) and the depth filter material was washed either with 50%
methanol in
30 mM ammonium acetate or 20 mM TEAB and centrifuged at 2500 g for 30 sec. 1.4
ug of
trypsin (Promega) was added in 20 uL of 100 mM ammonium bicarbonate and the
trapped
proteins were digested into peptides by incubation at 47 C for 1 hr. Post-
digest elution was
performed consecutively with 70 gl 300 mM ammonium bicarbonate and 70 I of 3%
formic
acid. The peptides were concentrated using C8 Stage tips for the downstream
analysis by mass
spectrometry; such C8 processing might be integrated underneath the trapping
matrix. This
example demonstrates the multiomic nature of SiTrap.
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Exantple 6
102121 Some experiments were analyzed on an Agilent 6546 QTOF using the
following
settings for peptide analysis, unless otherwise stated: 300 ¨ 1700 nilz
acquired in AutoMS2
positive mode with the Dual MS ESI source with 325 C gas temp at 13 L/min and
275 C sheath
gas temp. MSMSes were acquired a medium isolation width using 5000 MS absolute
precursor
threshold and 0.01% relative threshold with a target of 50,000 counts per
spectrum and active
exclusion enabled; VCap was set to 3500 and fragmentor to 175 V. An Agilent
1290 Infinity LC
system was used running at 0.3 mL/min on a 2.1 mm x 150 mm C18 column with a
gradient
between buffer A, water with 0.1% formic, and buffer B, 100% LCMS grade
acetoninile,
holding at 5% B for 2 minutes then ramping to 40% B over 50 minutes holding at
a 90% B was
for 5 min and ramping back down to 5% B. The column, an 2.1 x 150 mm Agilent
AdvanceBio
Peptide Mapping 2.7 urn column (cat #653750-902), was kept at 60 C.
102131 Metabolite analysis on an Agilent 6546 QTOF used the following settings
for
peptide analysis, unless otherwise stated: data was acquired over 300¨ 1700
m/z in AutoMS2
positive mode with the Dual MS ESI source with 350 C gas temp at 5 L/min and
350 C sheath
gas temp at 10 L/min. MSMSes were acquired a medium isolation width using 5000
MS
absolute precursor threshold and 0.01% relative threshold with a target of
50,000 counts per
spectrum and active exclusion disabled and isotope model set to common organic
molecules;
VCap was set to 3500 and fragmentor to 175 V. An Agilent 1290 Infinity LC
system was used at
0.8 mL/min on a 2.1 mm x 50 mm Agilent EclipsePlus C18 column (RRHD1.8um) with
a
gradient between buffer A, water with 0.1% formic, and buffer B, 100% LCMS
grade
acetonitrile (see table below for gradient). The column was kept at 40 C. The
gradient was as
follows:
Event Time (min) A (%) B (%) Flow (mL/inin) Pressure (bar)
1 2 95 5
0.8 600
2 4 60 40
0.8 600
3 5 50 50
0.8 600
4 10 50 50
0.8 600
15 30 70 0.8 600
6 19 20 80
0.8 600
7 20 20 80
0.8 600
8 21 95 5
0.8 600
102141 SiTrap columns of Figs. 10¨ 14 were made using plastic injection
molding
formed from TPX plastic. The inner column was loaded with a porous matrix
fashioned of
quartz, glass fiber, polymers, cellulose or cellulose with fillers such as
diatomaceous earth, using
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a pneumatic punching and pressing system. In some experiments, the matrix was
afforded
functional groups by derivatizations. In other experiments, the matrix was
layered atop a
chromatographic media like C18 or SCX. In other experiments, the inner vial
received a bottom
and top flit holding chromatographic media_
Example 7
Detection of SARS-CoV-2 using disclosed method and disclosed physical
embodiment
102151 The following example illustrates the use of the system to detect SARS-
CoV-2
nucleocapsid protein, one of the more abundant proteins in the SARS-CoV-2
virus. Due to the
BSL level of the laboratory in which the experimentation was performed,
infectious virus was
not directly analyzed. Rather, the nucelocapsid protein was made recombinantly
and spiked into
sputum. One skilled in the art will recognize that this example is not
limiting, is applicable to
live and infectious virus, within the limits of sensitivity of detection, and
serves to illustrate the
method's applicability to diagnosis. One skilled in the art will also
recognize that this example
can be extended to other viruses and pathogens, which have different genetic
sequences and thus
different protein sequences, by changing only the parameters of detection. The
steps of this
example are duplicated in other examples.
