phosphoprotein profiling by pa-gelc−ms/ms

Phosphoprotein Profiling by PA-GeLC-MS/MS

Kolbrun Kristjansdottir,†,‡ Donald Wolfgeher,‡ Nick Lucius,§ David Sigfredo Angulo,§ andStephen J. Kron*,†,‡

Department of Molecular Genetics and Cell Biology, and Ludwig Center for Metastasis Research, The Universityof Chicago, Chicago, Illinois 60637, and School of Computer Science, Telecommunications and Information

Systems, De Paul University, Chicago, Illinois 60604

Received December 5, 2007

A significant consequence of protein phosphorylation is to alter protein-protein interactions, leadingto dynamic regulation of the components of protein complexes that direct many core biologicalprocesses. Recent proteomic studies have populated databases with extensive compilations of cellularphosphoproteins and phosphorylation sites and a similarly deep coverage of the subunit compositionsand interactions in multiprotein complexes. However, considerably less data are available on thedynamics of phosphorylation, composition of multiprotein complexes or that define their interdepen-dence. We describe a method to identify candidate phosphoprotein complexes by combiningphosphoprotein affinity chromatography, separation by size, denaturing gel electrophoresis, proteinidentification by tandem mass spectrometry, and informatics analysis. Toward developing phosphop-roteome profiling, we have isolated native phosphoproteins using a phosphoprotein affinity matrix,Pro-Q Diamond resin (Molecular Probes-Invitrogen). This resin quantitatively retains phosphoproteinsand associated proteins from cell extracts. Pro-Q Diamond purification of a yeast whole cell extractfollowed by 1-D PAGE separation, proteolysis and ESI LC-MS/MS, a method we term PA-GeLC-MS/MS, yielded 108 proteins, a majority of which were known phosphoproteins. To identify proteins thatwere purified as parts of phosphoprotein complexes, the Pro-Q eluate was separated into two fractionsby size, <100 kDa and >100 kDa, before analysis by PAGE and ESI LC-MS/MS and the componentproteins queried against databases to identify protein-protein interactions. The <100 kDa fraction wasenriched in phosphoproteins indicating the presence of monomeric phosphoproteins. The >100 kDafraction contained 171 proteins of 20-80 kDa, nearly all of which participate in known protein-proteininteractions. Of these 171, few are known phosphoproteins, consistent with their purification byparticipation in protein complexes. By comparing the results of our phosphoprotein profiling with theinformational databases on phosphoproteomics, protein-protein interactions and protein complexes,we have developed an approach to examining the correlation between protein interactions and proteinphosphorylation.

Keywords: Protein complexes • phosphorylation • mass spectrometry • ESI LC-MS/MS • phosphoproteinaffinity enrichment • databases

Introduction

Systems biology is the study of the interactions betweencomponents of a biological system and how these interactionsbring about the function and behavior of that system (reviewedin ref 1). In contrast to the classical enzyme-substrate para-digm that underlies the biosynthesis of most metabolites, otherbasic biological processes such as replication, transcription,splicing, translation, or secretion require dynamic assembly ofmultiprotein complexes whose subunits function in concert to

perform highly regulated reactions that synthesize or modifymacromolecules (discussed in ref 2). Protein complexes bringproteins in close proximity and can facilitate sequential reac-tions on the same substrate. A relatively simple example is theproteasome, a large protein complex that breaks down poly-ubiquitinylated proteins via ATP-dependent proteolysis (re-viewed in ref 3). As a major mechanism by which cells regulatethe concentration of proteins and degrade misfolded proteins,the proteasome must recognize properly tagged substrates,unfold the polypeptide and thread it into the proteolyticchamber and degrade the protein to short peptides. Theproteasome itself is comprised of over 20 individual stablyassociated protein subunits that provide structural support orare involved in recognition, unfolding and/or proteolytic roles.

The limiting factor for identifying protein complexes is themethod for their isolation or enrichment. Large protein com-

* To whom correspondence should be addressed. Dr. Stephen J. Kron,Ludwig Center for Metastasis Research, 924 E. 57th St., Chicago, IL 60637,USA. Tel., 1-773-834-0250; fax, 1-773-702-4394; e-mail, [email protected].

† Department of Molecular Genetics and Cell Biology, The University ofChicago.

‡ Ludwig Center for Metastasis Research, The University of Chicago.§ De Paul University.

2812 Journal of Proteome Research 2008, 7, 2812–2824 10.1021/pr700816k CCC: $40.75 2008 American Chemical SocietyPublished on Web 05/30/2008

plexes and organelles such as nucleasome and centrosomescan be enriched on a sucrose gradient and analyzed by massspectrometry.4,5 Only a subset of protein complexes is largeenough in size and sufficiently dissimilar from other complexesto be suited for this type of analysis. Blue native polyacrylamidegel electrophoresis (BN-PAGE) has also been used to separatenative protein complexes in the first dimension followed bydenaturing SDS-PAGE electrophoresis in the second dimensioncreating a 2-D gel where the spots can be analyzed by massspectrometry.6–8 This method was recently applied to complexprotein samples.7 As with conventional denaturing, 2-D gelelectrophoresis limitations include reproducibility, recovery ofprotein and visualization and selection of proteins spots in gelfor mass spectrometry analysis. Alternatively, sedimentation ofprotein complexes in a rate zonal gradient allows estimationof the relative size of protein complexes as recently describedfor Arabidopsis thaliana.9

Methods for mass spectrometry analysis of protein com-plexes are reviewed in ref 10. A comprehensive genome-widestudy of protein complexes was completed in yeast where theauthors affinity purified individual tandem affinity purification(TAP) tagged proteins from whole-cell lysates.11,12 Each TAP-tagged protein and any co-purifying polypeptides were ana-lyzed by mass spectrometry from SDS-PAGE gel bands. Thisapproach identified over 500 distinct protein complexes con-taining two to dozens of proteins and showed that manyproteins serve as subunits of a number of different complexes.The interactions identified in this experiment were combinedin a protein complex database (http://yeast-complexes.em-bl.de). This rich database provides a blueprint of protein-proteininteractions in yeast; however, as these types of databases areconstantly being updated and re-evaluated, it is incomplete.For example the http://yeast-complexes.embl.de data set wasgenerated using exponentially growing cells and, thus, did notcapture cell cycle dynamics of protein abundance and interac-tions. Similar concerns affect the interpretation of databasescompiling interaction information including affinity based massspectrometry results, two-hybrid analysis and other approachesas found in the BOND (http://bond.unleashedinformatic-s.com/, Thomson Scientific) and BioGRID (http://www.the-biogrid.org/)13 databases.

The composition of protein complexes is determined bothby the availability of subunits and their post-translationalmodifications.12,14 A common motif is regulation by proteinphosphorylation.14,15 A third of eukaryotic proteins may besubject to phosphorylation,16 but low stoichiometry contributesto a low abundance of most phosphorylated species. Phos-phorylation is readily reversible and highly dynamic, whichfurther complicates its detection. Proteomic analysis of un-fractionated cellular proteins typically yields only a tiny fractionof the expected phosphorylated species. Beyond their lowabundance, factors such as increased hydrophilicity, lower pKand inefficient fragmentation conspire to limit detection ofphosphopeptides by conventional ESI LC-MS/MS approaches.

