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Research products for the study of the
important signal transduction pathways…
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VVHLTPEEH T ELIFE SCIENCELIFE SCIENCELIFE SCIENCE TECHNOLOGIESTECHNOLOGIESTECHNOLOGIES
HLTPEEKKPREPARING THE PROTEOME
FOR MASS SPECTROMETRY
Proteomics
Preparing samples for mass spectrometry analysis is not as simple as merelyisolating total protein from its biological source. They also need to be in a chemicalenvironment that is MS friendly and allows their interrogation. Often enzymaticdigestion and/or depletion/partitioning/enrichment are required to obtain ausable sample; but what are the best sample preparationmethods, and howmightthey impact experiments? By Gautam Thor
Until the early 1990s, mass spectrometry (MS) researchers were generallyrestricted to the study of small, thermostable molecules that could beeasily ionized. Two breakthroughs occurred in quick succession in thelate 1980s when John Fenn (who received part of the 2002 Nobel Prize in
Chemistry) developed electrospray ionization (ESI) and Koichi Tanaka introducedmatrix associated laser desorption ionization (MALDI) for large molecules, basedon the work of Franz Hillenkamp andMichael Karas. Thesemethodologies enabledMS analysis of a much broader range of biological molecules, without any need forchemical derivatization.
Finding the Needle in a HaystackNow, the way in which samples are prepared for MS experiments is frequentlydetermined by, and can have significant impact on, the subsequent steps in theprocedure.Sample preparation procedures prior to MS need to serve dual functions in
proteomic studies. First, they must provide adequate isolation of the protein(s)of interest, and second, the buffers used should provide a suitable environmentfor subsequent MS analysis. One of the biggest challenges, particularly in clinical,diagnostic, and prognostic applications such as those associated with biomarkerdiscovery, is dealing with both the sheer complexity and the large dynamic rangeof the samples used. Jerald Feitelson, business development, intellectual property,and alliance manager at Beckman Coulter, points out that “although any singleprotein preparation technology is unlikely to be ideal for all samples, the moreone can simplify the sample, reduce the dynamic range of protein concentrations,enrich for medium and low abundance proteins, and remove interfering peptidemass fingerprints the better the mass spectrometry results will be and the deeperone can dig into the proteome.”Fluids like blood, saliva, and urine can contain many thousands of proteins and
protein fragments that cover a dynamic range of 10 or more orders of magnitude,whereas most MS workflows are restricted to three or four logs of concentration,at best. Roughly 85 percent by mass of the human serum proteome is comprisedof only six highly abundant proteins, including albumin, immunoglobulins ofthe G and A subtypes, transferrin, haptoglobin, and α1-antitrypsin. But thishandful of components can mask the mass spectra of the low abundance (andoften more interesting) proteins. In fact, early research in biomarker discoveryusing low resolution, but rapid, MS technologies such as surface enhancedlaser desorption ionization (SELDI) was unable to establish any reproducibleor clinically valid biomarkers (most of the signature differences turned out tobe nonspecific components of the proteome). In order to overcome this hurdle,several approaches to sample preparation were developed that rely on depletingsamples of highly abundant proteins.Depletion procedures often involve immunoaffinity-based methods using
polyclonal or monoclonal antibodies—immobilized on a solid phase—to captureand retain themost abundant proteins. “TheMultiple Affinity Removal Systemwasdesigned to be highly selective for the removal of 14 highly abundant
“The more one can simplify
the sample, reduce the dynamic
range of protein conentrations,
enrich for medium and low
abundance proteins, and
remove interfering peptide
mass fingerprints the better the
results will be.”
