Proteomic Sample Preparation Techniques

Chapter 3
Proteomic Sample Preparation Techniques: Toward Forensic

Proteomic Applications
Carrie Nicora,1,‡ Marina Gritsenko,1,‡ Anna Lipton,1
Karen L. Wahl,*,2 and Kristin E. Burnum-Johnson*,1
Downloaded via CHALMERS UNIV OF TECHNOLOGY on November 16, 2019 at 15:45:01 (UTC).
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
1Biological Sciences Division, Pacific Northwest National Laboratory,

Richland, Washington 99352, United States
2Chemical and Biological Signature Science Group, Pacific Northwest National Laboratory,
Richland, Washington 99352, United States
‡These authors contributed equally to this work.
*E-mail: Kristin E. Burnum-Johnson, kristin.burnum-johnson@pnnl.gov; Karen L. Wahl,

karen.wahl@pnnl.gov.
Proteomic sample preparation techniques rely on dynamic multi-step workflows

that are sample classification dependent. As forensic proteomic applications
increase in popularity, they will require numerous sample preparation workflows
to account for the diverse sample types relevant to this field. As possible examples,
a sample may need pretreatment steps to solubilize proteins from a solid surface -
clothing, a swab, or a filter. A sample may need to be disrupted or homogenized
to liberate proteins trapped in cells. Or a sample may need additional processing
steps, such as organic extraction, to isolate proteins from small molecule
contaminants which interfere with mass spectrometry measurements. Following
these pretreatment steps, proteins will be reduced, denatured, alkylated, and
enzymatically digested. The resulting peptide mixture can be desalted by solid
phase extraction or fractionated prior to liquid chromatography-mass
spectrometry-based proteomic characterization. As a reference for the forensic
research community, herein the authors present possible workflows detailing each
step of this multi-stage sample type dependent processing to aid with future
forensic proteomic applications.
Proteomics is an emerging but powerful tool for forensic investigations (1). These forensic
proteomic applications go beyond static genomic measurements and have used expressed proteins
for identification of unknown bacterial species and strains (2–7), protein toxins such as ricin (8–11)
and botulinum neurotoxins (12–14), and species level identifications of tissues (15–17), body fluids
(18) and bones (19, 20) of unknown origin. For each of these applications, the proteins of interest are
© 2019 American Chemical Society

Merkley; Applications in Forensic Proteomics: Protein Identification and Profiling
ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
processed in multi-step workflows to generate peptides for mass spectrometric (MS) identification.
Sample preparation steps are challenging and critical for successful proteomic workflows. The
authors of this chapter are well-versed in the sample handling steps needed to achieve high quality
proteomic analyses. Therefore, the goal of this chapter is to provide researchers with versatile and
detailed sample preparation steps necessary to achieve high quality proteomic analyses of diverse
sample types relevant to forensic applications.
Figure 1. Proteomic sample dependent pretreatment analysis workflow. Possible sample pretreatment steps
A-D and subsequent processing steps prior to mass spectrometry analyses. Offline or online fractionation can
also be utilized prior to LC-MS/MS analysis for in-depth proteome coverage.
30
Preparing samples for proteomic analysis is a dynamic multi-step workflow that varies
depending on the sample matrix. For example, if the proteins of interest need to be liberated from a
solid surface such as an article of clothing, a medical swab, or a filter, the sample must be pretreated to
solubilize proteins (21). In other cases, the sample must first be disrupted or homogenized (22) and
then the solubilized proteins must be extracted from possible contaminants within the solution that
could potentially interfere with MS analyses. This step typically includes an organic extraction, which
precipitates protein, thereby separating proteins from small molecule contaminants which remain
in the solution (23–28). The sample is then centrifuged to pellet the protein, and the contaminant
is removed by decanting the supernatant. Following these pretreatment steps, isolated proteins are
reduced, denatured, alkylated, and enzymatically digested into peptides which are desalted through
solid phase extraction (SPE) prior to MS analysis (29–31).
Since proteomic samples can arrive in a laboratory in several forms, it is important to use sensible
judgment on how to process these diverse samples based on their classification. Four possible
proteomic sample types and appropriate pretreatment steps are detailed below and denoted with red
text in Figure 1.
Potential Pretreatment Steps
(A) The protein sample can be bound to a matrix and must first be liberated prior to further
processing. For instance, the proteins of interest may be integrated into a piece of cloth or
cotton swab, or reside at the surface of a solid material such as a rock, piece of plastic, or a
filter. (Pretreatment steps to solubilize proteins bound to a sample matrix are detailed in
Wilkins et al. (21)).
(B) The protein sample can be a solid material that must be prepared in its entirety either wet,
frozen, or dry; these types of samples can include a piece of human tissue, a piece of bone,
or plant tissue. Proper homogenization is of the utmost importance when optimizing for
protein extraction and ensuring proper reproducibility. Therefore, it is important to
consider the density of the material when deciding on the most appropriate
homogenization technique. Sample disruption techniques include cryogenic grinding,
mortar and pestle, and bead beating (22).
(C) The protein sample can be cellular in nature and must undergo a lysis step to break apart
the cell wall or membrane. These samples include gram-positive and gram-negative
bacterial and mammalian cells. Chemical lysis pretreatment steps are typically done on
viruses, mammalian cell lines, and cell cultures. Physical lysis (bead beating or sonication)
is generally done on gram-positive bacteria, gram-negative bacteria, yeast, fungus, soil, and
diatoms (29, 31).
(D) The protein sample can be a powder or biofluid that does not require lysis but needs to be
solubilized in a buffer compatible with subsequent enzymatic digestions steps. No lysis
pretreatment is needed on bodily fluids (plasma, serum, aqueous humor, saliva, urine,
nasal secretions, and tears), culture media, or vesicles (exosomes) (32). If the sample is
already in a high volume, a volume reduction technique must be used prior to digestion
(33–36).
In each possible scenario, samples must be processed to a point where proteins have been
liberated from the sample and are ready for either protein extraction from the sample followed by
protein digestion, or direct digestion clean up (desalting) and injection on a mass spectrometer.
31
Additional details into these pretreatment and subsequent processing steps used for proteomic
analyses are detailed below.
Possible Sample Pretreatment Scenarios
Solubilization of a Sample from a Matrix (Figure 1A)

Sample residing on the surface of a solid can be washed off with a buffer solution such as 100
mM ammonium bicarbonate (ambic), 100 mM tris(hydroxymethyl)aminomethane hydrochloride
(Tris-HCL), or 50 mM triethylammonium bicarbonate (TEAB) using as little solution as possible. If
feasible, place the material inside an appropriately sized tube and use a vortexing motion to liberate
proteins from the matrix with an appropriate volume of buffer. Alternative methods can be used,
such as water-bath sonication. If these actions are not enough to liberate the sample from the matrix,
the addition of detergents can be used to improve solubilization of the sample. However, additional
clean-up steps must usually be performed downstream to remove the detergents before samples are
analyzed on the mass spectrometer. Possible insoluble particulates can be removed by appropriate
centrifugation or filtration. Once the sample is in solution, the proteins can then be extracted and
digested for proteomic analysis (methods details 2 and 3).
Homogenization (Figure 1B) & Cell Lysis (Figure 1C)