One successful approach to increasing the coverage of thephosphoproteome has been to enrich phosphorylated speciesprior to proteomic analysis (reviewed in refs 17 and 18). Thebest characterized methodologies are based on isolation ofphosphopeptides after proteolysis. Anti-phosphotyrosine19,20

and other phosphoepitope antibodies21,22 are effective reagents,but as yet, a robust antibody-based approach to capturingserine or threonine phosphorylated peptides remains to bedescribed. A chemical approach exploits �-elimination of the

phosphate moieties of phosphoserine and phosphothreonineto permit chemical activation and tagging23–25 facilitatingtethering to a solid support. The phosphate moiety itself canbe used for affinity purification as by immobilized metal-affinitychromatography (IMAC)26,27 or on titanium dioxide (TiO2).28,29

A recent comparison of three common phosphopeptide isola-tion methods, �-elimination with covalent tethering, IMAC andTiO2, showed that, while each method recovered a large andreproducible population of phosphopeptides from a complexmixture, only a small fraction of these could be isolated by twoor more of the methods.25 These results argue that we remainfar from achieving comprehensive analysis of a phosphopro-teome in a single experiment, even with abundant samples.

A major limitation in this approach to phosphoproteomicsis the reliance on a single phosphopeptide as a tag foridentification of the phosphorylated protein. Even thoughmethods and equipment vary widely among laboratories, onlya small fraction of the expected peptides is reliably andreproducibly detected and from a complex mixture and theseare termed proteotypic peptides. Determinants of detectabilityinclude the length, hydrophobicity, charge and amino acidcomposition of these peptides.30,31 Importantly, phosphopep-tides fail to satisfy many of these criteria.

An alternative to enrichment of phosphopeptides is to purifythe phosphoproteins, exploiting one or another of the phospho-affinity approaches.19,32–38 Phosphoprotein profiling has thepotential practical advantage of improving the statisticalsignificance of protein identification. Intact proteins maintaincharacteristic properties such as their native molecular weight,allowing use of polyacrylamide gel separations. After proteoly-sis, the proteins can be recognized by multiple peptides,including any proteotypic peptides30 they may contain. How-ever, phosphoprotein enrichment suffers from the drawbackthat phosphorylation sites are unlikely to be identified, raisingthe possibility of misidentification. In turn, performing phos-phoprotein enrichment under native conditions is likely to co-purify any associated, nonphosphorylated proteins, furthercomplicating analysis. Nonetheless, phosphorylation site pre-diction and the emergence of large databases of phosphopep-tides as well as databases of known protein-protein interac-tions and protein complexes permit another method ofvalidation, through data mining approaches.

Several phosphopeptide enrichment strategies have beenapplied with limited success to enrichment of native ordenatured phosphoproteins. Chemical methods are likely tooffer only limited utility, while IMAC has demonstrated somevalue.32 Enrichment of phosphotyrosine containing proteinsvia anti-phosphotyrosine antibodies has been successful,19,33–35

but as with peptides, anti-phosphoserine and/or -threonineantibodies have proven to be less successful.36 Recently,phosphotyrosine immunoprecipitation was used to identifyproteins involved in erythropoietin receptor (EPOR) signaling.39

The phosphoprotein enriched sample was split and analyzedby two different proteomic strategies, 1-D electrophoresis withLC-MS/MS (1-D LC-MS/MS) or 2-D gel electrophoresis, silverstaining and identification of proteins by MALDI-TOF (2-DMALDI). A majority of proteins identified using the 2-D MALDItechnique were highly abundant housekeeping proteins andno proteins were identified from the EPOR-dependent path-ways. The 1-D LC-MS/MS method, however, allowed foridentification of multiple lower abundance proteins known tobe part of the EPOR-dependent pathways, but also a numberof new candidates.39

Phosphoprotein Profiling by PA-GeLC-MS/MS research articles

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Several commercial kits for native or denatured phosphop-rotein enrichment are available including PhosphoProteinPurification Kit (Qiagen), Pro-Q Diamond Phosphoproteinenrichment kit (Invitrogen/Molecular Probes) and BD Phos-phoprotein enrichment kit (BD Biosciences). Phosphoproteinscan be visualized on SDS-PAGE gels using the Pro-Q Diamondfluorescent stain from Invitrogen. Pro-Q Diamond detectsphosphate groups attached to tyrosine, serine or threonineresidues, with a sensitivity limit between 1 and 16 ng/proteinspot, depending on the phosphorylation state of the protein.40–42

Makrantoni et al.38 combined the PhosphoProtein PurificationKit from Qiagen with 2-D gel electrophoresis and Pro-QDiamond fluorescent phosphoprotein staining to show specificenrichment of phosphoproteins. Furthermore, the authorsexcised 13 protein spots from the gel and used matrix-assistedlaser desorption ionization (MALDI) mass spectrometry toidentify the proteins, 11 of which were known phosphopro-teins.38 Metodiev et al.37 used the PhosphoProtein PurificationKit from Qiagen and MALDI to identify several proteins fromhuman and yeast sources that were known phosphoproteins.Another study showed that phosphoproteins from undifferenti-ated and early differentiated mouse embryonic stem cells couldbe enriched using the Qiagen Phosphoprotein purification kitand identified over 30 proteins using 2-D PAGE and eitherMALDI-MS/MS or LC-MS/MS that exhibited differential re-covery from the column, indicating a change in phosphoryla-tion status.43

To test the potential for integrating phosphoprotein chem-istry, proteomics and bioinformatics to identify phosphopro-teins and phosphoprotein complexes on a systems level, weutilized the Pro-Q Diamond phosphoprotein enrichment kitand phosphoprotein stain from Invitrogen/Molecular Probes.We subjected a G2/M phase whole cell yeast extract tophosphoaffinity purification followed by gel electrophoresis andreverse phase HPLC combined with tandem electrospray massspectrometry (PA-GeLC-MS/MS). By querying several freelyavailable, large-scale databases, we found that most proteinshad previously been identified as phosphoproteins and/or ascomponents of protein complexes containing phosphoproteins.In summary, we have made progress toward a phosphoproteinprofiling method that permits phosphorylation to be studiedon a global scale and offers insight into the relationshipbetween phosphorylation of proteins and their association withother proteins in multisubunit complexes.

Materials and Methods

Reagents. The Pro-Q Diamond Phosphoenrichment kits usedfor this study were kindly provided by Invitrogen/MolecularProbes. Pro-Q Diamond stain and NuPAGE 2-12% gradientgels were obtained from Invitrogen/Molecular Probes, HALTphosphatase inhibitor cocktail from Pierce, Vivaspin centrifugalconcentrators from Vivascience, sequencing grade modifiedtrypsin from Promega, Lys-C protease from Princeton Separa-tions, and Zorbax 300SB-C18 reversed phase HPLC columns(dimensions: 3.5 µm packing, 150 mm × 75 µm) from Agilent.Other reagents were purchased from Sigma-Aldrich.