©ISTOCKPHOTO.COM\TARAMOL
LIFE SCIENCE TECHNOLOGIESAAAS/Science Business Office Feature
www.sciencemag.org/products 1081
Proteomics
proteins fromhumanserumorplasma,” statesScottO’Brien, productlinemanager at Stratagene, an Agilent Technologies division. The kitmakes use of a specifically formulated buffer “to minimize protein-protein interactions and ensure efficient capture and release oftargeted proteins,” says O’Brien.Immunoaffinity capture using avian antibodies may provide
higher selectivity and lower cross-reactivity than other comparableproducts, since chicken antibodies display less cross-reactivity withmammalian proteins. GenWay Biotech (recently acquired by Sigma-Aldrich) provides Seppro affinity depletion technology built from alibrary of 700 chicken-derived IgY antibodies, while Beckman Coulterhas been offering, for research use only, ProteomeLab IgY-12 spinand liquid chromatography column kits that are able to selectivelypartition 12 highly abundant proteins found in plasma or serum,enriching the sample up to 25-fold.Calbiochem (part of EMD Biosciences) makes use of a different
methodology: the company’s ProteoExtract kit includes an albumin/IgG removal component which uses a combination of the albumin-specific resin and an immobilized protein A polymeric resin. Afterremoval of the high abundance proteins, the kit allows for theselection of partial or full proteome extraction, a precipitation stepto concentrate the sample, and an enzymatic digestion if required.Novagen’s (also a part of EMD Biosciences) ProteoEnrich CAT-Xkit separates proteins under nondenaturing conditions based ontheir binding to the strong cation exchange resin. Almost the entireproteome should bind to the matrix under slightly acidic conditions,and partial proteomes can be eluted using a salt gradient.One profound drawback of depletion strategies results from the
tendency for abundant proteins, particularly albumin, to bind otherserum proteins and protein fragments. Thus it is possible thatdepletion of highly abundant proteins might also remove desirableones. Bio-Rad addresses this problem with its ProteoMinertechnology, which provides a large and highly diverse library ofhexapeptides bound to a chromatographic support, allowing eachunique hexapeptide to bind to a unique protein sequence. When thesample is added, the limiting binding capacity of the beads enableshigh abundance proteins to rapidly saturate their ligands, and excessprotein is washed out. In contrast, the low abundance proteins areconcentrated on their specific ligands. Following elution from thebeads, thedynamic rangeofproteins in thesample isgreatly reduced.Kate Smith, Bio-Rad’s product manager for expression proteomics,explains that “since our depletion strategy does not entirely discardall the high abundant proteins, we get a quantitative analysis ofthe low abundance proteins and limit the potential codepletion ofproteins associated with the highly abundant proteins.”The procedure described above not only depletes abundant
pro-teins but also provides a means to segment the sample,demonstrating an alternative strategy for protein sample prep-aration: enrichment.
Separating the Wheat from the ChaffIn contrast to depletion strategies for sample preparations, enrich-ment processes allow for the preparation of specific subsets of pro-teins. These can be achieved either through affinity purification usingposttranslational chemical modifications (for example, glycosylationor phosphorylation) or by the isolation of cellular subpopulations(such as organelles or plasma membrane fractions).“Most biofluid samples for clinical discovery experiments need to
be reduced, alkylated and trypsin digested, and then subjected toa sample cleanup by reverse phase HPLC [high performance liquidchromatography],” says Mary F. Lopez, director, biomarker researchinitiatives in mass spectrometry at Thermo Fisher Scientific. In her
opinion, the most successful approaches seem to be enrichmentstrategies for target pathways rather than depletion of interferingproteins. However, she also says that “the choice of targeted-enrichment strategy depends on how much is known about theprotein or family of proteins of interest.” Lopez indicates thatproteomics-based identification of relevant biomarkers is a veryactive area of research. “The objective is biomarker candidatesor signatures that can be used in a targeted, clinically relevantquantitative assay. However, the necessary sensitivity level in blood,urine, or even tissue extracts is often achievable only through anenrichment strategy.”Enrichment strategies frequently use solid-phase extraction
(SPE), a standard biochemical technique that uses the affinityof solutes within a liquid (mobile) phase for the solid (stationary)phase over which that sample is passed. Either the desiredanalytes of interest are collected in the flow through or, if they areretained on the stationary phase, they can then be removed with anappropriate eluent.Jeff Zonderman, director of clinical and toxicology LC/MC from
Thermo Fisher Scientific, points out that “the most widely usedpre-MS clinical sample preparation method is probably SPE due toits large selectivity.” However, he acknowledges that researchersfrequently use more than one selective technology to adequatelyreduce the complexity of a sample. One example is Thermo’sTranscend system with TurboFlow technology, which puts a uniquespinonpre-MSchromatographic separation. As Zondermanexplains,introducing turbulence into the system—by running cartridges atpredetermined high speeds—creates a more uniform velocity profileacross the column, and “provides a means to obtain high volume,high demand separation.”Varian’s OMIX product line uses SPE in a pipette tip to provide
a high throughput application for microextraction of proteins andpeptides. The company claims that the small sorbent bed makesfor a cleaner elution into a smaller volume. A similar product fromMillipore, the ZipTip, is made to be compatible with a number ofautomated liquid handling systems and, according to the companyliterature, “provides a reproducible, high recovery method forconcentrating and purifying femtomoles to picomoles of peptides,proteins, and oligonucleotides.”“Selective enrichment is an area we are pursuing vigorously,”
states Andrew Emili, professor at the Donnelly Centre for Cellularand Biomolecular Research, at the University of Toronto, Canada.“For example, we are using both HPLC and aptamer phage displayto physically isolate targets of interest from complex mixtures priorto MS detection.” Emili claims that their approach, which is still inthe research phase, is faster and cheaper for isolating proteins orpeptides than raising antibodies for immunoenrichment.