If the sample is a piece of tissue, bone, plant, or cellular material, it must first be homogenized
or cryopulverized (frozen and then ground into a powder) before addition of a solution and digestion
into peptides. The homogenization process consists of blending and mixing the sample to reduce
particle size and/or disrupting a cellular membrane to release the protein from inside a cell. This
is an important step in the processing workflow to reduce sample processing bias. The appropriate
homogenization method must be determined based on the physical properties of the sample.
For example, soft tissue such as brain, fat, and liver are tissues with little to no connective fibers,
and can be homogenized with bead homogenization (method details 1ai) (22) if preparing many
samples at once; or using a handheld pestle homogenizer (method details 1aii) if preparing one
sample at time. Tissues with tough connective material or dry, hard solids such as bone or fingernail
material first need to be cryogenically frozen and cryopulverized (method details 1bi) or ground in
a mortar and pestle (method details 1bii).
In mammalian cells, a plasma membrane separates the cell contents from the environment,
however in bacteria, fungi, and plants the plasma membrane is surrounded by a rigid cell wall.
Mammalian cells are relatively easier to lyse than most microbial cells. If the sample is a microbial
cell pellet or cell suspension, the membrane must first be sheared in order to release the proteins
from inside the cell membrane as microbes can withstand much harsher conditions than mammalian
cells. Methods include pressure cycling (method details 1ci), bead beating (method details 1cii),
or sonication (method details 1ciii).
Powder or No-Lysis Sample (Figure 1D)

The sample might come as a powder or in a form that does not require lysis; at this point the
sample can simply be solubilized in liquid chromatography (LC)-MS grade water or an appropriate
buffer solution.
32
Volume Reduction
Once the sample is solubilized, homogenized or lysed, the volume may or may not need to be
reduced. If the sample volume is more than a few milliliters, the volume can be reduced through
several methods including lyophilization or freeze-drying. This method sublimates the water content
in frozen samples by transitioning it from a solid state directly into a gaseous state without first
passing through the liquid phase. This method of removing water from the sample is ideal when
it is imperative that the sample remains frozen for stabilization purposes (method details 2a).
Centrifugal spin filter devices are another method for reducing the volume of a sample and can also
be used to exchange the sample from one buffer into another. These centrifugal spin filters retain the
protein in the upper portion of the device while the salt, detergents, and other contaminants filter
through (33, 35). The sample can then be brought up in any volume and with any buffer desired
(method details 2b).
Another method of removing contaminants and reducing volume is protein precipitation, which
is the process of separating a protein from a solution by adding a reagent to alter the protein solubility
(method details 2c-e). Protein precipitation can be enhanced using centrifugation and decantation,
followed by washing and lightly drying the pellet. The protein pellet can then be reconstituted for
digestion.
Multiple methods of protein precipitation exist based on the material being extracted, for
example phenol extractions may work best for plant tissues, which are rich in polysaccharides, lipids,
and phenolic compounds (37) (method details 2e). If the collection of metabolites and lipids is
necessary in addition to proteins, then the MPLex (metabolite, protein, lipid extraction) method (23,
38) can be used, which utilizes methanol and chloroform to induce the formation of two solvent
layers—an upper aqueous phase, containing hydrophilic metabolites, and a lower organic phase,
containing lipids and other hydrophobic metabolites. Proteins precipitate in the interphase (method
details 2d). All three classes of analytes can be collected for individual downstream analysis. Another
extraction technique is acidic precipitation, for example trichloroacetic acid/acetone extraction,
which lowers the pH of the solution and causes the proteins to achieve a net positive charge because
the amides gain an extra proton and reduce the solubility of the proteins (27, 28) (method details
2c).
In-Solution Protein Digestion

Urea and thiourea are chaotropic agents that increase the solubility of proteins by disrupting
hydrogen bonding and hydrophobic interactions both between and within proteins. These reagents
denature the three-dimensional structure of proteins, opening them up to allow more access for
tryptic digestion. Both reagents are uncharged and have no intrinsic effect on the charge of the
proteins (method details 3a). For complete protein unfolding it is necessary to reduce the intra- and
intermolecular disulfide bonds within and between protein subunits. Dithiothreitol (DTT) or tris(2-
carboxyethyl)phosphine (TCEP) are reductants that can be used for this purpose.
If denaturants such as urea are added to the digestion protocol, subsequent SPE processing
steps must be performed to “clean up” the resulting peptide solution, that is, to remove interfering
molecules prior to MS analysis (method details 4a). If the sample size is small, with limited biomass,
the urea and SPE can be avoided by utilizing 2,2,2-trifluoroethanol (TFE) digestion (method details
3b).
In order to avoid loss of protein due to aggregation and precipitation, it is important to prevent
hydrophobic interactions between the hydrophobic protein domains within and between proteins.
33
This is achieved using surfactants or surface-active agents. Surfactants have both a hydrophobic
tail and a hydrophilic head. The character of the polar head group gives rise to several surfactant
classifications including ionic (anionic or cationic), non-ionic, and zwitterionic (both positively and
negatively charged groups but with a net charge of zero). Zwitterionic surfactants can be removed
downstream using strong cation exchange (SCX) SPE (method details 4b). However, other
surfactants can be difficult to remove downstream and they can interfere with MS analysis. One
digestion method that can be used to counter this issue is Filter Aided Sample Preparation (FASP)
(39) (method details 3c).
In bottom-up proteomic approaches proteins are digested into peptides, which are typically in
the preferred mass range for MS analyses. Peptides also negate the physiochemical properties of
proteins that can be difficult to handle, such as insolubility and tendency to aggregate. Even a small set
of tryptic peptides from a specific protein can provide enough information for protein identification
and relative quantitation across multiple samples (40). The most commonly used protease for
proteomic analysis is trypsin since it cleaves peptide bonds that are C-terminal to the basic amino
acid residues arginine and lysine. This creates peptides in the preferred mass range for tandem MS
(MS/MS) sequencing and results in spectra that are information-rich and easily interpretable. Some
proteins have fewer or less widely distributed lysine and/or arginine residues and the presence of a
proline can also affect the cleavage efficiency of a lysine/arginine-proline bond. Trypsin digestion
generates peptides with an average size of 800 to 2000 Da. These tryptic peptides can be detected
with high sensitivity and are readily fragmented by collision-induced dissociation MS/MS methods
needed to obtain peptide amino acid sequence data.
High Performance Liquid Chromatography (HPLC)

