Global Identification of O-GlcNAc-ModifiedProteins

Animesh Nandi,† Robert Sprung,† Deb K. Barma,† Yingxin Zhao,† Sung Chan Kim,† John R. Falck,‡ andYingming Zhao†,‡,*

Departments of Biochemistry and Pharmacology, University of Texas Southwestern Medical Center,Dallas, Texas 75390-9038

The O-linked N-acetylglucosamine (O-GlcNAc) modifica-tion of serine/threonine residues is an abundant post-translational modification present in cytosolic and nuclearproteins. The functions and subproteome of O-GlcNAcmodification remain largely undefined. Here we report theapplication of the tagging-via-substrate (TAS) approach forglobal identification of O-GlcNAc-modified proteins. TheTAS method utilizes an O-GlcNAc azide analogue formetabolic labeling of O-GlcNAc-modified proteins, whichcan be chemoselectively conjugated for detection andenrichment of the proteins for proteomics studies. Ourstudy led to the identification of 199 putative O-GlcNAc-modified proteins from HeLa cells, among which 23 wereconfirmed using reciprocal immunoprecipitation. Func-tional classification shows that proteins with diversefunctions are modified by O-GlcNAc, implying that O-GlcNAc might be involved in the regulation of multiplecellular pathways.

The modification of nuclear and cytoplasmic proteins at serineand threonine residues with O-linked N-acetylglucosamine (O-GlcNAc) was first described over two decades ago.1 The modifica-tion was found in various classes of proteins including enzymes,transcription factors, cytoskeletal proteins, signaling proteins,receptors, nuclear pore complex proteins, and kinases.2 Similarto phosphorylation, the O-GlcNAc modification is dynamic with aturnover rate faster than that of the proteins it modifies.3 TheO-GlcNAc modification has been shown to affect protein-proteininteractions, protein-DNA interactions, protein stability andactivity, and cell signaling cascades.4 Disregulation of the O-GlcNAc modification has been implicated in the development ofdisease states including diabetes, cancer, and Alzheimer’s.2,4 Giventhe potentially broad regulatory influence of the O-GlcNAcmodification, a more comprehensive understanding of the targetsof O-GlcNAc transferase is needed to elucidate its functionalconsequences.

In this report, we describe the global detection and proteomicanalysis of O-GlcNAc-modified proteins in HeLa cells. An affinity-tagged version of the O-GlcNAc modification is metabolicallyincorporated onto proteins using an azide-tagged analogue ofN-acetylglucosamine. The azido-GlcNAc-modified proteins thuscontain an azide handle for chemoselective conjugation using abiotinylated phosphine reagent. The resulting conjugates wereaffinity-purified with streptavidin beads and subsequently digestedwith trypsin and analyzed by nano-HPLC-MS/MS. Using thisstrategy, we identified 199 azido-GlcNAc-modified proteins in HeLacells. We subsequently validated the presence of this modificationamong 10 previously reported and 13 newly identified O-GlcNAc-modified proteins using specific antibodies. Our results reveal thatproteins with a wide range of functions are modified by O-GlcNAc,implying its diverse cellular functions.

EXPERIMENTAL PROCEDURESMaterials. DMEM and glucose-free DMEM were purchased

from Life Technologies (Gaithesburg, MD). Bovine serum albu-min, trichloroacetic acid (TCA), sodium dodecyl sulfate (SDS),NP40, DMSO, and glucosamine were from Sigma (St. Louis, MO).Streptavidin agarose beads and D-biotin were from Pierce Bio-technology (Rockford, IL). Biotinylated phosphine capture reagent3 (Figure 1) and peracetylated N-(2-azidoacetyl)glucosamine 1(Figure 1) were synthesized in-house. Primary antibodies andProtein A/G agarose beads were from Santa Cruz BiotechnologyInc. (Santa Cruz, CA). Western Lighting plus chemiluminescencedetection kit was from Perkin-Elmer Life Sciences (Boston, MA).Bradford protein estimation reagent and Bio-gel P6 DG desaltingcolumn were from Bio-Rad (Hercules, CA). Streptavidin HRP wasfrom Amersham (Piscataway, NJ). Protease inhibitor cocktail wasfrom Calbiochem (San Diego, CA).