Teasing Apart the CellIf depletion strategies are too blunt an instrument and enrichmentprocedures too specific, the limitations introduced by the complexityof the MS samples could be addressed, at least in part, continued >
“The necessary
sensitivity level in
blood, urine, or even
tissue extracts is often
achievable only through
an enrichment strategy.”
©ISTOCKPHOTO.COM\ELKOR
LIFE SCIENCE TECHNOLOGIES AAAS/Science Business Office Feature
1082 www.sciencemag.org/products
Proteomics
by the use of strategies targeting information-rich subsets of theproteome. This includes using standard subcellular fractionationtechnologies such as Poppers kits from Pierce (now a part of ThermoFisher Scientific) and EMD Bioscience’s ProteoExtract SubcellularProteome Extraction kit, both of which are capable of enriching fornuclear, cytoplasmic, and mitochondrial proteins.The Qproteome kits from Qiagen, in contrast to straightforward
differential centrifugationmethods, promise to provide high purity ofisolated subcellular fractions and soluble proteins, with concomitantdepletion of high-abundant proteins such as IgG and albumin. “Atthe same time,” says Ute Boronowsky, global product manager, “theprocedure is very gentle, using only a benchtop centrifuge, leavingsubcellular organelles such asmitochondria and plasmamembranesintact, and retaining proteins in a native conformation with fullbiological activity.”Although it is predicted that nearly 30 percent of all proteins are
phosphorylated, phosphoproteins tend to be of low abundance andoften with multiple phosphorylation sites of varying stoichiometries,which presents several challenges for MS analysis. Enrichmentof phosphoproteins from complex mixtures can be performed byaffinity chromatography using immobilized antibodies specific forphosphoserine, phosphothreonine, and phosphotyrosine residues,obtainable as commercial kits. Another technique more amenable toMS analysis is the use of immobilized metal affinity chromatography(IMAC) as it relies on the affinity of phosphate groups for certainmetal ions (e.g., Fe3+ or Ga3+) bound to tethered chelating reagentspresented on solid phase supports. Pierce’s PhosphopeptideIsolation kit and Sigma-Aldrich’s PhosphoProfile I PhosphopeptideEnrichment kit are both designed to simplify the isolation andidentification of phosphopeptides from protein digests throughthe specific interaction of phosphate groups with immobilizedgallium. Using a different process involving magnetic beads as thecarrier and zirconium ions for capture, Calbiochem’s ProteoExtract
Phosphopeptide Capture kit enables isolation of phosphorylatedpeptides for downstream identification by MS. The company alsoproduces the ProteoEnrich ATP-Binders kit which uses a novel resinwith covalently bound ATP on a flexible linker to obtain samplesenriched for protein kinases and other ATP-binding proteins.Other subproteomes often targeted are those of the intracellular
and plasma membranes, of particular interest because they are thelocation of many proteins involved in cell signaling. However, theseproteins have proved difficult to analyse, as their hydrophilic regionsare preferentially detected by commonly used MS preparativemethods. This necessitates high sequence coverage in order toaccurately map sites of posttranslational modification and agonist/antagonist binding, elucidate stoichiometry, and identify sites ofprotein-protein interaction. Reagents that can increase sequencecoverage of hydrophobic proteins are therefore in great demand.Novagen’s ProteoExtract Native Membrane Protein Extraction kit isdesigned for the isolation of membrane proteins from mammaliancells and tissues. The extremely mild procedure yields a solutionof integral membrane and membrane-associated proteins in theirnondenatured state. Life Technology’s (previously Invitrogen)Invitrosol MALDI Protein Solubilizer includes the ability to prepareboth hydrophobic and hydrophilic proteins and peptides directly forMALDI time-of-flight MS analysis without the need for subsequentacid hydrolysis.