Phosphoprotein Enrichment. The lysate/sample was dilutedand loaded onto a column pre-equilibrated with Pro-Q Dia-mond resin. Fresh Pro-Q Diamond resin was used for eachexperiment. The column was washed and phosphoproteinswere eluted in buffers supplied with the Pro-Q Diamondphosphoenrichment kit, with all steps performed at 4 °C. Theprotein sample or lysate, flow-through from wash step and

eluate were concentrated by centrifugation in 10 kDa MWCOVivaspin concentrators at 4 °C and washed with 50 mM Tris,pH 7.5. The proteins were precipitated using methanol/chloroform/water as described in the Pro-Q Diamond phos-phoenrichment kit, resuspended in 4× Laemmli buffer andboiled for 10 min before loading on NuPAGE 2-12% gradientgels. The gel was stained with Coomassie for proteins and/orwith Pro-Q Diamond stain for phosphoproteins, followingmanufacturer’s instructions. Coomassie-stained gels werescanned with a Microtek Scan-Maker 6800. Pro-Q Diamond-stained gels were visualized with a Bio-Rad Molecular ImagerFX.

Phosphorylated GST-SH3n-Abltide. Purification of Abl ty-rosine kinase substrate, GST-SH3n-Abltide, and in vitro phos-phorylation by c-Abl kinase was as described,44 except that 100µCi of [γ-32P]-ATP was added and the reaction was scaled up7-fold. The reaction was loaded onto a Vivaspin centrifugalconcentrator with a molecular weight cutoff (MWCO) of 10 kDa.The retentate was collected and subjected to phosphoproteinenrichment as above. Fractions from the lysate, flow-through,wash and eluate of the Pro-Q Diamond column were scannedfor radioactivity by Geiger counter, pooled, precipitated andsubjected to SDS-PAGE on NuPAGE 2-12% gradient gels.Protein was imaged using Pierce Imperial Coomassie Stain andincorporated radioactivity was imaged with a GE Storm 860phosphorimager.

K562 Lysate Preparation. K562 cells were grown in suspen-sion at 37 °C and 5% CO2 in RPMI 1640 medium (Sigma)containing 10% heat-inactivated fetal bovine serum (Gemini),1% penicillin/streptomycin and 0.3 mg/mL L-glutamine. Wholecell lysates were prepared from 5 × 106 cells in 0.5 mL of Pro-QDiamond phosphoenrichment kit lysis buffer with 1:1000dilution of Pierce HALT phosphatase inhibitor cocktail, aproprietary mixture of sodium fluoride, sodium orthovanadate,sodium pyrophosphate and sodium glycerophosphate. Thesupernatant was collected, and protein yields were determinedby Bradford analysis using Bio-Rad protein assay reagent.Phosphoprotein enrichment was performed as described above.

Yeast Lysate Preparation. Yeast cells were treated with 30µg/mL nocodazole for 3 h at 30 °C to arrest cells in G2/M phaseand then harvested by centrifugation at 3000g for 5 min. Thepellet was resuspended in ice-cold Pro-Q Diamond phospho-enrichment kit lysis buffer or a buffer composed of 50 mMHEPES, pH 7.5, 10% glycerol, 150 mM NaCl, and 0.1% NP-40supplemented with 1 µM okadaic acid. Either buffer wassupplemented with a protease inhibitor mix from the Pro-QDiamond phosphoenrichment kit. The sample was subjectedto bead beating (3 × 30 s with 60 s on ice) on a BioSpecProducts Mini-Bead-Beater-8. After centrifugation at 14 000gfor 30 min at 4 °C, the supernatant was collected, yield wasdetermined with Bio-Rad protein assay reagent and phosphop-rotein enrichment was performed as described above.

Pro-Q eluate from nocodazole-arrested yeast extract (1.2 mg)was further fractionated on a 100 kDa MWCO Vivaspincentrifugal concentrator. The retentate was collected as the>100 kDa sample. The flow-through was concentrated on a10 kDa MWCO Vivaspin centrifugal concentrator and theretentate was collected as the <100 kDa sample. Both the >100kDa and <100 kDa samples were precipitated and loaded ontoa NuPAGE 2-12% gradient gel.

Western Blotting. Samples were separated on 12% NuPAGEgels and transferred to nitrocellulose membranes according tostandard methods. Uniform sample loading and transfer were

research articles Kristjansdottir et al.

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confirmed using Pierce Memcode reversible protein stain kit.Membranes were blocked in 5% bovine serum albumin (BSA)for 1 h at 25 °C. The membrane containing K562 cell extractwas probed with 1:1000 4G10 anti-phosphotyrosine primaryantibody (Upstate Cell Signaling Solutions) in 5% BSA at 25 °Cfor 1 h and detected with 1:5000 horseradish peroxidase (HRP)-conjugated anti-mouse IgG secondary antibody (GE-Amer-sham) in 5% BSA for 30 min. The membrane from yeast cellextract was probed with 1:1000 anti-phosphothreonine primaryantibody (Upstate Cell Signaling Solutions) in 5% Carnationdried milk at 25 °C overnight and 1:3000 HRP-conjugatedsecondary antibody in 5% milk for 1 h. Blots were developedusing Supersignal WestPico chemiluminescent substrate (Pierce),recorded on autoradiography film and scanned with a MicrotekScan-Maker 6800 at 600ppi resolution.

In-Gel Tryptic Digestion and Mass Spectrometry. Gel lanesto be analyzed were excised from SDS-PAGE gels by razor bladeand divided into 12 ∼1-cm slices. Each slice was then furtherdivided into ∼1-mm3 pieces. Each section was washed in waterand completely destained using 100 mM ammonium bicarbon-ate in 50% acetonitrile. A reduction step was performed byaddition of 100 µL of 50 mM ammonium bicarbonate, pH 8.9,and 10 µL of 10 µM TCEP (Tris(2-carboxyethyl)phosphine HCl)at 37 °C for 30 min. The proteins were alkylated by adding 100µL of 50 mM iodoacetamide and allowed to react in the darkat 20 °C for 40 min. Gel sections were first washed in water,then acetonitrile, and finally dried by SpeedVac for 30 min.

Digestion was carried out using 20 µg/mL sequencing grademodified trypsin in 50 mM ammonium bicarbonate. Sufficienttrypsin solution was added to swell the gel pieces, which werekept at 4 °C for 45 min and then incubated at 37 °C overnight.Sections containing proteins larger than 150 kDa were predi-gested with 0.25 mg/mL Lys-C in 6-8 M urea overnight at 25°C, diluted to final concentration of <2 M urea, then digestedwith trypsin as described above. Peptides were extracted fromthe gel pieces with 5% formic acid.

For Agilent XCT (Trap): The peptides were loaded onto theZorbax 300SB-C18 reversed phase column on and onlineAgilent XCT nanoHPLC system, separated with a gradient of5-60% acetonitrile in 0.1% trifluoroacetic acid over 110 min-utes, and then analyzed by electrospray tandem mass spec-trometry (LC-MS/MS) on an Agilent XCT ion trap.