Due to the massive complexity of proteomes, another important step in processing proteomics
samples is the reduction of complexity through analytical separations, such as HPLC. While
fractionation via HPLC is not always necessary, it can help improve peptide and protein
identifications. For two-dimensional proteomics analyses online fractionation can provide high
measurement sensitive for peptides from small (biomass limited) samples (41). Offline high pH
reversed-phase liquid chromatography (RPLC) followed by fraction concatenation and online low
pH RPLC can yield more peptide identifications than the offline strong cation exchange (SCX)
chromatography (first dimension) and online low pH RPLC (second dimension) (42) (method
details 5).
As detailed above, there is a multi-step framework to MS-based proteomic methods that exist
for a variety of sample classifications and proper sample preparation is crucial to attaining high quality
proteomic analyses. This chapter provides a comprehensive description of various pre-analytical
proteomic sample preparation methodologies which have the potential to be adapted to forensic
applications and provide a new tool to complement traditional forensic DNA analysis. It is
recognized that forensic proteomics is still in its early stages and research proteomic methods will
have to go through intensive revision and validation processes prior to routine use in forensic
research. However, the authors believe that the MS-based research proteomic workflows described
here have the potential to advance forensic proteomic applications.
34
Method Details – Options For Each Step
1) Homogenization (22):
a) Soft tissue homogenization
i) Homogenization system – Place the samples in pre-chilled to 4°C

centrifuge tubes. The samples can be processed dry after lyophilization
or frozen in liquid nitrogen and then ground into a powder or in a
buffer (such as 100mM ambic, pH 8) to create a slurry. The tissues are
homogenized using high speed shaking with stainless steel, tungsten
carbide, or glass beads at recommended speed and duration according
to the manufacturer with pre-chilled to -20°C adapters (43). The size
and material of the bead should be selected based on the
manufacturer’s instructions.
ii) A handheld homogenizing pestle motor can be used to complete
homogenization by placing the pestle into the tube and manually
homogenizing until no visibly large pieces remain in the tube. The
sample should remain on ice or inside an ice block (44).
b) Hard or frozen tissue cryopulverization
i) Tissue cryopulveriazation or cryofracture is a highly effective method

for sample processing in which tissue samples are shattered into tiny
pieces by controlled high-impact mechanical force. Frozen material
can be cryopulverized into a powder using a cryopulverization system
according to manufacturer’s instructions. Briefly, collected dry tissues
are flash frozen in liquid nitrogen and then disrupted by a selected
controlled force, where the impact level is carefully chosen based on
the tissue type and sample size. Next, the cryopulverized tissue sample
is transferred into an appropriate receptacle suitable for downstream
applications. Transfer of the cryopulverized material is performed on
dry ice, which keeps it frozen for the preservation of sample stability
(45–47).
ii) A mortar and pestle can also be used to grind frozen samples, such as
plant material, by pouring liquid nitrogen into the mortar and
thoroughly freezing the sample while gently grinding the sample with
the pestle. The sample can then be scooped out directly into sample
tubes for extraction or digestion. A mortar and pestle can also be used
without liquid nitrogen on tissue that has been lyophilized (48, 49).
c) Cellular homogenization
i) Pressure cycling uses an instrument for cell lysis based on alternating

levels of hydrostatic pressure to break apart cells by destabilizing
intermolecular interactions. The samples are placed in specialized
35
tubes equipped with a lysis disk and placed inside the instrument
which generates cycles of pressure from ambient up to 35kpsi with
nearly instantaneous depressurization time and dwell times of 5 to
60 seconds at any pressure in between. Pressurization and
depressurization forces the sample through the lysis disk providing
a shearing action while high hydrostatic pressure acts preferentially
on the compressible constituents of the sample. Lipids are the most
compressible sample components and dissociate upon
depressurization. Selective energy distribution results in
destabilization of molecular interactions in the lipid bilayers and other
cellular components (21, 23, 50–52).
ii) Bead beating is a form of cellular disruption that utilizes small beads of
various size and material inside a sample tube while the sample is in
solution. The sample and bead mix are subjected to high agitation
through shaking and the beads break apart the cellular membrane,
releasing the intercellular components.
The type of beads, buffer and tubes should be decided according to
sample type and volume. An example of bead beating a bacterial cell
pellet is briefly described below. Solubilize pellet with an equal volume
of 100mM ambic, pH 8.0 or desired lysis buffer or water. Transfer cell
suspensions to 1.5-mL safe-locking tube and add double the volume
with 0.1 mm zirconia/silica beads. Rinse the initial tube with 50-100
µL of lysis buffer and add to beads. Gently vortex to mix. Evenly space
tubes in a bead beater and process for an appropriate time and speed.
After lysis, place tubes on ice for a few minutes to cool. Carefully
poke a hole in the base of the tubes containing sample/beads mixture
using a 26-gauge needle and place the tubes into empty 15-mL tubes.
Centrifuge using a swing bucket rotor. Wash beads by adding 100-
200 µL lysis buffer to the beads and centrifuging once more. Transfer
the entire lysate to new microcentrifuge tubes and perform a
bicinchoninic acid (BCA) assay to get a total protein mass estimate.
If unable to continue processing the sample, flash freeze with liquid
nitrogen and store at -80°C, or proceed directly to digestion (23, 41,
53–58).
iii) Probe sonication is a common mechanical cell disruption method
based on the high shear force created by a high-frequency ultrasound.
Place an appropriately sized probe tip tightly on the sonicator.
Keeping the sample on ice, place the probe tip halfway down inside the
sample and pulse the sonicator at settings according to manufacturer’s
instruction based on sample type and volume (23, 25).
2) Volume reduction / sample concentration:
a) Lyophilization is performed on the sample by freezing it in liquid nitrogen,

placing it inside freeze-dry glassware, and sublimating it under vacuum with heat
energy applied.
36
b) Centrifugal filter concentrators - Molecular weight cut off (MWCO) centrifugal
filters can be used to reduce the volume of samples, such as biofluids or exosome
media samples. Pipette the sample directly into a 3 kDa to 10 kDa MWCO filter
(30K filters can be used for protein samples that have already been denatured)
and centrifuge the sample according to manufacturer’s instruction. Discard the
flow-through, as the contaminants, such as salts, filter through the device while
the protein remains above. This is repeated until the entire sample has been
reduced in volume. The sample can then be removed for digestion or buffer
exchanged by adding the desired buffer into the filter and centrifuging again (34,
35, 59).
c) Trichloroacetic acid / acetone extraction - Proteins can be precipitated by adding
20% trichloroacetic acid and incubating at -20 °C for 1.5 hours to overnight. The
samples are then thawed on ice and centrifuged at 5,000 x g for 20 mins at 4°C.
The supernatant is removed, and the protein pellets are washed twice with 2 mL
pre-chilled (-20 °C) acetone, vortexed, and centrifuged at 5,000 x g for 5 mins at
4 °C. The acetone is discarded and the pellet lightly dried under a nitrogen
stream, ready for digestion or storage at -80 °C.
d) Metabolite, protein, lipid extraction or MPLex – This method of protein
precipitation is used to induce a tri-phasic partitioning of the sample into
metabolite, protein, and lipid fractions all of which can be collected for
individual analyses. Polar metabolites are in the upper aqueous layer, whereas
lipids are in the lower organic layer after centrifugation, with a protein disc
situated between the two phases. The chloroform/methanol protein/
metabolite/lipid extraction is performed by adding pre-chilled (-20 °C)
chloroform:methanol mix (prepared 2:1 v/v), in a 5:1 ratio over sample volume.
Samples remain on ice for 5 mins followed by rigorous vortexing and
centrifuging at 12,000 x g for 10 mins. The upper water-soluble metabolite phase
is transferred into a glass vial and dried completely for metabolomic analysis; the
lower lipid soluble portion is added to a separate glass vial and dried completely
for lipidomics analysis and the protein layer is then washed with multiple
aliquots of pre-chilled -20°C methanol and finally allowed to dry slightly. The
protein pellet is then ready for digestion or storage at -80 °C (23, 38).
e) Phenol extraction – Plant tissues tend to contain relatively low levels of protein
and they also contain multiple compounds (such as polysaccharides and
phenolic compounds) that can complicate proteomic analysis. Briefly, ground
plant tissue is placed into an extraction buffer. An equal volume of phenol
saturated with Tris-HCL, pH 7.5 is added and allowed to shake at 4˚C for 30
mins. The sample is then centrifuged, and the upper phenolic phase is collected.
An equal volume of extraction buffer is then added again, shaken and centrifuged
as before. The upper phase is collected again, and the protein is precipitated with
5 volumes of pre-chilled -20°C 0.1 M ammonium acetate in methanol and
allowed to incubate at -20°C overnight. The precipitated protein is then pelleted
in a centrifuge and washed with pre-chilled -20°C methanol. The protein pellet
is allowed to gently dry and is then ready for digestion or storage at -80°C.
37
3) Protein digestion methods:
a) Urea digestion - A protein pellet or protein solution can be resuspended in 8 M