Methods. Cell Culture and Metabolic Labeling. Hela cells werecultured in DMEM (4.5 g of glucose/L) supplemented with 10%FBS and antibiotics at 37 °C with 5% CO2. For labeling, culturemedium was replaced at 70% cell confluency with DMEM (1 g ofglucose/L) containing 250 µM peracetylated GlcNAc or peracety-lated azido-GlcNAc in DMSO. The cells were labeled for 24 h.Three hours before harvest, glucosamine was added to thecultures to a final concentration of 4 mM as an inhibitor ofO-GlcNAcase during the harvest procedure.

Isolation of Nucleocytoplasmic Proteins. Fifty dishes (15 cm) oflabeled cells were harvested by scraping in chilled PBS containing4 mM glucosamine. The cells were collected by centrifugation at

* Corresponding author. E-mail: yzhao@biochem.swmed.edu. Fax: (214) 648-2797. Tel: (214) 648-7947.

† Department of Biochemistry.‡ Department of Pharmacology.

(1) Torres, C. R.; Hart, G. W. J. Biol. Chem. 1984, 259, 3308-3317.(2) Wells, L.; Vosseller, K.; Hart, G. W. Science 2001, 291, 2376-2378.(3) Comer, F. I.; Hart, G. W. J. Biol. Chem. 2000, 275, 29179-29182.(4) Vosseller, K.; Wells, L.; Hart, G. W. Biochimie 2001, 83, 575-581.

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1000g. Four milliliters of hypotonic lysis buffer (10 mM HEPES,10 mM KCl, 1.5 mM MgCl2, and protease inhibitor cocktail) wasadded to the pellet and the resultant mixture incubated on ice for30 min. The sample was Dounce homogenized using a “B”-ratedpestle, and NaCl was added drop by drop to a final concentrationof 400 mM to lyse nuclei as previously described.5 The samplewas ultracentrifuged at 100000g for 1 h at 4 °C. The supernatant(nucleocytosolic fraction) was carefully removed, and the proteinwas estimated by the Bradford method.

Conjugation with Biotinylated Capture Reagent and AffinityPurification. The nucleocytosolic protein extract was precipitatedwith eight volumes of ice-cold acetone and one volume of TCAfor 2 h at -20 °C. The protein pellet was obtained by centrifugationat 20000g, washed with ice-cold acetone, and resuspended in PBScontaining 2% SDS at a concentration of 4-8 µg/µL. For conjuga-tion, biotinylated phosphine capture reagent was added to a finalconcentration of 50 µM. The samples were kept agitating for 10-12 h in the dark at room temperature. Unconjugated capturereagent was removed using a Bio-gel P6 DG desalting column.The samples were diluted with PBS to a final SDS concentrationof 0.2% and mixed with streptavidin beads (for protein affinitypurification and subsequent identification) or avidin monomerbeads (for verification of azido-GlcNAc modification by Westernblot analysis) for 1 h at room temperature. For protein identifica-tion, the streptavidin beads were washed with PBS containing 2%SDS three times followed by 8 M urea three times and finallywith 1 M KCl three times. The beads were then washed with 50mM NH4HCO3 (pH 8.0) and digested overnight at 37 °C with 0.5µg of trypsin. The peptides were collected, and the beads werewashed with buffer (ACN/HOAc/water, 40:1:59, v/v/v). Theeluates were pooled and dried in a Speed-Vac for protein

identification. For Western blotting analysis, avidin monomerbeads were collected by centrifugation and washed with PBScontaining 0.5% NP40 five times. The beads were then boiled in1× SDS sample buffer prior to SDS-PAGE.