102161 Human embryonic kidney cells 11EK293 (approximately 1.5E6 cells/well, 6
well
plate) were transfected with plasmid pCI-SARS-CoV-2-nucleoprotein (2 ug
plasmid per well)
using Lipofectamine 2000. The ORF of SARS-CoV-2 nucleoprotein was PCR
amplified from a
synthetic DNA clone. The nucleoprotein ORF sequence contained in the pCI-SARS-
CoV-2-
nucleoprotein plasmid is as follows:
ATGTCTGATAATGGACCCCAAAATCAGCGAAATGCACCCCGCATTACGITTIGGTGG
ACCCTCAGATTCAACTGGCAGTAACCAGAATGGAGAACGCAGTGGGGCGCGATCAA
AACAACGTCGGCCCCAAGGITTACCCAATAATACTGCGTCTTGGITCACCGCTCTCA
CTCAACATGGCAAGGAAGACCTTAAATTCCCTCGAGGACAAGGCGTTCCAATTAAC
ACCAATAGCAGTCCAGATGACCAAATTGGCTACTACCGAAGAGCTACCAGACGAAT
TCGTGGTGGTGACGGTAAAATGAAAGATCTCAGTCCAAGATGGTATTTCTACTACCT
AGGAACTGGGCCAGAAGCTGGACTTCCCTATGGTGCTAACAAAGACGGCATCATAT
GGGTTGCAACTGAGGGAGCCITGAATACACCAAAAGATCACATTGGCACCCGCAAT
CCTGCTAACAATGCTGCAATCGTGCTACAACTTCCTCAAGGAACAACATTGCCAAA
AGGCTTCTACGCAGAAGGGAGCAGAGGCGGCAGTCAAGCCTCTTCTCGTTCCTCAT
CACGTAGTCGCAACAGTTCAAGAAATTCAACTCCAGGCAGCAGTAGGGGAACTTCT
CCTGCTAGAATGGCTGGCAATGGCGGTGATGCTGCTCTTGCTTTGCTGCTGCTTGAC
AGATTGAACCAGCTTGAGAGCAAAATGTCTGGTAAAGGCCAACAACAACAAGGCC
AAACTGTCACTAAGAAATCTGCTGCTGAGGCTTCTAAGAAGCCTCGGCAAAAACGT
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ACTGCCACTAAAGCATACAATGTAACACAAGCTTIUGGCAGACGTGGTCCAGAACA
AACCCAAGGAAATTITGGGGACCAGGAACTAATCAGACAAGGAACTGATTACAAA
CATTGGCCGCAAATTGCACAATTTGCCCCCAGCGCTTCAGCGTTCTTCGGAATGTCG
CGCATTGGCATGGAAGTCACACCITCGGGAACGTGGTTGACCTACACAGGTGCCAT
CAAATTGGATGACAAAGATCCAAATTTCAAAGATCAAGTCATTTTGCTGAATAAGC
ATATTGACGCATACAAAACATTCCCACCAACAGAGCCTAAAAAGGACAAAAAGAA
GAAGGCTGATGAAACTCAAGCCITACCGCAGAGACAGAAGAAACAGCAAACTGTG
ACTCTTCTTCCTGCTGCAGATITGGATGATITCTCCAAACAATTGCAACAATCCATG
AGCAGTGCTGACTCAACTCAGGCCTAA
10217] The expressed nucleoprotein amino acid sequence is as follows:
MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQ
HGICEDLICFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGICMICDLSPRWYFYYLGTG
PEAGLPYGANKDGIIWVATEGALNTPICDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEG
SRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESIC_M
SGKGQQQQGQTVTICKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQEL
IRQGTDYICHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDICDPNFICDQ
VILLNICHIDAYKTFPPTEPICKDKICKICADETQALPQRQICKQQTVTLLPAADLDDFSKQL
QQSMSSADSTQA
10218] One well was transfected with 1 ug pCI-SARS-CoV-2-nucleocapsid protein
+ 1
ug plasinid pTM2-GFP. One well was transfected with 2 ug plasinid pTM2-GFP.