For LTQ-FT (Thermo): The peptides were loaded onto theZorbax 300SB-C18 reversed phase column on and onlineDionex Ultimate nanoHPLC system, separated with a gradientof 5-55% acetonitrile in 0.1% formic acid over 120 minutesand then analyzed by electrospray tandem mass spectrometry(LC-MS/MS) on a LTQ hybrid ion-trap/FTICR mass spectrom-eter.

The Agilent XCT ion trap was operated in Ultrascan Modeat a speed of 26 000 m/z/s with the following settings: Skimmerat 40 V, Cap exit at 65 V, Capillary at 1700 V, and End PlateOffset at -500 V. After a 150 ms MS scan over 200-2200 m/zwith 3 averages, the 5 most intense ions above an intensity of6000 were subjected to MS/MS. The MS/MS was set to excludesingle charged ions and prefer double charged ions. Activeexclusion was used excluding ions after 2 scans for 1 min.

The LTQ FT was operated in positive ion mode, and parentions were selected for fragmentation by data-dependent analy-sis (five most abundant ions in each cycle): 1 scan LTQ FT-MS(m/z 400-2000) and maximum 5 scan LTQ-MS/MS (m/z

50-2000), 60-s dynamic exclusion. A normalized collisionenergy of 35 was used for low energy CID MS/MS of peptideions.

Database Searching and Criteria for Protein Identification.Mascot (version 2.1.01, Matrix Science) and X! Tandem (version2007.01.01.1, www.thegpm.org) were used to identify proteinsbased on MS/MS spectra. Mascot was set up to search theNCBInr_20060910 database (selected for Saccharomyces cer-evisiae, 11 101 entries) assuming the digestion enzyme trypsin.A fragment ion mass tolerance of 1.0 Da and a parent iontolerance of 0.6 Da were specified for the Agilent XCT data. Afragment ion mass tolerance of 1.0 Da and a parent iontolerance of 0.2 Da were specified for the LTQ FT data.Oxidation of methionine, N-formylation of the amino terminusand iodoacetic acid derivative of cysteine were specified asvariable modifications.

X! Tandem was set up to search the scd.fasta.pro database(selected for S. cerevisiae, 6794 entries) also assuming trypsin.X! Tandem was searched with a fragment ion mass toleranceof 0.60 Da and a parent ion tolerance of 10.0 ppm. Iodoaceta-mide derivative of cysteine was specified as a fixed modifica-tion. Deamidation of asparagine and glutamine, oxidation ofmethionine and tryptophan, sulfonation of methionine, tryp-tophan oxidation to formylkynurenin of tryptophan and acety-lation of lysine and the amino terminus were specified asvariable modifications.

Scaffold (version Scaffold-01_06_00, Proteome Software) wasused to visualize and validate MS/MS based peptide andprotein identifications. Peptide identifications were acceptedif they could be established at greater than 90% probability asspecified by the Peptide Prophet algorithm.45 Protein identi-fications were accepted if they could be established at greaterthan 95% probability and contained at least one identifiedpeptide. Proteins identified with only one peptide were manu-ally confirmed. Protein probabilities were assigned by theProtein Prophet algorithm.46 Proteins that contained similarpeptides and could not be differentiated based on MS/MSanalysis alone were grouped to satisfy the principles ofparsimony.

Results

Phosphorylated Proteins Are Bound to and Eluted fromPro-Q Diamond Resin. To evaluate the Pro-Q Diamond affinitymedia (Invitrogen/Molecular Probes) as a phosphoproteinenrichment tool, we first tested the resin with a single modelprotein, GST-SH3n-Abltide.44 We have previously shown thatGST-SH3n-Abltide,44 a synthetic 35 kDa protein affinity purifiedfrom bacteria consisting of glutathione-S-transferase, the 56residue SH3n domain of CrkL and the substrate peptide Abltide(EAIYAAPFAKKK) can be phosphorylated quantitatively by thetyrosine kinase c-Abl in vitro. Our prior work suggests that thisprotein can only be phosphorylated by c-Abl on a single site,the Tyr residue in the Abltide peptide. By including [γ-32P]-ATP in the reaction as a tracer, the phosphorylated proteincould be followed through Pro-Q Diamond fractionation.Aliquots of the kinase reaction, column flow-through, wash,and eluate were subjected to SDS-PAGE and protein detectionby Coomassie stain and phosphorimaging (Figure 1). 32P-labeled GST-SH3n-Abltide formed in the kinase reaction wasapplied to the resin. No radioactivity was detected in the flow-through, indicating that the Pro-Q Diamond resin bound mostif not all of the phosphorylated protein. Nearly all the radio-activity was recovered in the eluate and very little remained



on the beads after elution. These results show that the Pro-QDiamond resin selectively and quantitatively binds GST-SH3n-Abltide, a model singly phosphorylated protein.

Enrichment of Phosphotyrosine and PhosphothreonineContaining Proteins from Complex Samples. To determine ifthe Pro-Q Diamond resin enriches for phosphoserine/threonineproteins, we tested whole cell extracts of yeast, which has littleor no tyrosine phosphorylation. The extract was passed overthe Pro-Q Diamond resin and tested for the presence ofphosphothreonine (Figure 2A) by Western blotting. The yeastextract shows enrichment of phosphoproteins in the eluate andlittle if any phosphoproteins in the flow-through fraction. Todetermine if Pro-Q Diamond also enriches for phosphoty-rosine-containing proteins, we tested whole cell extract fromK562 leukemia cells. Whole cell extract was passed over thePro-Q Diamond resin and tested for the presence of phospho-tyrosine (Figure 2B) by Western blotting. Like the yeast extract,the K562 extract shows enrichment of phosphoproteins in theeluate and little if any phosphoproteins in the flow-throughfraction. These experiments were repeated twice resulting inidentical staining patterns (data not shown). Our data suggestthat Pro-Q Diamond resin provides a reasonably efficientenrichment of both phosphothreonine- and phosphotyrosine-containing proteins from complex samples.

To evaluate the degree of nonspecific binding, we loadedyeast extract onto the beads used to make Pro-Q Diamondresin, CL-4B, and then eluted with the Pro-Q elution buffer.Pro-Q Diamond and Coomassie staining revealed abundantphosphoproteins in the flow-through fraction and no discern-ible protein bands in the eluate (data not shown), indicatingthat the Pro-Q resin is specific for phosphoproteins and theirbinding partners.

Phosphatase Inhibitors. Many phosphatase inhibitors com-pete with phospho-amino acids for binding to the catalytic site.One concern is that the Pro-Q affinity column may also besubject to saturation by such phospho-amino acid mimics.Indeed, we found that excess phosphatase inhibitors apparentlybound to the column competitively with phosphoproteins. Forexample, adding the recommended amount of Halt Inhibitorcocktail (Pierce), a proprietary mixture of sodium fluoride,sodium orthovanadate, sodium pyrophosphate and sodiumglycerophosphate, to K562 extracts resulted in significantdetection of phosphotyrosine proteins in the flow-through anddecreased amounts in the eluate (data not shown). Nonethe-less, omission of phosphatase inhibitors could result in loss ofbinding due to dephosphorylation, suggesting the need forbalancing phosphatase inhibition against loss of resin capacity.