Protein Identification by Nano-HPLC-MS/MS. Tryptic peptidesobtained above were cleaned with ZipTip C18 (Millipore, Bedford,MA) prior to nano-HPLC/tandem mass spectrometry analysis.Nano-HPLC/tandem mass spectrometry analysis was performedin an LCQ DECA XP ion trap mass spectrometer (ThermoFinni-gan, San Jose, CA) equipped with a nano-ESI source (Ther-moFinnigan). The electrospray source was coupled online withan Agilent 1100 series nano flow HPLC system (Agilent, Palo Alto,CA). A 2-µL aliquot of the peptide solution in buffer A (2%acetonitrile/ 97.9% water/0.1% acetic acid (v/v/v)) was manuallyloaded into a capillary HPLC column (50 mm length × 75 µmi.d., 5-µm particle size, 300-Å pore diameter) packed in-house withLuna C18 resin (Phenomenex, St. Torrance, CA). The peptideswere eluted from the column with a gradient of 5-80% buffer B(90% acetonitrile/9.9% water/0.1% acetic acid (v/v/v)) in buffer Aover 30 min. The eluted peptides were electrosprayed directly intothe LCQ mass spectrometer. The MS/MS spectra were acquiredin a data-dependent mode that determined the masses of theparent ions and fragments of the three strongest ions.

Protein Sequence Database Search. Tandem mass spectra weresearched against NCBI-nr database with MASCOT search engine(Matrix Science, London, U.K.). Enzyme was specified as trypsinwith one or two missing cleavages. Mass error for parent ion masswas set as (4 Da and for fragment ion as (0.5 Da. Spectra with+1, +2, and +3 charge states were considered. If more than onespectrum were assigned to one peptide, each spectrum was givena Mascot score and only the spectrum with the highest score wasused for fragmentation analysis. Peptides identified with a Mascotscore higher than 30 were considered as potential positive

(5) Comer, F. I.; Vosseller, K.; Wells, L.; Accavitti, M. A.; Hart, G. W. Anal.Biochem. 2001, 293, 169-177.

Figure 1. Schematic representation of the TAS technology. (A) Metabolic incorporation of O-GlcNAc into proteins. PeracetylatedN-(2-azidoacetyl)glucosamine 1 is converted in cells to UDP-azido-GlcNAc 2, which is used by O-GlcNAc transferase for O-GlcNAc modificationof target proteins. Protein is represented by the ribbon structure. (B) Conjugation reaction between azido-GlcNAc-modified protein and biotinylatedphosphine capture reagent 3 for subsequent detection and isolation.

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identifications, and each of them was manually verified by themethod specified as following.

Manual Verification of Protein Identification. Strict manualanalysis was applied to validate protein identification results usinga procedure previously described.6 The following criteria wereused for manual verification. y, b, and a ions as well as their waterloss or amine loss peaks are considered. All the major isotope-resolved peaks should match fragment masses of the identifiedpeptide. The isotope-resolved peaks were emphasized because asingle peak could come from an electronic spark and are less likelyto be relevant to peptide fragments. The major isotope-resolvedpeaks are defined as (1) those isotope-resolved daughter ions withm/z higher than parent m/z and intensity higher than 5% of themaximum intensity or (2) those isotopically resolved peaks withintensities higher than 20% of the maximum intensity and m/zvalues between one-third of the parent m/z and the parent m/z.Typically >7 isotope-resolved peaks were matched to theoreticalmasses of the peptide fragments.