After 40 hours
of transfection, cells were washed with PBS, scarped into PBS, collected by
centrifugation at
300 x g for 2 minutes and stored at -80 C. After 40 hours of transfection,
cells were washed with
PBS, scraped into PBS and collected by centrifugation at 300 x g for 2
minutes. Cotransfection
with GFP and nucleocapsid protein did not show any difference to GFP alone,
indicating that
expression of the nucleocapsid protein does not affect the growth of HEK293
cells.
10219] Cell pellets were processed as follows: to the cells was added either
1.8%
ammonium hydroxide at approximately a 1:20 v/v ratio. Samples were sonicated
either on a
sonication water bath or a Covaris ultrasonicator. Samples were treated as a
suspension and were
sonicated and vortexed prior to any removal of sample. Protein concentration
was assayed by
BCA and set to mg/mL by dilution with 1.8% ammonium hydroxide. Sputum was
obtained
ammonium hydroxide added to a final concentration of 1.8% from a 32% stock
solution; sputum
concentration was roughly 3 ing/mL. The sputum sample was similarly
(ultra)sonicated and 1
uL of nucleocapsid protein solution was added to 99 uL sputum solution for a
total protein load
of roughly 300 ug in 100 uL; multiple replicate mixtures were prepared. To the
samples was
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added 100 tiL of 1 M acetic acid and 2 or 4 volumes of methanol was added in
different SiTrap
tubes loaded with cellulosic matrix in their lower parked position.
[0220] The SiTrap assemblies were raised to their upper parked positions and
the sample
was propelled through the matrix by centrifugation at 4,000 g for 5 min and
the flow thorough,
containing metabolites and small molecules, kept in the outer vial, The SiTrap
inner vials were
placed in fresh outer vial tubes in the lower parked position. Al this point,
five replicates were
left at room temperature for 4 days and five other replicates were kept at -80
C. At the end of 4
days all samples were brought to room temperature. Proteins were reduced and
alkylated for 10
min at 80 C with 10 mAil TCEP and 25 inM chloroacetamide in a 50 inNI tiis
buffer at pH 8 in
the lower parked position. The reduction and alkylation reagents were removed
by
centrifugation in the upper solution, the proteins were washed with 75% Me0H
and the washes
and flow through discarded. The inner vial was placed in the lower parked
position of the outer
vial. Four samples, two stored at room temperature and two at -80 C, were
subjected to
accelerated digestion by exposure to ultrasonication. Because the
ultrasonciation treatment was
serial, to each of the four samples was first added 30 ug of trypsin added and
the samples were
immediately placed in a Covaris M220 and suspended via an improvised wire rack
fashioned
from straightened paperclip wire and held in place with lab tape. Each sample
received
ultrasonication treatment for 20 minutes with settings of 50 peak power, 20
duty factor and 300
burst/cycle. Samples were placed on ice to limit trypsin's activity. Two other
samples received
30 ug of trypsin and four samples 30 ug of trypsin followed by a 1 hr 47 C
incubation. Two
samples were incubated overnight at 37 C with 15 ug of trypsin. Digestions
were in 50 rnM
TEAB using Worthington trypsin.