We analyzed the binding of K562 extract to Pro-Q Diamondresin in the presence of Halt Inhibitor cocktail diluted 10-fold.A strong phosphotyrosine signal was detected in the eluate ofProQ resin with a very faint signal in the flow-through (datanot shown, and Figure 2B). We next analyzed the binding ofyeast extract to Pro-Q Diamond resin in the presence of severalspecific inhibitors of the serine/threonine phosphatases ProteinPhosphatase 1 (PP1) and 2 (PP2).47–49 The inhibitors wereadded at values 5-100-fold above the experimentally deter-mined IC50 values. Calyculin at 10 nM, Microcystin at 20 nM,and Cantharadin at 2 µM each individually resulted in reducedbinding and elution of phosphoproteins from the column.Okadaic acid at 1 µM, however, did not impair binding to thecolumn (data not shown, and Figure 2A). Okadaic acid inhibitsPP2A at concentrations as low as 1-2 nM but PP1 typephosphatases are much less sensitive; complete inhibition isobserved only at 1 µM Okadaic acid.50 Thus, Okadaic acid at 1µM was used in yeast whole cell extract preparations and 10-

Figure 1. Pro-Q Diamond phosphoenrichment resin selectivelybinds phosphorylated protein. Bcr-Abl kinase was incubated withGST- SH3n-Abltide and [γ-32P]-ATP to allow for phosphorylationof GST- SH3n-Abltide. The solution was passed over a columncontaining Pro-Q Diamond resin and samples were taken fromthe loaded sample (L), flow-through (FT), elution (E) and remain-ing beads after elution (Beads). (a) Each fraction was subjectedto SDS-PAGE and the phosphoprotein detected using a phos-phorimager. The two E lanes represent the same sample loadedtwice, first a 5 µL aliquot of the sample (1×) and then with a 4-foldhigher amount of sample or 20 µL (4×). (b) Total phosphorylationin each sample after accounting for sample dilution and loading.The asterisk (*) represents that only a small sample (3%) of theloaded sample (L) was loaded on the gel. The remainder ofsample (L) was used for the Pro-Q phosphoenrichment columnand is represented in lanes FT, E, E2 and Beads.

Figure 2. Enrichment of phosphothreonine- and phosphotyrosine-containing proteins from complex samples using Pro-Q Diamondresin. Cell lysates (L) were passed over a column containingPro-Q Diamond resin, the flow-through (FT) was collected andphosphoproteins were eluted (E). Each sample was subjected toSDS-PAGE and Western blotting with (A) anti-phosphothreonine(R-pThr) for the yeast sample or (B) anti-phosphotyrosine (R-pTyr)for the K562 human cell line. In each case, phosphorylatedspecies were bound to and eluted from the Pro-Q resin withminimal amounts of the phosphorylated species in the flow-through sample.



fold diluted Halt Inhibitor Cocktail was used for phosphoty-rosine-containing extracts such as K562.

Identification of Phosphoproteins and Associated ProteinsEnriched from G2-Arrested Yeast. To visualize phosphopro-teins enriched from the G2-arrested yeast, the eluate wassubjected to 1-D SDS-PAGE and stained for phosphoproteinsusing Pro-Q Diamond stain and then for total protein withCoomassie stain. Pro-Q staining revealed a scarceness ofphosphoproteins in the flow-through fraction versus abun-dance in the eluate, while Coomassie-stained proteins werepresent in both flow-through and eluate (Figure 3). To identifyproteins, the gel was sliced into 12 even sections that weresubjected to tryptic digestion and extraction of peptides. Thepeptides were separated and detected on an Ion Trap XCTLC-MS/MS (Agilent) and data analysis was performed withMascot (Matrix Science) and X!Tandem (The Global ProteomeMachine Organization) software. Figure 4 shows a massspectrum of a peptide originating from protein Cor1 (inset) andthe tandem mass spectrum of this peptide. Four other peptideswere identified from Cor1, confirming the identification. Theruns from each section were compiled and analyzed withScaffold (Proteome Software) software, yielding identificationof 131 proteins with 90% protein confidence levels based upon95% peptide confidence with a minimum of 1 peptide peridentification (Supplemental Table 1). More stringent criteriaof 99% protein confidence levels with a minimum of 2 peptidesper identification resulted in identification of 108 proteins. Theidentification software (Mascot, X!Tandem) suggested the pres-ence of several phosphopeptides. However, manual inspectionrevealed low quality spectra and/or poor fragmentation in allof the proposed phosphopeptide MS/MS spectra (data notshown). Instead, mining proteomic databases and literature

revealed that over half of the 131 identified proteins havepreviously been described as phosphoproteins based on iden-tification of the site of phosphorylation (Supplemental Table1 and refs 51–55). The remaining proteins could be novelphosphoproteins since phosphoenrichment protocols are notcomprehensive as evidenced by the nonoverlapping results ofpublished phosphopeptide studies.25

Enrichment of Phosphoprotein Complexes. Phosphopro-tein enrichment was performed under native conditions toretain proteins that are components of phosphoprotein com-plexes. Thus, it is likely that several of the proteins identifiedare isolated as nonphosphorylated components of phosphop-rotein complexes. To investigate whether phosphoproteincomplexes remained intact under the experimental conditions,we first performed a gene annotation analysis using theBioGRID protein interaction database. Osprey software wasused for visualization of protein-protein interaction net-works.56 The analysis reveals several functional clusters andmany potential protein-protein interactions within a subsetof our 131 identified proteins (Figure 5A). The functionalsubsets include ribosomal proteins, metabolic enzymes, pro-teins involved in translation, RNA polymerase components,heat shock proteins, and proteasomal proteins.

Utilizing protein-protein interactions databases, we thenqueried our data against protein-protein interactions withinthe proteasome. One of the components, Pre2, has 39 knownbinding partners in BioGRID, encompassing several proteincomplexes. Our data set contains 11 of these binding partners,including Pre1, Pre3, Pre5, Pre7, Pre8, Pre9, Pre10, Pup2, Scl1and Rpn5 (Supplemental Table 1, Figure 5B). When studyingindividual affinity purifications of a Pre proteins complex, weobtained even better coverage. Our data set contained 10 of13 identified binding partners for Pre2 in the BOND databaseand 9 out of 13 interactions in the http://yeast-complexes.em-bl.de database for Pre10.57 Examining the components of the20S core particle of the proteasome on the SaccharomycesGenome Database (http://www.yeastgenome.org) reveals 15proteins of which we found 14 in this study. The core particlecan be divided into the alpha and beta subunit complexes. Allof the components of these subunit complexes are identifiedin our study with the alpha subunit containing Pre10, Pre5,Pre6, Pre8, Pre9, Pup2 and Scl1 and the beta complex contain-ing Pre1, Pre2, Pre3, Pre4, Pre7, Pup1 and Pup2. Thus, ouraffinity purification is highly enriched in proteasome compo-nents even though only three of these proteins have previouslybeen described as phosphoproteins: Pre5, Pre7 and Pup2. It islikely that phosphorylated Pre5, Pre7 and/or Pup2 proteinsbound to the Pro-Q Diamond resin and other components ofthe protein complex were co-purified in the phosphoproteinenrichment.