Identified proteins were functionally classified based on theirannotation in the NCBI protein database (www.ncbi.nlm.nih.gov)and NIAIDs DAVID.7,8

Verification of Azido-GlcNAc Modification. Cells were harvestedin PBS containing 0.5% NP40. The cell lysate was centrifuged at20000g for 1 h at 4 °C, and the supernatant was carefully removed.For immunoprecipitation, 5 mg of whole cell lysate was mixedwith 4 µg of specific antibody and 20 µL of protein A/G agarosebeads for 2 h at room temperature. The beads were collected bycentrifugation and washed with PBS containing 0.5% NP40 fivetimes. The proteins were eluted by boiling for 5 min in 2% SDScontaining PBS. The supernatant was removed and then mixedwith biotinylated capture reagent 3 (final concentration ∼200 µM).The conjugation reaction was carried out at room temperaturefor 12 h. The unconjugated capture reagent was removed using aBio-gel P6 DG desalting column. SDS sample buffer was addedto the eluted biotin-conjugated protein sample and analyzed byWestern blot using streptavidin-HRP. Alternatively, protein O-GlcNAc modification was confirmed by avidin/Western blottinganalysis. In this experiment, the azido-O-GlcNAc-modified proteinswere conjugated using the same method as described above andisolated by avidin-conjugated agarose beads. The isolated proteinswere eluted by boiling in SDS sample buffer and analyzed byWestern blotting analysis using an antibody of interest.

RESULTSSelective Metabolic Labeling of O-GlcNAc-Modified Pro-

teins. The effective proteomic analysis of proteins bearing specificposttranslational modifications requires selective enrichment ofthe proteins of interest from a complex protein mixture. Withrespect to the O-GlcNAc modification, the TAS strategy involvesmetabolic labeling of cells with an azide-derivatized analogue ofperacetylated N-acetylglucosamine 1 (Figure 1). This compoundis modified by the cell’s endogenous metabolic machinery intoUDP-azido-GlcNAc 2, which is appended onto proteins in place

of the O-GlcNAc modification. Incorporation of the azide-taggedanalogue of O-GlcNAc onto proteins would then allow the selectiveisolation, detection, and characterization of O-GlcNAc-modifiedproteins via the enhanced Staudinger ligation between the azideand a phosphine probe 3 engineered with an affinity tag, such asbiotin.9 In this study, we applied the previously characterized TAStechnology toward the proteomic analysis of O-GlcNAc-modifiedproteins in HeLa cells. This strategy takes advantage of thepromiscuity of metabolic enzymes in tolerating small perturbationsin the structure of the modification substrate.9-11

Detection of Azido-GlcNAc Modification in Nucleocyto-plasmic Proteins. Metabolic incorporation of an azide-taggedGlcNAc molecule onto O-GlcNAc-modified proteins would allowthe efficient detection, isolation, and characterization of O-GlcNAc-modified proteins. To demonstrate our ability to label and detectO-GlcNAc-modified proteins, HeLa cells were grown to 70%confluency and labeled for 24 h with either peracetylated GlcNAcor peracetylated azido-GlcNAc. Nucleocytoplasmic proteins wereextracted from the labeled cells and conjugated with biotinylatedphosphine capture reagent 3. As shown in Figure 2, O-GlcNAc-modified proteins can be detected in samples prepared from azido-GlcNAc-labeled HeLa cells, only after conjugation with 3 (lane 3,Figure 2). The Western blotting signal can be competitivelyinhibited by performing the capture reaction in the presence of0.1 mM concentration of an exogenous azide-containing substrate,azido-farnesyl diphosphate (FPP-N3) (lane 2). The signal can alsobe competitively inhibited by probing the nitrocellulose membranein the presence of 0.1 mM D-biotin (lanes 4-6). In addition, there

(6) Chen, Y.; Kwon, S. W.; Kim, S. C.; Zhao, Y. J. Proteome Res. 2005, 4, 998-1005.

(7) Dennis, G., Jr.; Sherman, B. T.; Hosack, D. A.; Yang, J.; Gao, W.; Lane, H.C.; Lempicki, R. A. Genome Biol. 2003, 4, P3.

(8) Hosack, D. A.; Dennis, G., Jr.; Sherman, B. T.; Lane, H. C.; Lempicki, R. A.Genome Biol. 2003, 4, R70.