urea and a BCA assay is performed to determine total protein mass. Then 5-10
mM DTT is added and the sample is incubated at 37-60˚C for 30 min – 1 hour
with constant shaking on a Thermomixer. Samples are then diluted 4-10 fold
with a dilution buffer and sequencing-grade modified porcine trypsin is added
to all protein samples at a 1:50 (w/w) trypsin-to-protein ratio for 3 hours at
37˚C incubation. The samples are then desalted via SPE (C-18 resin). The
samples are concentrated in vacuo using a Speed Vac concentrator and a final
BCA assay is performed on the clean peptides to determine the final peptide
concentration. The peptide samples are then diluted to the same concentration
and characterized by MS analysis (44, 60–62).
b) Small sample, no final SPE - TFE – Very small protein pellets are reconstituted
in 100 mM ambic, pH 8.0 and assayed with BCA to determine the protein
concentration. TFE is added to the sample for a final concentration of 50% TFE.
The sample is sonicated in an ice-water bath for 1 min and incubated at 60°C for
2 hours with gentle shaking at 300 rpm on a Thermomixer. The sample is then
reduced with 2 mM DTT with incubation at 37 °C for 1 hour with gentle shaking
at 300 rpm. Next, samples are diluted 5-fold with 100 mM ambic; 1 M calcium
chloride for preparation for digestion. Sequencing-grade modified porcine
trypsin is added to the samples at a 1:50 (w/w) trypsin-to-protein ratio for 3
hours at 37 °C incubation. The sample is concentrated in vacuo to a low volume
and centrifuged at 12,000 x g to clear the peptide solution. The supernatant can
be characterized by MS analysis (61, 63).
c) Filter Aided Sample Preparation or FASP - Kits for FASP protein digestion can
be purchased through Expedeon (San Diego, CA). To each cell pellet, 100 µl of
4% sodium dodecyl sulfate (SDS), 100mM DTT in Tris-HCL buffer is added
and water-bath sonicated into solution. Each sample is incubated at 95°C for 5
mins to ensure solubilization as well as reduction of the protein. The samples are
then vortexed and sonicated for 2 mins, lightly spun to collect condensate and
allowed to cool at 4 °C for 45 mins. The samples are then centrifuged at 15,000
x g for 10 mins and prepared according to the manufacturer’s instructions.
Briefly, 400 µL of 8 M urea (all reagents included in the kit) are added to each
500 µL 30 kDa MWCO FASP spin column and up to 30 µL of the sample in SDS
buffer is added, centrifuged at 14,000 x g for 30 mins to bring the sample all the
way to the dead volume. The waste is removed from the bottom of the tube and
another 400 µL of 8 M urea is added to the column and centrifuged again at
14,000 x g for 30 mins and repeated once more. 400 µL of 50 mM ambic
(provided) is added to each column and centrifuged for 20 mins, done twice.
The column is placed into a new fresh, clean, and labeled collection tube.
Digestion solution is made by dissolving 4 μg trypsin in 75 μL 50 mM ambic
solution and added to the sample. Each sample is incubated for 3 hours at 37 °C
with 800 rpm shaking on a Thermomixer with a thermotop to reduce
condensation into the cap. The resultant peptides have 40 µL of 50 mM ambic
solution added and then centrifuged through the filter and into the collection
38
tube at 14,000 x g for 15 mins. The collected clean peptides are concentrated
to ~30 µL using a Speed Vac concentrator. Final peptide concentrations are
determined using a BCA assay. Each sample is diluted and vialed for MS analysis
(39).
4) Solid phase extraction (SPE): SPE is a commonly adapted sample preparation technique
that uses chromatographic packing material to chemically separate and purify different
compounds of the liquid sample. This technique is an effective tool for the selective
removal of sample matrix interferences, making samples suitable for MS applications.
a) C-18 SPE – C-18 SPE is used for reversed-phase extraction of peptides from
protein digested matrix. An SPE C-18 protocol consists of the series of the
sequential steps, where the first step is preparation of the SPE C-18 cartridge –
conditioning of it with the organic solvent (methanol) and proper equilibration
with acidified water (0.1% trifluoroacetic acid (TFA)). Liquid sample containing
peptides is slowly applied to the column during the loading step and unwanted
interferences are washed away during the washing step. Properly selected
amount of C-18 sorbent and sample loading speed is important for retaining of
the peptides on the SPE column. The washing step is designed to remove
compounds that will interfere with downstream analysis using an appropriate
washing solution (95:5 water:acetonitrile, 0.1% TFA). Lastly, the peptides
retained on the SPE C-18 sorbent are eluted with a stronger solvent (80:20
acetonitrile:H2O, 0.1% TFA) during the elution step. Volume of the eluted
peptide sample can then be reduced via lyophilization.
b) SCX SPE – SCX SPE is used when the digested protein sample (peptide)
solution contains detergents (such as CHAPS, NP-40, Tween-20, and Triton X-
100) that are not compatible with MS analysis and must be removed from the
sample matrix. Condition the SCX SPE column with the organic solvent
(methanol) followed by washing with acidified water (0.1% TFA) and then with
the same amount of 1% TFA solution. Appropriate amount of 100% TFA is
added to the digested protein sample solution to reach a concentration of 1%
TFA and centrifuged in order to remove any possible precipitates. The acidified
peptide sample is slowly loaded onto the SCX SPE column. Undesirable
compounds are washed away during the washing step with 70% methanol, 0.1%
TFA solution. Purified peptides are eluted off the SCX sorbent with the elution
buffer (5% sodium hydroxide in 30% methanol) and concentrated by
lyophilization to the desired volume.
5) HPLC fractionation: High pH C-18 reversed-phase - Samples are diluted with 10 mM