We repeated the phosphoprotein enrichment using a lysisbuffer (50 mM HEPES, pH 7.5, 10% glycerol, 150 mM NaCl,and 0.1% NP-40) similar in composition to the buffers usedin Tandem Affinity Purifications (TAP) for identification ofprotein complexes in yeast.58 This lysis buffer is not asoptimized for the phosphoprotein resin, and did result inan increase of phosphoproteins in the flow-through fraction(data not shown). However, since this lysis buffer is opti-mized to retain protein-protein interactions at neutral pHand a sizable number of phosphoproteins were still boundand eluted from the resin, we decided to accept this decreasein binding. To identify which proteins are components ofprotein complexes, we added a fractionation step based on

Figure 3. PA-GeLC-MS/MS results in apparent enrichment ofphosphoproteins and identification of several known phosphop-roteins. Cell lysates (L) were passed over a Pro-Q Diamondcolumn, the flow-through (FT) collected and phosphoproteinseluted (E) and subjected to SDS-PAGE. The gel was stained withCoomassie for proteins and Pro-Q Diamond stain for phosphop-roteins. The peptides were eluted from the gel with trypsin andsubjected to LC-MS/MS. Phosphoproteins are selectively boundand eluted from the phosphoaffinity column. A sampling ofproteins is listed to the right, grouped by the fraction in whichthey were identified and the range of molecular weight ofproteins identified within that fraction. Asterisk indicates knownphosphoproteins.



size. Protein complexes composed of multiple proteins aretypically large and would be expected to be retained by a100 kDa molecular weight cutoff (MWCO) centrifugal con-centrator, while monomeric proteins and protein complexessmaller than 100 kDa should filter through. The retentateand flow-through samples were subjected to 1-D SDS-PAGEand analyzed by Coomassie staining (Figure 6). Preparing

the samples for SDS-PAGE by boiling in SDS denatured theproteins and resulted in dissolution of all protein complexesallowing each protein to migrate on the gel according to themonomeric molecular weight. Most proteins were found inthe retentate fraction despite that fact that a vast majorityof the proteins have a monomeric MW of less than 100 kDa(Figure 6).

Figure 4. An example of peptide mass spectra used for protein identification. The inset (top right) shows the mass spectrum of theprecursor ion. The remainder of the figure depicts the tandem mass spectrum of the same peptide, LAAQIFGSYNAFEPASR. The identifiedy- and b-fragments are indicated in the sequence above the spectrum. Four additional peptides identified the Cor1 protein.

Figure 5. Gene annotation analysis of enriched proteins reveals clusters of functionalities. Protein interactions within a subset of the Pro-QDiamond enriched proteins were obtained from the BioGRID database13 and are indicated by solid lines (http://www.thebiogrid.org/). Ospreysoftware was used to generate the figures, where each node represents a single protein.56 (A) Functionalities identified include the proteasome(lower left), ribosome (upper left), heat shock proteins (lower right), RNA polymerase (upper right) and proteins involved in translation(middle). The nodes in (A) are color coded (grayscale) according to gene annotation. (B) Protein protein interactions between a subset of theproteins identified from the 20S subunit of the proteasome.



Proteins were identified by mass spectrometry as describedabove, except a newer instrument was used, the FT LTQ(Thermo Finnigan). When these methods were used, 237proteins were identified in the retentate (>100 kDa fraction)using 90% protein confidence levels based upon 95% peptideconfidence with a minimum of two peptides per identification(Figure 6, Supplemental Table 2). To avoid the need for manualinterpretation and increase confidence levels, at least twopeptides were required for each protein identification. Applyingthe same conditions to the flow-through sample (<100 kDafraction) resulted in identification of 45 proteins (SupplementalTable 3). The total amount of proteins identified was 250 withsome proteins identified in both sections. This number is morethan double the amount of proteins identified in the originalenriched sample (108 proteins, Figure 3, Supplemental Table1). Several factors could have contributed to the increase inproteins identified. First, we moved the mass spectrometryanalysis to a newer, higher performance instrument (from anAgilent XCT Ion Trap to a Thermo LTQ FT). Second, we useda greater amount of starting material, and finally, we increasedthe fractionation of the sample (additional separation into >100kDa and <100 kDa fractions). Comparing the two experimentsrevealed that over 80% of the proteins were identified in bothexperiments for two peptide identifications and 89% whenidentifications with one peptide were allowed. Thus, our PA-GeLC-MS/MS method is highly reproducible.

As expected, the retentate (>100 kDa) contained several largeproteins, including 23 proteins ranging between 100-250 kDa(Supplemental Table 2). Only three proteins in the flow-throughfraction were larger than 100 kDa: Kap123, Yef3 and Hsp104

at 123, 116 and 102 kDa, respectively (Supplemental Table 3).It is not surprising that proteins close in size to 100 kDa areable to travel through the membrane given that the molecularweight cutoff (MWCO) is assigned based on 90% retention ofa standard with that molecular weight. Indeed, these threeproteins are detected in both the flow-through and the reten-tate fractions.

Proteins in the retentate fraction are highly connected, withseveral known binding partners (Figure 7). Noncomplexedproteins in the retentate would include monomeric and ho-modimeric proteins with molecular weights around 100 kDaincluding Arg5,6, Vtc2, Vtc3, Ala1 and Cdc3. The heterodimericproteins Pdi1-Ero1, Eno2-YIL091C, Cor1-Qcr2 and Ses1-Dre2are around 100 kDa in size. Individual nodes with no linesconnecting to them indicate that the protein has not beendescribed to bind to any of the other proteins identified in thisstudy. Several individual nodes, however, including Rps18B,Rpl27B, Bfr2 and Qcr7 are small proteins and thus should nothave been retained as monomers in the retentate. This couldbe due to the fact that the proteins are a part of a proteincomplex that has not been previously reported. Alternatively,they might be part of a known complex, but the interactingpartner(s) was not identified in our sample. One caveat to ourexperimentation is that the sample is concentrated on a 10 kDaMWCO centrifugal concentrator, resulting in loss of anyproteins smaller than 10 kDa. Several ribosomal proteins aresmaller than 10 kDa including Rpl29, Rpl39, Rpl41A/B, Rps29A/Band RPS30A/B. Indeed, one of the individual nodes mentionedabove has been found to bind to both Rpl29 and Rps30A.

Since monomeric proteins should be purified based on theirphosphorylation state, we would expect the <100 kDa fractionto contain a high percentage of phosphoproteins. This can beseen visually in Figure 8, where black nodes represent proteinspreviously reported to be phosphorylated and gray nodesrepresent nonphosphorylated proteins. Indeed, over 78% of theproteins identified in the <100 kDa fraction have previouslybeen identified as phosphoproteins (Supplementary Table 3).On the other hand, the >100 kDa fraction should containphosphoprotein complexes and as such should have a higherpercentage of nonphosphorylated proteins and thus a loweroverall percentage of phosphorylated proteins. Indeed, thepercentage of phosphoproteins in the >100 kDa fraction ismuch lower at 43% (Figure 7, Supplemental Table 2).