(9) Saxon, E.; Luchansky, S. J.; Hang, H. C.; Yu, C.; Lee, S. C.; Bertozzi, C. R.J. Am. Chem. Soc. 2002, 124, 14893-14902.

(10) Kho, Y.; Kim, S. C.; Jiang, C.; Barma, D.; Kwon, S. W.; Cheng, J.; Jaunbergs,J.; Weinbaum, C.; Tamanoi, F.; Falck, J.; Zhao, Y. Proc. Natl. Acad. Sci. U.S.A.2004, 101, 12479-12484.

(11) Sprung, R.; Nandi, A.; Chen, Y.; Kim, S. C.; Barma, D.; Falck, J. R.; Zhao, Y.J. Proteome Res. 2005, 4, 950-957.

Figure 2. Western blot demonstrating specificity of detection ofazido-GlcNAc-labeled proteins. HeLa nucleocytoplasmic protein fromcells treated with peracetylated GlcNAc or peracetylated azido GlcNAcwas harvested and conjugated with 50 µM compound 3 in thepresence (lanes 2, 5, 8, 11) or absence (lanes 3, 6, 9, 12) ofexogenous azide-containing substrate (FPP-N3). Lanes 1, 4, 7, and10 omit the conjugation step. Western blot membranes were probedwith streptavidin-HRP in the presence (lanes 4-6, 10-12) orabsence (lanes 1-3, 7-9) of exogenous D-biotin. Twenty microgramsof protein was loaded in each lane.

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is little cross-reactivity for proteins from those cells labeled withperacetylated O-GlcNAc (lanes 7-12). These results demonstratethat the endogenous cellular machinery can incorporate azido-GlcNAc into proteins, which can then be selectively conjugatedwith biotinylated phosphine capture reagent. The resulting bioti-nylated, azido-O-GlcNAc-modified proteins can be specificallydetected using streptavidin-HRP.

Isolation and Identification of O-GlcNAc-Modified Pro-teins. The selective enrichment and identification of proteinsbearing the O-GlcNAc modification can give insight into theregulatory roles of the modification. Having shown our ability toselectively detect azido-GlcNAc-modified proteins, we wanted toapply the TAS strategy toward the proteomic profiling of azido-GlcNAc-modified proteins using an affinity purification strategyand subsequent protein identification. Nucleocytoplasmic proteinsof HeLa cells labeled with peracetylated GlcNAc or peracetylatedazido-GlcNAc were isolated. The proteins were conjugated withbiotinylated phosphine capture reagent 3. The biotinylated, azido-

GlcNAc-modified proteins were then selectively affinity-purifiedusing streptavidin beads. The strong interaction between biotinand streptavidin permitted the use of harsh washing conditionsto facilitate removal of any nonspecifically bound proteins. Theremaining proteins bearing the azido-GlcNAc modification wereidentified by nano-HPLC-MS/MS analysis of tryptic digests fromthe affinity-purified samples. A representative chromatogram andmass fingerprint spectrum are presented in Figure 3. In a parallelexperiment, we also labeled the cells with peracetylated GlcNAcand performed the conjugation reaction and affinity purificationusing the same conditions as described above in order to identifypotential contaminant proteins.

Subtractive analysis of proteins identified from peracetylatedazido-GlcNAc- and peracetylated GlcNAc-treated cells led toidentification of 199 azido-GlcNAc-modified proteins (Figure 4 andSupporting Information). Significantly, 21 of the identified proteinswere previously reported to be O-GlcNAc-modified.12

Figure 3. HPLC-MS/MS analysis of tryptic peptides from affinity-purified azido-O-GlcNAc-modified HeLa cell proteins. (A) Total ion current(TIC) chromatogram of a capillary-HPLC-MS/MS of the tryptic digests. (B) MS spectrum at retention time of 25.95 min and (C) MS/MS spectrumof peptide AHSSMVGVNLPQK, unique to phosphoglycerate kinase. The analysis led to the identification of 199 proteins.