ammonium formate buffer (pH 10.0) and resolved on an appropriate C-18 HPLC column.
Separations are performed using an offline HPLC system with mobile phases (A) 10 mM
ammonium formate, pH 10.0 and (B) 10 mM ammonium formate, pH 10.0/acetonitrile
(10:90). The gradient is adjusted from 100% A to 95% A over the first 10 min, 95% A to
65% A over mins 10 to 70, 65% A to 30% A over mins 70 to 85, maintained at 30% A over
mins 85 to 95, re-equilibrated with 100% A over mins 95 to 105, and held at 100% A until
minute 120. Fractions are collected every 1.25 mins (96 fractions over the entire gradient)
and every 12th or 24th fraction is combined for a total of 12 or 24 samples (each with n=8
39
or n=4 fractions pooled). All fractions are dried under vacuum and 20 µl of LC-MS grade
water is added to each fraction for storage at -20°C until liquid chromatography-tandem
mass spectrometry (LC-MS/MS) analysis (44, 64).
Acknowledgments
This work was supported by the Department of Homeland Security Science and Technology
Directorate (HSHQPM-14-X-00088/P00005). A portion of this research was performed using
EMSL, a national scientific user facility sponsored by the Department of Energy’s (DOE) Office
of Biological and Environmental Research and located at Pacific Northwest National Laboratory
(PNNL). The authors would like to thank PNNL Graphic Designer Nathan Johnson for assistance in
40
preparing the figure. PNNL is a multi-program national laboratory operated by Battelle for the DOE
under Contract DE-AC05-76RLO 1830.
References
1. Merkley, E. D.; Wunschel, D. S.; Wahl, K. L.; Jarman, K. H. Applications and Challenges of
Forensic Proteomics. Forensic Sci. Int. 2019, 297, 350–363.
2. Karlsson, R.; Davidson, M.; Svensson-Stadler, L.; Karlsson, A.; Olesen, K.; Carlsohn, E.;
Moore, E. R. Strain-Level Typing and Identification of Bacteria Using Mass Spectrometry-
Based Proteomics. J. Proteome Res. 2012, 11, 2710–2720.
3. Boulund, F.; Karlsson, R.; Gonzales-Siles, L.; Johnning, A.; Karami, N.; Al-Bayati, O.; Ahren,
C.; Moore, E. R. B.; Kristiansson, E. Typing and Characterization of Bacteria Using Bottom-up
Tandem Mass Spectrometry Proteomics. Mol. Cell. Proteomics 2017, 16, 1052–1063.
4. Clowers, B. H.; Wunschel, D. S.; Kreuzer, H. W.; Engelmann, H. E.; Valentine, N.; Wahl, K.
L. Characterization of Residual Medium Peptides from Yersinia Pestis Cultures. Anal. Chem.
2013, 85, 3933–3939.
5. Jabbour, R. E.; Deshpande, S. V.; Wade, M. M.; Stanford, M. F.; Wick, C. H.; Zulich, A. W.;
Skowronski, E. W.; Snyder, A. P. Double-Blind Characterization of Non-Genome-Sequenced
Bacteria by Mass Spectrometry-Based Proteomics. Appl. Environ. Microbiol. 2010, 76,
3637–3644.
6. Pfrunder, S.; Grossmann, J.; Hunziker, P.; Brunisholz, R.; Gekenidis, M. T.; Drissner, D.
Bacillus Cereus Group-Type Strain-Specific Diagnostic Peptides. J Proteome Res 2016, 15,
3098–3107.
7. Dworzanski, J. P.; Dickinson, D. N.; Deshpande, S. V.; Snyder, A. P.; Eckenrode, B. A.
Discrimination and Phylogenomic Classification of Bacillus Anthracis-Cereus-Thuringiensis
Strains Based on Lc-Ms/Ms Analysis of Whole Cell Protein Digests. Anal Chem 2010, 82,
145–155.
8. Jarman, K. H.; Heller, N. C.; Jenson, S. C.; Hutchison, J. R.; Kaiser, B. L. D.; Payne, S.
H.; Wunschel, D. S.; Merkley, E. D. Proteomics Goes to Court: A Statistical Foundation for
Forensic Toxin/Organism Identification Using Bottom-up Proteomics. J. Proteome Res. 2018,
17, 3075–3085.
9. Fredriksson, S. A.; Hulst, A. G.; Artursson, E.; de Jong, A. L.; Nilsson, C.; van Baar, B. L.
Forensic Identification of Neat Ricin and of Ricin from Crude Castor Bean Extracts by Mass
Spectrometry. Anal. Chem. 2005, 77, 1545–1555.
10. Schieltz, D. M.; McGrath, S. C.; McWilliams, L. G.; Rees, J.; Bowen, M. D.; Kools, J. J.;
Dauphin, L. A.; Gomez-Saladin, E.; Newton, B. N.; Stang, H. L.; Vick, M. J.; Thomas, J.;
Pirkle, J. L.; Barr, J. R. Analysis of Active Ricin and Castor Bean Proteins in a Ricin Preparation,
Castor Bean Extract, and Surface Swabs from a Public Health Investigation. Forensic Sci. Int.
2011, 209, 70–79.
11. Merkley, E. D.; Jenson, S. C.; Arce, J. S.; Melville, A. M.; Leiser, O. P.; Wunschel, D. S.; Wahl,
K. L. Ricin-Like Proteins from the Castor Plant Do Not Influence Liquid Chromatography-
Mass Spectrometry Detection of Ricin in Forensically Relevant Samples. Toxicon 2017, 140,
18–31.
41
12. Boyer, A. E.; Moura, H.; Woolfitt, A. R.; Kalb, S. R.; McWilliams, L. G.; Pavlopoulos, A.;
Schmidt, J. G.; Ashley, D. L.; Barr, J. R. From the Mouse to the Mass Spectrometer: Detection
and Differentiation of the Endoproteinase Activities of Botulinum Neurotoxins a-G by Mass
Spectrometry. Anal. Chem. 2005, 77, 3916–3924.
13. Kalb, S. R.; Goodnough, M. C.; Malizio, C. J.; Pirkle, J. L.; Barr, J. R. Detection of Botulinum
Neurotoxin a in a Spiked Milk Sample with Subtype Identification through Toxin Proteomics.
Anal. Chem. 2005, 77, 6140–6146.
14. Kalb, S. R.; Barr, J. R. Mass Spectrometric Identification and Differentiation of Botulinum
Neurotoxins through Toxin Proteomics. Rev. Anal. Chem. 2013, 32, 189–196.
15. Parker, G. J.; Leppert, T.; Anex, D. S.; Hilmer, J. K.; Matsunami, N.; Baird, L.; Stevens, J.;
Parsawar, K.; Durbin-Johnson, B. P.; Rocke, D. M.; Nelson, C.; Fairbanks, D. J.; Wilson,
A. S.; Rice, R. H.; Woodward, S. R.; Bothner, B.; Hart, B. R.; Leppert, M. Demonstration
of Protein-Based Human Identification Using the Hair Shaft Proteome. PLoS One 2016, 11,
e0160653.
16. Buckley, M. Species Identification of Bovine, Ovine and Porcine Type 1 Collagen; Comparing
Peptide Mass Fingerprinting and Lc-Based Proteomics Methods. Int. J. Mol. Sci. 2016, 17,
445.
17. Laatsch, C. N.; Durbin-Johnson, B. P.; Rocke, D. M.; Mukwana, S.; Newland, A. B.; Flagler,
M. J.; Davis, M. G.; Eigenheer, R. A.; Phinney, B. S.; Rice, R. H. Human Hair Shaft Proteomic
Profiling: Individual Differences, Site Specificity and Cuticle Analysis. PeerJ 2014, 2, e506.
18. Van Steendam, K.; De Ceuleneer, M.; Dhaenens, M.; Van Hoofstat, D.; Deforce, D. Mass
Spectrometry-Based Proteomics as a Tool to Identify Biological Matrices in Forensic Science.
Int. J. Legal Med. 2013, 127, 287–298.
19. Buckley, M.; Collins, M.; Thomas-Oates, J.; Wilson, J. C. Species Identification by Analysis
of Bone Collagen Using Matrix-Assisted Laser Desorption/Ionisation Time-of-Flight Mass
Spectrometry. Rapid Commun. Mass Spectrom. 2009, 23, 3843–3854.
20. Mason, K. E.; Anex, D.; Grey, T.; Hart, B.; Parker, G. Protein-Based Forensic Identification
Using Genetically Variant Peptides in Human Bone. Forensic Sci. Int. 2018, 288, 89–96.
21. Wilkins, M. J.; Wrighton, K. C.; Nicora, C. D.; Williams, K. H.; McCue, L. A.; Handley, K.