[0221] All samples were analyzed in targeted MS mode on the Agilent 6546 set
to
perform targeted MSMS on the following miz for +2 charges for expected tryptic
peptides of the
SARS-CoV-2 protein: 375.180466, 403.193573, 443.706317,458.742368, 471.784567,
563,78563, 564.785827, 573,751461, 601.809833, 741.330469, 835.948346,
842,948869,
894.929196, 912.411368, 931.48073, 1013.021708, 1030.578571,
1091.013989,11118.541465,
1134.044029, 1162.598357, 573.751461, 912_411368, 1162.598357, 1091_013989,
1134.044029, 842.948869, 1030.578571, 443.706317, 375.180466, 403.193573,
835.948346,
601.809833, 563.78563, 894.929196, 1118.541465, 1013.021708, 471_784567,
458.742368,
564.785827, 931.48073, 741.330469. Data files were subsequently exported and
loaded into
Scaffold using its internal deconvolution and data search algorithms. Of the
include list, the
following peptides were detected: ITFGGPSDSTGSNQNGER at 912.411368,
WYFYYLGTGPEAGLPYGANK at 1134.041029, DGI1WVATEGALNTPK at 842.948869,
NPANNAAIVLQLPQGTTLPK at 1030.578571, MAGNGGDAALALLLLDR at 835.948346,
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AYNVTQAFGR at 563.78563, GPEQTQGNFGDQELIR at 894.929196,
IGMEVTPSGTWLTYTGAIK at 1013.021708, ADETQALPQR at 564.785827,
QQTVTLLPAADLDDFSK at 931.48073 and QLQQSMSSADSTQA at 741.330469; fragment
ions observed are listed in the below table and representative traces are
shown in Figs. 23-24.
Peptide sequence Fragment
ions observed
ITFGGPSDSTGSNQNGER y12, y8,
y7, y6, y5, y3,131, b2, b6, b7, b8, b9, b12
y15, y14, y13, y12, y11, y6, y4, y2, b2, b3, b4, b5,
WYFYYLGTGPEAGLPYGANK
b6, b8, b9, b12
DGIIWVATEGALNTPK yll,
y10, y8, yl, b6, b9, b10
NPANNAAIVLQLPQGTTLPK y10, y8,
y2, yl, b2, b3
MAGNGGDAALALLLLDR y13,
y12, yll, y9, y8, y7, y6, y3, b2, b3
AYNVTQAFGR y8, y7,
y6, y5, y3, b2, b3, b4
GPEQTQGNFGDQELIR y5, y4-,
y3, yl, b6, bll
IGMEVTPSGTWLTYTGAIK y14,
y13, y8, y7, y6, y4, b6
ADETQALPQR y2, yl,
b3, b7
QQTVTLLPAADLDDFSK y12, y6,
bl, b7, b9, b14
QLQQSMSSADSTQA y4, bl,
b2, b8
[0222] There were no significant differences in the ability to detect the SARS-
CoV-2
protein between 2 and 4 X methanol additions, between storage at room
temperature for four
days and storage at room temperature, or between overnight 37 C, 1 hr 47 C or
20 min RT
ultrasonication accelerated digestion. These results indicate that the SiTrap
method is applicable
to detect viruses and pathogens; that the inner and outer vial assembly is
compatible with
treatments of ultrasonication, and that ultrasonication speeds enzymatic
processing, and
importantly that sample, once bound, is stable at room temperature without
desiccation and in
the presence of oxygen for at least four days.