Pushing the Limits of TechnologySample preparationmethods are highly dependent on the proteomicquery of interest. Generally, protein purification procedures requirenonvolatile buffers, solubilizing agents, EDTA, and detergents toensure that the proteins retain their native conformation. Some MSapplications might require relatively harsh treatments to exposecertain structures or hydrophobic components, while others,such as ESI-MS, might require more gentle buffers that retainmacromolecular associations, but could require meticulous cleanupof interfering reagents prior to MS analysis. Although approachesrelying on the depletion of potentially interfering, highly abundantproteins in clinical samples are often used, the approach runs therisk of throwing out the proverbial baby with the bathwater, sincesome of these high abundance proteins might have proteins ofinterest associated with them.These issues can be overcome by partitioning procedures that
not only separate out the unwanted proteins, but also allow for theanalysis of the bound sets of fragments, too. The combination ofproteome partitioning and fractionation allows for the enrichmentof low abundant proteins, as well as abrogation of the maskingeffect created by peptides derived from highly abundant proteins.Complementary biochemical manipulations for sample preparativemethods include enrichment strategies that target subsets of theproteome such as cellular organelles or functional groups—thechoice being determined by the specific target in mind.Although preparative technologies for MS have improved
dramatically in the past decade, ironically the extraordinary abilityandpower ofMS instruments is still being held back by the seeminglymundane biochemical limitations imposed by the procedures forsample preparation.
Agilent Technologies
www.agilent.com
Beckman Coulter
www.beckman.com
Bio-Rad
www.bio-rad.com
Calbiochem
www.calbiochem.com
EMD Biosciences
www.emdbiosciences.com
GenWay Biotech
www.genwaybio.com
Life Technologies
www.lifetech.com
Millipore
www.millipore.com
Novagen
www.novagen.com
Pierce
www.piercenet.com
Qiagen
www.qiagen.com
Sigma-Aldrich
www.sigmaaldrich.com
Stratagene
www.stratagene.com
Thermo Fisher Scientific
www.thermofisher.com
University of Toronto
www.utoronto.ca
Varian
www.varian.com
Featured Participants
Guatam Thor has a Ph.D. in Neurobiology and is currently a freelance writerbased in San Diego, CA.
DOI: 10.1126/science.opms.p0900032
Mass SpectrometerThe AB Sciex Qtrap 5500 System is a next-generation mass spectrometer that combines highsensitivity, scanning speed, and capacity to analyze a greater number of compounds, proteins, andcontaminants in a single run than previously possible. The system integrates triple quadrupolecapabilities with an innovation in ion trap technology called Linear Accelerator Trap for combinedquantitative and qualitative analysis. The system supports multiple experiments on the sameinstrument through a proprietary innovation, called TripleTrap Scanning, that allows scientists tomove from sensitive, specific, triple quadrupole scan modes to highly sensitive full-scan ion trapmode in less than one millisecond.
Applied BiosystemsFor information 800-327-3002www.appliedbiosystems.com
SFC ColumnsAnew line of supercritical fluid chromatography (SFC) columns bringsnewphasesandselectivities toSFCseparations, expanding theuseofthis technology to analytical and preparative applications. SFC offersseveral advantages over high-performance liquid chromatography,including improved resolution, faster separations, and higherthroughput. SFC also reduces the use of toxic solvents. SFC columnshave applications in pharmaceutical and academic research as wellas medicinal chemistry. Phenomenex’s new SFC offerings include theLuna HILIC (hydrophilic interaction liquid chromatography) columnswith unique, cross-linked diol chemistry for analysis of polarmetabolites; Synergi Polar-RP (ether-linked phenyl phase) for polarand aromatic analysis; and Lux chiral phases.PhenomenexFor information 310-212-0555www.phenomenex.com
Liquid Chromatography SystemThe AKTAready liquid chromatography system is designed forprocess scale-up and production for drug development and full-scale production following good laboratory practice and good manu-facturing practice standards. The system can improve cost efficiencyand productivity by saving time and expenditures for startup, labor,and consumables. AKTAreadyoperateswith ready-to-use, disposableflow paths, eliminating the risk of cross-contamination and the needfor cleaning and validation of cleaning procedures. The systemincludesthechromatographyunit,Unicornsoftware,andadisposableReadyToProcess Flow Kit including sensors and detection flow cells.The software includes an installation wizard with instructions andreports for column installation. The system is supported by extensiveregulatory product documentation and services, including validationdocumentation, product documentation, and more.GE HealthcareFor information 800-526-3593www.gelifesciences.com