M.; Miller, C. S.; Giloteaux, L.; Montgomery, A. P.; Lovley, D. R.; Banfield, J. F.; Long, P.
E.; Lipton, M. S. Fluctuations in Species-Level Protein Expression Occur During Element and
Nutrient Cycling in the Subsurface. PLoS One 2013, 8, e57819.
22. Bryant, A. E.; Aldape, M. J.; Bayer, C. R.; Katahira, E. J.; Bond, L.; Nicora, C. D.; Fillmore, T.
L.; Clauss, T. R.; Metz, T. O.; Webb-Robertson, B. J.; Stevens, D. L. Effects of Delayed Nsaid
Administration after Experimental Eccentric Contraction Injury - a Cellular and Proteomics
Study. PLoS One 2017, 12, e0172486.
23. Nakayasu, E. S.; Nicora, C. D.; Sims, A. C.; Burnum-Johnson, K. E.; Kim, Y. M.; Kyle, J.
E.; Matzke, M. M.; Shukla, A. K.; Chu, R. K.; Schepmoes, A. A.; Jacobs, J. M.; Baric, R. S.;
Webb-Robertson, B. J.; Smith, R. D.; Metz, T. O. Mplex: A Robust and Universal Protocol for
Single-Sample Integrative Proteomic, Metabolomic, and Lipidomic Analyses. mSystems 2016,
1, e00043–00016.
24. Burnum-Johnson, K. E.; Baker, E. S.; Metz, T. O. Characterizing the Lipid and Metabolite
Changes Associated with Placental Function and Pregnancy Complications Using Ion Mobility
42
Spectrometry-Mass Spectrometry and Mass Spectrometry Imaging. Placenta 2017, 60 (Suppl
1), S67–S72.
25. Nicora, C. D.; Burnum-Johnson, K. E.; Nakayasu, E. S.; Casey, C. P.; White, R. A., III; Roy
Chowdhury, T.; Kyle, J. E.; Kim, Y. M.; Smith, R. D.; Metz, T. O.; Jansson, J. K.; Baker, E. S.
The Mplex Protocol for Multi-Omic Analyses of Soil Samples. J. Vis. Exp. 2018, e57343.
26. Khadempour, L.; Burnum-Johnson, K. E.; Baker, E. S.; Nicora, C. D.; Webb-Robertson,
B. M.; White, R. A., III; Monroe, M. E.; Huang, E. L.; Smith, R. D.; Currie, C. R. The
Fungal Cultivar of Leaf-Cutter Ants Produces Specific Enzymes in Response to Different Plant
Substrates. Mol. Ecol. 2016, 25, 5795–5805.
27. Santa, C.; Anjo, S. I.; Manadas, B. Protein Precipitation of Diluted Samples in Sds-Containing
Buffer with Acetone Leads to Higher Protein Recovery and Reproducibility in Comparison
with Tca/Acetone Approach. Proteomics 2016, 16, 1847–1851.
28. Crowell, A. M.; Wall, M. J.; Doucette, A. A. Maximizing Recovery of Water-Soluble Proteins
through Acetone Precipitation. Anal. Chim. Acta 2013, 796, 48–54.
29. Sowell, S. M.; Wilhelm, L. J.; Norbeck, A. D.; Lipton, M. S.; Nicora, C. D.; Barofsky, D.
F.; Carlson, C. A.; Smith, R. D.; Giovanonni, S. J. Transport Functions Dominate the Sar11
Metaproteome at Low-Nutrient Extremes in the Sargasso Sea. ISME J. 2009, 3, 93–105.
30. Burnum, K. E.; Callister, S. J.; Nicora, C. D.; Purvine, S. O.; Hugenholtz, P.; Warnecke,
F.; Scheffrahn, R. H.; Smith, R. D.; Lipton, M. S. Proteome Insights into the Symbiotic
Relationship between a Captive Colony of Nasutitermes Corniger and Its Hindgut
Microbiome. ISME J. 2011, 5, 161–164.
31. Callister, S. J.; Nicora, C. D.; Zeng, X.; Roh, J. H.; Dominguez, M. A.; Tavano, C. L.; Monroe,
M. E.; Kaplan, S.; Donohue, T. J.; Smith, R. D.; Lipton, M. S. Comparison of Aerobic and
Photosynthetic Rhodobacter Sphaeroides 2.4.1 Proteomes. J. Microbiol. Methods 2006, 67,
424–436.
32. Duijvesz, D.; Burnum-Johnson, K. E.; Gritsenko, M. A.; Hoogland, A. M.; Vredenbregt-van
den Berg, M. S.; Willemsen, R.; Luider, T.; Pasa-Tolic, L.; Jenster, G. Proteomic Profiling of
Exosomes Leads to the Identification of Novel Biomarkers for Prostate Cancer. PLoS One 2013,
8, e82589.
33. Shi, T.; Gao, Y.; Quek, S. I.; Fillmore, T. L.; Nicora, C. D.; Su, D.; Zhao, R.; Kagan, J.;
Srivastava, S.; Rodland, K. D.; Liu, T.; Smith, R. D.; Chan, D. W.; Camp, D. G., 2nd; Liu,
A. Y.; Qian, W. J. A Highly Sensitive Targeted Mass Spectrometric Assay for Quantification of
Agr2 Protein in Human Urine and Serum. J. Proteome Res. 2014, 13, 875–882.
34. Brown, J. N.; Brewer, H. M.; Nicora, C. D.; Weitz, K. K.; Morris, M. J.; Skabelund, A. J.;
Adkins, J. N.; Smith, R. D.; Cho, J. H.; Gelinas, R. Protein and Microrna Biomarkers from
Lavage, Urine, and Serum in Military Personnel Evaluated for Dyspnea. BMC Med Genomics
2014, 7, 58.
35. Sigdel, T. K.; Mercer, N.; Nandoe, S.; Nicora, C. D.; Burnum-Johnson, K.; Qian, W. J.;
Sarwal, M. M. Urinary Virome Perturbations in Kidney Transplantation. Front Med (Lausanne)
2018, 5, 72.
36. Sigdel, T. K.; Salomonis, N.; Nicora, C. D.; Ryu, S.; He, J.; Dinh, V.; Orton, D. J.; Moore,
R. J.; Hsieh, S. C.; Dai, H.; Thien-Vu, M.; Xiao, W.; Smith, R. D.; Qian, W. J.; Camp, D. G.,
2nd; Sarwal, M. M. The Identification of Novel Potential Injury Mechanisms and Candidate
43
Biomarkers in Renal Allograft Rejection by Quantitative Proteomics. Mol. Cell. Proteomics
2014, 13, 621–631.
37. Faurobert, M.; Pelpoir, E.; Chaib, J. Phenol Extraction of Proteins for Proteomic Studies of
Recalcitrant Plant Tissues. Methods Mol. Biol. 2007, 355, 9–14.
38. Burnum-Johnson, K. E.; Kyle, J. E.; Eisfeld, A. J.; Casey, C. P.; Stratton, K. G.; Gonzalez, J.
F.; Habyarimana, F.; Negretti, N. M.; Sims, A. C.; Chauhan, S.; Thackray, L. B.; Halfmann,
P. J.; Walters, K. B.; Kim, Y. M.; Zink, E. M.; Nicora, C. D.; Weitz, K. K.; Webb-Robertson,
B. M.; Nakayasu, E. S.; Ahmer, B.; Konkel, M. E.; Motin, V.; Baric, R. S.; Diamond, M. S.;
Kawaoka, Y.; Waters, K. M.; Smith, R. D.; Metz, T. O. Mplex: A Method for Simultaneous
Pathogen Inactivation and Extraction of Samples for Multi-Omics Profiling. Analyst 2017, 142,
442–448.
39. Wisniewski, J. R.; Zougman, A.; Nagaraj, N.; Mann, M. Universal Sample Preparation Method
for Proteome Analysis. Nat. Methods 2009, 6, 359–362.
40. Webb-Robertson, B. J.; Matzke, M. M.; Jacobs, J. M.; Pounds, J. G.; Waters, K. M. A Statistical
Selection Strategy for Normalization Procedures in Lc-Ms Proteomics Experiments through
Dataset-Dependent Ranking of Normalization Scaling Factors. Proteomics 2011, 11,
4736–4741.
41. Aylward, F. O.; Khadempour, L.; Tremmel, D. M.; McDonald, B. R.; Nicora, C. D.; Wu, S.;
Moore, R. J.; Orton, D. J.; Monroe, M. E.; Piehowski, P. D.; Purvine, S. O.; Smith, R. D.;
Lipton, M. S.; Burnum-Johnson, K. E.; Currie, C. R. Enrichment and Broad Representation
of Plant Biomass-Degrading Enzymes in the Specialized Hyphal Swellings of Leucoagaricus
Gongylophorus, the Fungal Symbiont of Leaf-Cutter Ants. PLoS One 2015, 10, e0134752.
42. Yang, F.; Shen, Y.; Camp, D. G., 2nd; Smith, R. D. High-Ph Reversed-Phase Chromatography
with Fraction Concatenation for 2d Proteomic Analysis. Expert Rev. Proteomics 2012, 9,
129–134.
43. Hasin-Brumshtein, Y.; Khan, A. H.; Hormozdiari, F.; Pan, C.; Parks, B. W.; Petyuk, V. A.;
Piehowski, P. D.; Brummer, A.; Pellegrini, M.; Xiao, X.; Eskin, E.; Smith, R. D.; Lusis, A. J.;
Smith, D. J. Hypothalamic Transcriptomes of 99 Mouse Strains Reveal Trans Eqtl Hotspots,
Splicing Qtls and Novel Non-Coding Genes. Elife 2016, 5.