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Summary of Identified Azido-GlcNAc-Modified Proteins.Identified proteins were functionally classified based on theirannotation in the NCBI protein database (www.ncbi.nlm.nih.gov)and NIAIDs DAVID.7,8

The O-GlcNAc modification has the potential to be a regulatorymechanism connected to energy availability due to its position inthe hexosamine biosynthetic pathway.13 It is estimated that 25%of the energy generated by a cell is used for protein synthesisand processing.14 Thus, one might expect these processes to betightly regulated by energy availability. Significantly, more thana quarter of the proteins identified in this study are annotated asbeing involved in protein synthesis and processing, representingthe largest functional group among the identified proteins.Numerous factors involved in transcription and RNA processingwere also identified. The prevalence of O-GlcNAc on these proteinsprovides a mechanism through which energy availability may becoupled directly to protein metabolism and gene expressionpatterns.

The next largest functional group is cellular housekeeping andmaintenance proteins. This group is composed of structural andsignal transduction proteins involved in the regulation of cellulargrowth, motility, and division. Fifteen proteins among the variouscategories were annotated as being involved in the regulation ofthe cell cycle. Thirty proteins were identified with signal trans-duction activity. These include mediators of G-protein signaling,the MAP kinase pathway, tyrosine kinases, IkB/NFkB signaling,calmodulin-binding signal transducers, and components of thecaspase cascade as well as Wnt and Jak/Stat signaling proteins.These processes may be regulated in part by O-GlcNAc modifica-tions.

Proteins involved in intracellular transport were also wellrepresented among identified proteins. This group includesnuclear transport proteins as well as proteins associated with thetransport of membrane-bound vesicles. Such trafficking is essentialfor many metabolic and cell housekeeping functions and isregulated through various cell-signaling cascades. The O-GlcNAcmodification may influence trafficking by mediating protein-protein interactions or through modulation of the signalingcascades leading to the transport process.

O-GlcNAc was also found on many enzymes involved in aminoacid, carbohydrate, lipid, cofactor, and nucleotide metabolism.Such enzymes mark a primary interface between energy genera-tion and energy use and represent ideal targets for a regulatorymodification associated with energy status.

Five proteins involved in the cellular response to DNA damagewere identified, suggesting that the O-GlcNAc modification mayplay a role in mediating cell survival pathways. It is already knownthat O-GlcNAcase is a target for caspase cleavage. Although thein vivo consequences of this cleavage are unknown, it does notaffect the catalytic activities of the enzyme in vitro.15 Cleavagemay impact the enzyme’s regulation or substrate recognition.

The O-GlcNAc modification has been implicated as a regulatorymodification related to the stress response in cells.12 In additionto the DNA-damage responsive proteins identified, 16 proteinswere annotated as playing a role in redox homeostasis. A numberof chaperone proteins important in the unfolded protein responsewere also identified. These results are in agreement with thehypothesis that the O-GlcNAc modification is involved in theregulation of stress-induced pathways.

An additional 17 proteins were identified for which a functionalannotation was not available.

The identification of 21 previously known O-GlcNAc-modifiedproteins represents almost 20% coverage of previously reportedO-GlcNAc-modified proteins. This serves as a good positive controland helps confirm that the TAS technology can be successfullyapplied to studies of the O-GlcNAc modification. We are unawareof any single study to yield identification of so many previouslyreported O-GlcNAc-modified proteins. In addition, we have nearlytripled the list of putative O-GlcNAc-modified proteins, extendingthe list to >200 (Figure 4 and Supporting Information).

Verification of the O-GlcNAc Modification in Proteins. Tofurther validate the presence of the azido-GlcNAc modification inthe identified proteins, we used reciprocal immunoprecipitationto confirm some previously known and putative O-GlcNAc-modified proteins from the list of identified proteins.