A global analysis of protein expression levels in yeast hasbeen performed in which the authors TAP- and/or GFP-taggedevery protein and calculated the copy number of proteins in acell.59 Using these measurements on the proteins identifiedfrom Figure 3 reveals an average abundance of 123 000 copiesper cell (Supplemental Table 1). Separating the enrichedphosphoproteins into two fractions (Figure 6) and analyzingeach fraction separately resulted in a decrease of averageabundance to 73 000 copies per cell (Supplemental Table 2).The lowest abundant protein was Rpl3 at 450 copies per cell(Supplemental Table 1) and from the fractionated sample,YIL091C at 172 copies per cell (Supplemental Tables 2 and 3).Comparing the abundance of proteins within the two sectionsrevealed a similar distribution of abundances with the excep-tion of an absence of low-abundance proteins (<1000 copies)in the <100 kDa fraction (Lane FT from Figure 6).

Discussion

Recent proteomic studies have populated databases withextensive compilations of cellular phosphoproteins and phos-

Figure 6. Separation of proteins and protein complexes by size.Yeast extract was enriched for phosphoproteins and associatedproteins using Pro-Q Diamond resin (see Figure 3) and thenseparated by size using a Vivaspin centrifugal concentrator witha 100 kDa MW cutoff. The supernatant (S) or retentate of theconcentrator was collected and contains protein complexes andproteins larger than 100 kDa. The flow-through (FT) contains theproteins and protein complexes that are smaller than 100 kDa.The samples were denatured and subjected to SDS-PAGE andthe gel was stained with Pro-Q Diamond stain (A) and Coomassiestain (B). Molecular weight markers are indicated between gels.



phorylation sites and similarly deep coverage of the subunitcompositions and interactions in multiprotein complexes. Wedescribe a method, PA-GeLC-MS/MS, to identify candidatephosphoprotein complexes by combining phosphoaffinitychromatography, size separation, 1-D SDS-PAGE, mass spec-trometry and informatics analysis. On a global scale, we showsuccessful enrichment of 108 phosphoproteins and associatedproteins from G2-phase yeast using the commercially availablePro-Q Diamond phosphoprotein affinity resin. Comparing theidentified proteins to several large-scale phosphoproteomicstudies reveals that about half was previously identified asphosphoproteins, many in several different studies. To identifycomponents of phosphoprotein complexes, we fractionated theenriched sample under nondenaturing conditions using acentrifugal concentrator into components larger (retentate) orsmaller (flow-through) than 100 kDa. The results show that theconcentrator provided good separation with only three proteinslarger than 100 kDa found in the flow-through. Furthermore,these proteins were also found in the retentate, suggesting that

their size was too close to the molecular weight exclusion sizeof the membrane for efficient separation. As expected, all largerproteins were found in the retentate, including Acc1 and Ura2at around 250 kDa. In accordance with isolation of proteincomplexes, more than 200 of the proteins identified in theretentate are smaller than 100 kDa. It appears that, under theexperimental conditions used in this study, a majority ofproteins are not single, monomeric proteins but componentsof known protein complexes. Indeed, the percentage of knownphosphoproteins was higher in the flow-through (78%) thanin the retentate (43%), which correlates with an abundance ofmonomeric phosphoproteins in the flow-through. We tookadvantage of the extensive protein interaction databases (Bio-GRID, BOND, http://yeast-complexes.embl.de) to identify pro-teins previously identified as components of protein complexes.We noticed enrichment of several functional groups includingchaperones. Chaperones are abundant proteins that bind tounfolded proteins and help them fold correctly. Chaperonesthus participate in protein-protein interactions and each

Figure 7. Protein interactions within the >100 kDa sample reveal a high density of protein-protein interactions. Phosphoproteins andphosphoprotein complexes were obtained by passing yeast extract over the Pro-Q Diamond resin. The sample was then separated bysize using a 100 kDa centrifugal concentrator. The retentate contained proteins and protein complexes larger than 100 kDa. Figure 8shows the proteins found in the flow-through indicating a size smaller than 100 kDa. Osprey software was used to generate a figuredepicting all possible interactions within the sample, where each node represents a single protein.56 Proteins are represented by grayand black nodes where black nodes represent proteins previously reported to be phosphorylated (see Supplemental Table 2). Proteininteractions from the entire retentate of the 100 kDa centrifugal concentrators were obtained from the BioGRID database13 and areindicated by solid lines (http://www.thebiogrid.org/).



chaperone has several potential binding partners. Indeed, wefind several chaperones in our retentate including Hsc82,Hsp104, Hsp60, Hsp82, Ssa1, Sse1, Sti1, and Ydj1. We also findseveral chaperones in the flow-through fraction representingeither small chaperones bound to small proteins or monomericchaperones. To further analyze the oligomeric status of theseproteins, a more comprehensive size separation experiment isin order. Several methodologies exist to study the size of proteincomplexes. Centrifugal concentrators with different MWCO orgel filtration/size exclusion columns can be used to obtain agradual series of molecular weights. Blue native polyacrylamidegel electrophoresis (BN-PAGE) has also been used to separatenative protein complexes in the first dimension followed bydenaturing SDS-PAGE electrophoresis in the second dimensioncreating a 2-D gel where the spots can be analyzed by massspectrometry.6,7 Alternatively, sedimentation of protein com-plexes in a rate zonal gradient allows estimation of the relativesize of protein complexes as recently described for A. thaliana.9

Any of these methods could be combined with PA-GeLC-MS/MS for a more comprehensive analysis of protein complexes.

Reproducibility was evaluated both on the phosphoaffinitylevel and the mass spectrometry level. On the phosphoaffinitylevel, Pro-Q staining revealed a paucity of phosphoproteins inthe flow-through fraction versus abundance in the eluate, whileCoomassie-stained proteins were present in both flow-throughand eluate fractions. This staining pattern was consistentlyproduced in replicate experiments (data not shown). Twoindependent experiments (108 proteins, Figure 3, SupplementalTable 1 versus 250 proteins, Figure 6, Supplemental Tables 2and 3) using different lysis conditions and with or without asize separation step still have 89% overlap of proteins identifiedwhen 1 peptide was required for protein identification showingthe reproducibility of the PA-GeLC-MS/MS method.

Phosphoprotein enrichment results in identification of sev-eral peptides from each protein and a corresponding highconfidence of protein identification. However, the enrichedfraction is much more complex than phosphopeptide-enrichedfractions because of the surplus of nonphosphorylated peptidesboth in number and abundance. Thus, our method does a poorjob of identifying phosphorylation sites within the proteinsidentified. To improve the identification of phosphorylationsites, the sample complexity needs to be reduced. First, theexperiment could be done under denaturing conditions toeliminate the presence of protein complexes containing non-phosphorylated components. Second, the sample could befurther fractionated. A promising approach was described byHung et al.60 in which tryptic peptides are subjected toisoelectric focusing (IEF), which is a gel-based method thatseparates species based on overall charge or pI value. Phos-phopeptides and peptides composed of more acidic than basicamino acid residues have low pI values (2.1-6.5). Peptidescontaining a higher or equal number of basic compared toacidic residues have high pI values (5.8-9.8). Acidic aminoacids were neutralized by esterification increasing the pI valueof the corresponding peptides resulting in their separation fromphosphopeptides (low pI). This method allows isolation of ahighly enriched phosphopeptide fraction in addition to allow-ing isolation of nonphosphorylated proteins. It would likelyresult in a large increase in identification of phosphorylationsites.