44. Gritsenko, M. A.; Xu, Z.; Liu, T.; Smith, R. D. Large-Scale and Deep Quantitative Proteome
Profiling Using Isobaric Labeling Coupled with Two-Dimensional Lc-Ms/Ms. Methods Mol.
Biol. 2016, 1410, 237–247.
45. Mertins, P.; Yang, F.; Liu, T.; Mani, D. R.; Petyuk, V. A.; Gillette, M. A.; Clauser, K. R.; Qiao,
J. W.; Gritsenko, M. A.; Moore, R. J.; Levine, D. A.; Townsend, R.; Erdmann-Gilmore, P.;
Snider, J. E.; Davies, S. R.; Ruggles, K. V.; Fenyo, D.; Kitchens, R. T.; Li, S.; Olvera, N.; Dao,
F.; Rodriguez, H.; Chan, D. W.; Liebler, D.; White, F.; Rodland, K. D.; Mills, G. B.; Smith,
R. D.; Paulovich, A. G.; Ellis, M.; Carr, S. A. Ischemia in Tumors Induces Early and Sustained
Phosphorylation Changes in Stress Kinase Pathways but Does Not Affect Global Protein Levels.
Mol. Cell. Proteomics 2014, 13, 1690–1704.
46. Mertins, P.; Tang, L. C.; Krug, K.; Clark, D. J.; Gritsenko, M. A.; Chen, L.; Clauser, K. R.;
Clauss, T. R.; Shah, P.; Gillette, M. A.; Petyuk, V. A.; Thomas, S. N.; Mani, D. R.; Mundt, F.;
Moore, R. J.; Hu, Y.; Zhao, R.; Schnaubelt, M.; Keshishian, H.; Monroe, M. E.; Zhang, Z.;
Udeshi, N. D.; Mani, D.; Davies, S. R.; Townsend, R. R.; Chan, D. W.; Smith, R. D.; Zhang,
44
H.; Liu, T.; Carr, S. A. Reproducible Workflow for Multiplexed Deep-Scale Proteome and
Phosphoproteome Analysis of Tumor Tissues by Liquid Chromatography-Mass Spectrometry.
Nat. Protoc. 2018, 13, 1632–1661.
47. Cha, J.; Burnum-Johnson, K. E.; Bartos, A.; Li, Y.; Baker, E. S.; Tilton, S. C.; Webb-
Robertson, B. J.; Piehowski, P. D.; Monroe, M. E.; Jegga, A. G.; Murata, S.; Hirota, Y.; Dey, S.
K. Muscle Segment Homeobox Genes Direct Embryonic Diapause by Limiting Inflammation
in the Uterus. J. Biol. Chem. 2015, 290, 15337–15349.
48. Valdes-Lopez, O.; Batek, J.; Gomez-Hernandez, N.; Nguyen, C. T.; Isidra-Arellano, M. C.;
Zhang, N.; Joshi, T.; Xu, D.; Hixson, K. K.; Weitz, K. K.; Aldrich, J. T.; Pasa-Tolic, L.; Stacey,
G. Soybean Roots Grown under Heat Stress Show Global Changes in Their Transcriptional and
Proteomic Profiles. Front. Plant Sci. 2016, 7, 517.
49. Brechenmacher, L.; Nguyen, T. H.; Hixson, K.; Libault, M.; Aldrich, J.; Pasa-Tolic, L.; Stacey,
G. Identification of Soybean Proteins from a Single Cell Type: The Root Hair. Proteomics 2012,
12, 3365–3373.
50. Zhou, J. Y.; Dann, G. P.; Shi, T.; Wang, L.; Gao, X.; Su, D.; Nicora, C. D.; Shukla, A. K.;
Moore, R. J.; Liu, T.; Camp, D. G., 2nd; Smith, R. D.; Qian, W. J. Simple Sodium Dodecyl
Sulfate-Assisted Sample Preparation Method for Lc-Ms-Based Proteomics Applications. Anal.
Chem. 2012, 84, 2862–2867.
51. Wilkins, M. J.; Callister, S. J.; Miletto, M.; Williams, K. H.; Nicora, C. D.; Lovley, D. R.;
Long, P. E.; Lipton, M. S. Development of a Biomarker for Geobacter Activity and Strain
Composition; Proteogenomic Analysis of the Citrate Synthase Protein During Bioremediation
of U(Vi). Microb. Biotechnol. 2011, 4, 55–63.
52. Smith, D. P.; Thrash, J. C.; Nicora, C. D.; Lipton, M. S.; Burnum-Johnson, K. E.; Carini,
P.; Smith, R. D.; Giovannoni, S. J. Proteomic and Transcriptomic Analyses of “Candidatus
Pelagibacter Ubique” Describe the First Pii-Independent Response to Nitrogen Limitation in a
Free-Living Alphaproteobacterium. MBio 2013, 4, e00133–00112.
53. Liberton, M.; Chrisler, W. B.; Nicora, C. D.; Moore, R. J.; Smith, R. D.; Koppenaal, D. W.;
Pakrasi, H. B.; Jacobs, J. M. Phycobilisome Truncation Causes Widespread Proteome Changes
in Synechocystis Sp. Pcc 6803. PLoS One 2017, 12, e0173251.
54. Pomraning, K. R.; Kim, Y. M.; Nicora, C. D.; Chu, R. K.; Bredeweg, E. L.; Purvine, S. O.;
Hu, D.; Metz, T. O.; Baker, S. E. Multi-Omics Analysis Reveals Regulators of the Response to
Nitrogen Limitation in Yarrowia Lipolytica. BMC Genomics 2016, 17, 138.
55. Orellana, R.; Hixson, K. K.; Murphy, S.; Mester, T.; Sharma, M. L.; Lipton, M. S.; Lovley, D.
R. Proteome of Geobacter Sulfurreducens in the Presence of U(Vi). Microbiology 2014, 160,
2607–2617.
56. Bagwell, C. E.; Hixson, K. K.; Milliken, C. E.; Lopez-Ferrer, D.; Weitz, K. K. Proteomic and
Physiological Responses of Kineococcus Radiotolerans to Copper. PLoS One 2010, 5, e12427.
57. Lewis, N. E.; Hixson, K. K.; Conrad, T. M.; Lerman, J. A.; Charusanti, P.; Polpitiya, A. D.;
Adkins, J. N.; Schramm, G.; Purvine, S. O.; Lopez-Ferrer, D.; Weitz, K. K.; Eils, R.; Konig, R.;
Smith, R. D.; Palsson, B. O. Omic Data from Evolved E. Coli Are Consistent with Computed
Optimal Growth from Genome-Scale Models. Mol. Syst. Biol. 2010, 6, 390.
45
58. Ding, Y. H.; Hixson, K. K.; Aklujkar, M. A.; Lipton, M. S.; Smith, R. D.; Lovley, D. R.;
Mester, T. Proteome of Geobacter Sulfurreducens Grown with Fe(III) Oxide or Fe(III) Citrate
as the Electron Acceptor. Biochim Biophys Acta 2008, 1784, 1935–1941.
59. Park, J.; Piehowski, P. D.; Wilkins, C.; Zhou, M.; Mendoza, J.; Fujimoto, G. M.; Gibbons, B.
C.; Shaw, J. B.; Shen, Y.; Shukla, A. K.; Moore, R. J.; Liu, T.; Petyuk, V. A.; Tolic, N.; Pasa-
Tolic, L.; Smith, R. D.; Payne, S. H.; Kim, S. Informed-Proteomics: Open-Source Software
Package for Top-Down Proteomics. Nat. Methods 2017, 14, 909–914.
60. Piehowski, P. D.; Petyuk, V. A.; Orton, D. J.; Xie, F.; Moore, R. J.; Ramirez-Restrepo, M.;
Engel, A.; Lieberman, A. P.; Albin, R. L.; Camp, D. G.; Smith, R. D.; Myers, A. J. Sources of
Technical Variability in Quantitative Lc-Ms Proteomics: Human Brain Tissue Sample Analysis.
J. Proteome Res. 2013, 12, 2128–2137.
61. Matzke, M. M.; Brown, J. N.; Gritsenko, M. A.; Metz, T. O.; Pounds, J. G.; Rodland, K.
D.; Shukla, A. K.; Smith, R. D.; Waters, K. M.; McDermott, J. E.; Webb-Robertson, B. J. A
Comparative Analysis of Computational Approaches to Relative Protein Quantification Using
Peptide Peak Intensities in Label-Free Lc-Ms Proteomics Experiments. Proteomics 2013, 13,
493–503.
62. Dominy, S. S.; Brown, J. N.; Ryder, M. I.; Gritsenko, M.; Jacobs, J. M.; Smith, R. D. Proteomic
Analysis of Saliva in Hiv-Positive Heroin Addicts Reveals Proteins Correlated with Cognition.
PLoS One 2014, 9, e89366.
63. Piehowski, P. D.; Petyuk, V. A.; Sontag, R. L.; Gritsenko, M. A.; Weitz, K. K.; Fillmore, T.
L.; Moon, J.; Makhlouf, H.; Chuaqui, R. F.; Boja, E. S.; Rodriguez, H.; Lee, J. S. H.; Smith,
R. D.; Carrick, D. M.; Liu, T.; Rodland, K. D. Residual Tissue Repositories as a Resource for
Population-Based Cancer Proteomic Studies. Clin. Proteomics 2018, 15, 26.
64. Wang, Y.; Yang, F.; Gritsenko, M. A.; Wang, Y.; Clauss, T.; Liu, T.; Shen, Y.; Monroe, M.
E.; Lopez-Ferrer, D.; Reno, T.; Moore, R. J.; Klemke, R. L.; Camp, D. G., 2nd; Smith, R. D.
Reversed-Phase Chromatography with Multiple Fraction Concatenation Strategy for Proteome
Profiling of Human Mcf10a Cells. Proteomics 2011, 11, 2019–2026.
46