Stain-Free ImagingThe Criterion Stain Free imaging system for detection andquantitation of proteins consists of a new formulation of Bio-Rad’sCriterion precast gels, a Criterion Stain Free imager, and Image-Labsoftware. The new system enables scientists to bypass the stainingand destaining steps of sodium dodecyl sulfate-polyacrylamide gelelectrophoresis and visualize protein samples in 2.5 minutes afterelectrophoresis. In addition, the system captures digital images
of gels to make record-keeping easy, with no need for a gel dryingstep. Benefits of the system include one-touch instrument operation,equal or improved sensitivity compared with a Coomassie stain,compatibility with protein immunoblotting and mass spectrometry,and digital images and data that are easy to share, print, and store.There is no organic waste.Bio-Rad LaboratoriesFor information 800-424-6723www.bio-rad.com
Antibody Purification ToolsThe PureProteome Protein A and Protein G Magnetic Beads enableresearchers to perform small-scale purifications faster and moreeasily. The beads provide high binding capacity for high recoveryof protein. The beads also ensure reproducible separation ofimmunoglobulins (IgG) by allowing for total removal of buffers andcomplete recovery of beads. Magnetic beads were developed toensure rapid and reproducible purifications by immobilizing thebeads to the side of the tube allowing for total removal of buffers withno loss of sample. In the past, magnetic beads have had significantlylower binding capacities than other bead-based purification media,but PureProteome beads overcome this by binding higher amountsof IgG. PureProteome beads are available with an optional magneticrack that immobilizes the beads against the side of the tube inseconds, allowing for reproducible purifications to be performedquickly and easily.MilliporeFor information 978-762-5170www.millipore.com
Fluorescent Detection KitThe DetectX Glutathione Fluorescent Detection Kit is designed toquantitatively measure free and oxidized glutathione in a singlesample. The kit makes use of a proprietary molecule, ThioStar, thatcovalently binds to the free thiol group on glutathione to yield ahighly fluorescent product. After mixing the sample with ThioStar for15 minutes, the user reads the glutathione-generated signal at 510nm in a fluorescent plate reader. Addition of the provided reactionmixture to the wells converts all the glutathione disulfide into freeglutathione, which then reacts with the excess ThioStar during asecond 15-minute incubation to detect total glutathione.LuminosFor information 734-677-1774www.LuminosAssays.com
AAAS/Science Business Office Feature
Newly offered instrumentation, apparatus, and laboratory materials of interest to researchers in all disciplines in academic, industrial, and governmental organizationsare featured in this space. Emphasis is given to purpose, chief characteristics, and availabilty of products andmaterials. Endorsement by Science or AAAS of any productsor materials mentioned is not implied. Additional information may be obtained from the manufacturer or supplier.
Electronically submit your new product description or product literature information! Go to www.sciencemag.org/products/newproducts.dtl for more information.
LIFE SCIENCE TECHNOLOGIESAAAS/Science Business Office Feature
www.sciencemag.org/products 1083
Proteomics
Ser1254
Tyr1571
PI3K
Akt
Rheb
LKB1
Growth Factors,Insulin, etc.Amino
Acids
WntSignaling AMP:
ATP
Rapamycin
mTORC1: mTORG�L (mLST8)Raptor
mTORC2: mTORG�L (mLST8)RictorSin1
p70 S6K 4E-BP1
Translation andCell Growth
AMPK
Rag A/B
Rag C/D
TSC2
TSC1
P
Ser2481Ser2448
P
P
P
P
Thr37/46/70
P
Thr389
P
Ser371
PRAS40
PThr246
S6P
Ser235/236/240/244
PP P
PSer473
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Thr308
PDCD4P Ser67
GSK-3mTORC2
mTORC1
Ser65
Ser939Ser1462
PP
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P P
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Cutting-edge
mTOR Signaling AntibodiesUnparalleled product quality, validationand technical support.
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Immunohistochemical analysis of paraf-fin-embedded human breast carcinomausing Phospho-S6 RibosomalProtein (Ser235/236) (D57.2.2E)Rabbit mAb #4858 in the presenceof control peptide (left) or Phospho-S6Ribosomal Protein (Ser235/236) Block-ing Peptide #1220 (right).
Immunohistochemical analysis of paraffin-embedded humanbreast carcinoma using Phospho-PRAS40 (Thr246)(C77D7) Rabbit mAb #2997.
Confocal immunofluorescent analysis of HeLa cells usingRagC Antibody #3360 (green). Actin filaments have beenlabeled with DY-554 phalloidin (red). Blue pseudocolor =DRAQ5™ (fluorescent DNA dye).
Confocal immunofluorescent analysis of HeLa cells using4E-BP1 (53H11) Rabbit mAb #9644 (green). Actinfilaments have been labeled with Alexa Fluor® 555 phalloidin(red). Blue pseudocolor = DRAQ5™ (fluorescent DNA dye).
Antibodies against all of the targets in this pathway areavailable from Cell Signaling Technology.
©C
ellSignaling
Technology,Inc./D
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Q5
®is
aregistered
trademark
ofB
iostatusLim
ited./A
lexaFluor
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aregistered
trademark
ofM
olecularProbes,Inc./
Selected
Rabbit
Monoclonals
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rights,includingthose
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Innovative Discovery Tools for Signal Transduction Research