Example 8
Accelerated serum processing
[0223] 100 ug of serum in 50 uL was denatured and dissolved in 1.8% ammonium
hydroxide, mixed with 50 uL of 1 M acetic acid and provided with 2 volumes of
methanol. This
solution was applied to the inner and outer vial assembly in the upper parked
position and the
flow through recovered after centrifugation for 5 min at 4,000 g. The flow
through fraction was
taken for analysis via MSMS in the metabolite analysis mode. Serum was
reduced, alkylated and
digested as described for nucleocapsid protein by sonication in a Covaris
M220. The small
molecule fraction was analyzed by Agilent's MassHunter Qualitative Analysis
version 10.0
searching against all METLIN and metabolite databases. 231 compounds were
detected in
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metabolite mode. Peptides from the digestion were analyzed and searched using
SpectrumMill
against the human UniProt database. 344 protein groups including 1207 total
proteins were
detected. In some experimental iterations, the flow through fraction was dried
and exposed to a
4:2:1 mixture v/v/v of 2-propanol/methanol/chloroform containing 7.5 mm
ammonium acetate
to generate a lipid fraction. 95% water with 0.1% formic plus 5% acetonitrile
was then added to
the outer vial and sonicated. In other experimental iterations, to the
neutralized sample, 100 uL
of chloroform followed by 300 uL of water and 400 uL of methanol was added and
mixed. The
inner vial was placed in a new vial and the solution centrifuged through. The
mixture phase
separated, with the upper layer containing more hydrophilic moieties and the
lower more
hydrophobic moieties like lipids. These approaches generated separate
lipidornics and
metabolomics fractions for higher ID rates. This sample illustrates that the
SiTrap method and
assembly can rapidly produce samples for metabolomics, lipidoinics and
proteornics analysis.
The many metabolites are hydrophilic, and the use of additional chromatography
such as HILIC
will afford more identifications, and that the solvents described here are not
limiting, but can be
chosen to match the solubility properties of the analytes or classes of
analytes of interest. It is
specifically noted that combinations of solvents which phase partition are of
special use, because
their relative hydrophobicity can be altered, allowing tuning to the specific
needs of analysis or
treatment or processing.
Example 9
In-trap cell and tissue processing
[0224] ¨10 uL of red blood cells (RBCs) or ¨10 mg of mouse liver were added to
50 uL
of 1.8% ammonium hydroxide directly in the assembly in the lower parked
position. The
assembly was exposed to 5 ¨ 10 min of ultrasonication on the M220 to lyse the
cells or tissue.
RBCs appeared to be fully dissolved and the liver appeared to be either fully
disaggregated. 50
uL of 1 M acetic acid was then added, followed by 250 uL of HPLC grade
methanol. The inner
vial was moved to the upper parked position and the small molecule fraction
recovered via
centrifugation. In other experimental variations the processed RBCs or mouse
liver was
provided with chloroform as well to yield phase separated fractions for
lipidomics (bottom
layer) and metabolomics (top layer). The liver sample inner vial was placed in
a clean new outer
vial and 50 uL of TE was added and incubated for 30 min_ This fraction,
containing surviving
RNA as well as DNA sheered by the ultrasonication, was eluted in the upper
parked position,
the matrix washed with 100 uL of TE and the sample was placed in anew outer
vial. SDS-
PAGE analysis showed very little protein. The inner vial was placed in a new
outer vial and 10
uL of PNGAse F at 500 units/mL was added in 50 mM sodium phosphate at pH 8.6
to a total
volume of 50 uL. The sample was processed at 37 C for 5 lus and this fraction,
containing
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glycans, was eluted by movement of the inner vial to the upper parked position
and
centrifugation. SDS-PAGE analysis showed no protein in this fraction and it
was glycan positive
by periodic acid and alcian blue tests. Finally, in a new outer vial, the
sample was reduced,
digested and alkylated as described above but with an overnight 37 C
incubation. After further
elution of peptides with 50 inM TEAB and 50% ACN, no material was observed on
the column,
indicating that the procedure was sufficient to fully process the tissue. In
the event that had the
tissue been contaminated with blood, an appropriately designed assembly with
pore sizes great
enough to allow passage of RBCs presents a mechanism for a manual or automated
cleaning
system.