Proteomic Sample Preparation Techniques

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Proteomic Sample Preparation Techniques

Uploaded by

Copyright:

Available Formats

Chapter 3

Proteomic Sample Preparation Techniques: Toward Forensic

1Biological Sciences Division, Pacific Northwest National Laboratory,

*E-mail: Kristin E. Burnum-Johnson, kristin.burnum-johnson@pnnl.gov; Karen L. Wahl,

Proteomic sample preparation techniques rely on dynamic multi-step workflows

© 2019 American Chemical Society

Potential Pretreatment Steps

Possible Sample Pretreatment Scenarios

Solubilization of a Sample from a Matrix (Figure 1A)

Homogenization (Figure 1B) & Cell Lysis (Figure 1C)

Powder or No-Lysis Sample (Figure 1D)

In-Solution Protein Digestion

High Performance Liquid Chromatography (HPLC)

a) Soft tissue homogenization

i) Homogenization system – Place the samples in pre-chilled to 4°C

b) Hard or frozen tissue cryopulverization

i) Tissue cryopulveriazation or cryofracture is a highly effective method

i) Pressure cycling uses an instrument for cell lysis based on alternating

2) Volume reduction / sample concentration:

a) Lyophilization is performed on the sample by freezing it in liquid nitrogen,

a) Urea digestion - A protein pellet or protein solution can be resuspended in 8 M

5) HPLC fractionation: High pH C-18 reversed-phase - Samples are diluted with 10 mM

You might also like