This proteomic analysis of phosphoprotein complexes is notmeant to be comprehensive; however, comparing our resultsto the global proteome reveals interesting over- and under-represented subpopulations. We analyzed the gene ontology(GO) of the proteins identified in this study and compared themto the yeast proteome. Analysis of protein localization revealsa large overrepresentation of cytosolic proteins (5.5-fold) andto a lesser extent cytoplasmic (1.7-fold), mitochondrial (1.4-fold) and endoplasmic proteins (1.4-fold). On the other hand,our protein set is slightly under-populated by nuclear proteins(1.1-fold). A quarter of the proteome is defined as having“cellular component unknown”. We do not identify any of theseproteins, which might reflect that they are either low abun-dance or only expressed under special conditions. Additionally,a large portion of the proteins with “cellular compartmentunknown” are classified as dubious open reading frames andmight thus not be expressed as proteins.

Analysis of GO terms for proteins associated with differentprocesses revealed that the trend remained the same since themost populated category in the proteome, cellular processes,was also the most populated in our sample and so forth.However, metabolic processes and the general category ofcellular processes were overrepresented in our results by about35% probably due to these proteins being relatively highabundance. Transport, transcription, signal transduction andamino acid metabolic processes were between 55 and 75%underrepresented. Cell cycle related proteins were only 6%underrepresented.

GO analysis of protein complexes reveals an overrepresen-tation of components of 69 complexes including ribonucle-oprotein complex, ribosome, proteasome, translation initiationfactor complexes, vacuolar transporter chaperone complex andmitochondrial respiratory chain complex. Six complexes identi-fied in our study are underrepresented compared to theproteome: DNA-directed RNA polymerase II complex, tran-scription factor complex small nuclear ribonucleoprotein com-

Figure 8. A majority of proteins in the <100 kDa sample areknown phosphoproteins. Phosphoproteins and phosphoproteincomplexes were obtained by passing yeast extract over the Pro-QDiamond resin. The sample was then separated by size using a100 kDa centrifugal concentrator. The figure shows proteinsfound in the flow-through of the concentrator indicating a sizesmaller than 100 kDa. Figure 7 depicts the retentate, which shouldcontain proteins and protein complexes larger than 100 kDa.Osprey software was used to generate a figure depicting allpossible interactions within the sample, where each noderepresents a single protein.56 Proteins are represented by grayand black nodes where black nodes represent proteins previouslyreported to be phosphorylated (see Supplemental Table 3).Protein interactions within the entire flow-through of the 100 kDacentrifugal concentrator were obtained from the BioGRID data-base (http://www.thebiogrid.org/)13 and are indicated by solidlines.



plex, kinetochore, nuclear pore, and histone acetyltransferasecomplex. A majority of these complexes are localized in thenucleus. This is not surprising since the sample preparationwas not targeted toward nuclear proteins and based on the GOanalysis of protein localization mentioned previously. A largenumber of complexes (220) were not identified in our sample.This could be due to several factors such as the absence of aphosphorylated protein in the complex and would thus not beisolated via the PA-GeLC-MS/MS method, formation of com-plexes under certain conditions (for example cell cycle stageand stress conditions) and/or low abundance of the proteincomplex.

Most of the proteins identified in this study, including thechaperones, are highly abundant proteins. This is a recognizedproblem in proteomics, mostly stemming from the dynamicrange of protein concentrations being much larger than thedynamic range of the mass spectrometer. The limit of detectionfor Pro-Q Diamond phosphostain in an SDS-PAGE gel is around1 ng, which is similar to silver stain; however, the limit forCoomassie stain is much higher at about 50 ng.40–42,61 Pro-Qstaining is proportional to the number of phosphates on theprotein; thus, more highly phosphorylated proteins will bedetected at low levels. The method of subjecting samples to1-D PAGE, trypsin digestion, and identification using reversephase chromatography on an LC-MS/MS has been shown tobe more sensitive than, for example, 2-D SDS-PAGE coupledwith MALDI.39 The level of sensitivity highly depends on thetype and model of mass spectrometer. The FT-LTQ, which isused for the size separation experiment in Figure 6, has beenshown to identify proteins at levels as low as 20 fmol (about0.5 ng for a 40 kDa protein).62 Thus, we should be identifyingall proteins that are detected by Coomassie stain and the vastmajority of Pro-Q stained proteins depending on the phos-phorylation status and peptide fragmentation pattern bytrypsin.

We were able to detect some low-abundance proteins, aslow as 200 copies per cell, and as mentioned previously, weidentified several signal transduction and cell cycle regulatedproteins. However, there is significant room for improvement,and for purposes of studying cell cycle regulated events, thenumber of low-abundance proteins detected needs to beincreased. To reach this goal, we are scaling up the experiment,performing more extensive fractionation and improving iden-tification software. Another approach that could be taken is tocombine our PA-GeLC-MS/MS method with targeting ofcertain cellular compartments and/or organelles, which hasproven to be very successful and is reviewed in ref 63.

We have demonstrated that PA-GeLC-MS/MS can be usedto catalog potential phosphoproteins and phosphoproteincomplexes at a given time and/or condition. The challengeremains to create software to analyze and predict the presenceof protein complexes based on experimental criteria andprotein interaction databases. Our method relies greatly on thequality and density of protein-protein interaction databases.We anticipate much improvement in density, sensitivity andaccuracy of the protein-protein interaction networks as moreand more data and databases are combined and re-evaluated.

Acknowledgment. Proteomics on the Thermo LTQ FTinstrument were performed in the CBC-UIC ResearchResources Center Proteomics and Informatics ServicesFacility, which was established by a grant from The SearleFunds at the Chicago Community Trust to the Chicago

Biomedical Consortium. Proteomics on the Agilent XCTinstrument were performed at the University of ChicagoProteomics Core. We thank Tamara Nyberg and BrianAgnew at Molecular Probes-Invitrogen for generouslyproviding Pro-Q Diamond phosphoprotein enrichmentmedia, other reagents and helpful suggestions for their use.We thank Donald L. Helseth, Jr. for technical support andhelpful discussions. We thank Samuel L. Volchenboum forcritical reading of the manuscript. This work was supportedby NIH R01s GM60443 and HG003864. K.K. was supportedby University of Chicago Cancer Biology NRSA TrainingGrant CA09594 and S.J.K. was a Leukemia & LymphomaSociety Scholar.

Supporting Information Available: Supporting Tables1-3 list the proteins identified in each proteomic experiment.This material is available free of charge via the Internet athttp://pubs.acs.org.

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phosphoprotein profiling by pa-gelc−ms/ms

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