Example 10
FFPE
[0225] 1 min cores of formalin fixed paraffin embedded mouse livers stored at
room
temperature were punched and homogenized in trap hydroxide as with in-trap
cell and tissue
processing for 10 min on the M220 following an overnight incubation in 1.8%
ammonium to
rehydrate the sample. The methanol and chloroform extraction protocol used on
serum was
applied, affording an upper layer free of paraffin which was taken for further
metabolomics
analysis. Protein processing with trypsin was performed as with serum to yield
MSMS ready
peptides.
Example 11
RNA capture visualization
[0226] The ability of the SiTrap system to capture and release RNA was
visualized with
RNA. Yeast tRNA (Roche 10109495001) was labeled every tenth amine with
fluorescein
isothiocyanate (FITC, Sigma cat. no. 46951); the resulting labeled RNA was
precipitated with
ethanol and samples were washed until the supernatant was free of color,
indicating removal of
non-covalently-bound FITC. Labeled RNA was resuspended with or without the
presence of 5%
SDS in a final volume of 50 uL (see table in Fig. 25). As expected, the FITC-
labeled tRNA
glowed under UV light (Fig. 25A), allowing immediate visualization.
Importantly, the SiTrap
spin columns, here within a different vesicle to the assembly herein
presented, but identical in its
binding matrix, do not glow (Fig. 258). To each sample was added 5 uL of 10 M
ammonium
acetate and 350 uL of either 90% methanol ("M" in Fig. 25) with 100 inM TEAB
or straight
ethanol ("E" in Fig. 25). Samples were mixed and immediately passed through
SiTrap columns.
All conditions trapped RNA (Fig. 25C). Columns were washed with 350 uL of the
specified
organic. Straight ethanol was more effective at retaining RNA on the column
(Fig. 25D). RNA
was eluted with 50 mM TEAB and ethanol-bound RNA was quantitatively released
(Fig. 25E,
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conditions 3 and 4; note the lack of glow in the SiTrap binding matrix). This
experiment
demonstrates the reversible capture and release RNA for downstream processing.
Example 12
Multifraction analyses with combined matrices
[0227] The assembly was afforded the same binding matrix and beneath it a
layer of
either SCX or C18 flexible capture media (Affinisep). Samples were processed
as described for
serum. For SCX SiTraps, the sample was diluted 10X with 21 Me0H/water to
effect better
binding and the initial flow through kept. The SCX matrix was then eluted with
250 rnM
ammonium acetate in a new outer vial. Subsequently the proteins were digested
as with serum
using 1 hr 47 C digestions in new outer vials. The flow through from the 50
niM was removed
250 mlvl arrunonium acetate provided to the inner vial in the parked position.
After brief
sonication, the inner vial was placed in the upper parked position to retrieve
the bound peptides.
SCX fractions so generated were highly complementary with relatively few IDs
shared between
the flow through and eluted fractions for either metabolites or peptides. For
C18, the same
procotol was repeated with the following changes: no dilution of the sample
was initially
performed and elutions were with 75% ACN rather than ammonium acetate. Peptide
fraction
C18 flow through had very little sample, as did metabolite flow through. These
results are
anticipated however because C18 chromatography was used subsequent to SiTrap
processing.
Thus, the C18 served as a cleanup step. Alternatively, elution "cuts" of 20%
ACN were applied.
These had noticeable amounts of peptides and metabolites and lipids.
[0228] While various embodiments have been described above, it should be
understood
that such disclosures have been presented by way of example only and are not
limiting. Thus,
the breadth and scope of the subject compositions and methods should not be
limited by any of
the above-described exemplary embodiments, but should be defined only in
accordance with the
following claims and their equivalents.
102291 The above description is for the purpose of teaching the person of
ordinary skill
in the art how to practice the present invention, and it is not intended to
detail all those obvious
modifications and variations of it which will become apparent to the skilled
worker upon
reading the description. It is intended, however, that all such obvious
modifications and
variations be included within the scope of the present invention, which is
defined by the
following claims. The claims are intended to cover the components and steps in
any sequence
which is effective to meet the objectives there intended, unless the context
specifically indicates
the contrary.
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