For those proteins whose only antibodies for Western blottinganalysis (but not immunoprecipitation antibody) are available,nucleocytoplasmic proteins were isolated from cells labeled withperacetylated GlcNAc (lanes 1 and 2, Figure 5) or peracetylated

(12) Zachara, N. E.; Hart, G. W. Biochim. Biophys. Acta 2004, 1673, 13-28.(13) Wells, L.; Vosseller, K.; Hart, G. W. Cell. Mol. Life Sci. 2003, 60, 222-228.(14) Inoki, K.; Zhu, T.; Guan, K. L. Cell 2003, 115, 577-590.

(15) Wells, L.; Gao, Y.; Mahoney, J. A.; Vosseller, K.; Chen, C.; Rosen, A.; Hart,G. W. J. Biol. Chem. 2002, 277, 1755-1761.

Figure 4. Functional classifications of proteins. Pie chart showing functional categories of the 199 identified proteins.

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azido-GlcNAc (lanes 3 and 4, Figure 5) and conjugated withbiotinylated phosphine capture reagent 3. Biotinylated, azido-GlcNAc proteins were affinity purified using avidin (monomer)agarose and resolved by SDS-PAGE. After transfer to a nitro-cellulose membrane, proteins were probed with specific antibodiesto verify their presence among the azido-GlcNAc-modified pro-teins.

For those proteins where immunoprecipitating antibodies wereavailable, the protein of interest was isolated from cells labeledwith peracetylated GlcNAc or peracetylated azido-GlcNAc byimmunoprecipitation. The immunoprecipitated samples wereconjugated with biotinylated phosphine capture reagent 3 and theresulting conjugate was subjected to Western blot analysis usingstreptavidin-HRP.

This analysis confirmed O-GlcNAc modification of all of the23 proteins assayed including 10 known and 13 newly identifiedO-GlcNAc-modified proteins (Figure 5). This result offers furtherevidence that the TAS technology can be applied to the accurate,selective identification of O-GlcNAc-modified proteins.

DISCUSSION

The proteomic analysis of all proteins expressed in a cell ischallenging due to the large number and wide dynamic range ofproteins expressed at any given time. There are over 200 knownposttranslational modifications of proteins, which add a layer ofcomplexity to the proteome with important functional conse-

quences.16 Unfortunately, there are few methods currently avail-able that are able to detect and enrich samples for proteins bearingspecific modifications. The strategy presented here attempts toaddress this problem by providing a global approach to thedetection and proteomic analysis of O-GlcNAc-modified proteinsin HeLa cells under steady-state resting conditions. Identificationof novel O-GlcNAc-modified proteins will help elucidate thefunctional consequences of the modification.

We believe that the TAS technology represents a usefulstrategy for the detection, enrichment, and identification ofproteins bearing specific posttranslational modifications. Forexample, no enzymatic purification, mutation, or overexpressionis necessary using TAS. Also, the TAS strategy is not contingenton the presence of a specific sequence motif. Rather, the cell’sendogenous enzymatic machinery is utilized in vivo. The chem-istry used in the TAS strategy is mild; the reaction conditions donot perturb the peptide backbone structure. The chemistry is alsohighly selective; there is no detectable cross reactivity withunmodified proteins (lanes 7-12, Figure 2). One reason for thisselectivity is that neither phosphines nor azides occur in anyknown biomolecules, making them truly bioorthogonal. As aresult, the TAS strategy does not directly affect posttranslationalmodifications beyond the modification of interest. The resultingcovalent linkage forms specifically between the phosphine andazide. This opens up the possibility of tandem analysis of proteinglycosylation and phosphorylation on a single sample. Additionally,TAS can be employed in the detection of dynamic changes inpatterns of modification in response to a stimulus. This advantagestems from the fact that only those proteins posttranslationallymodified after introduction and conversion of the substrateanalogue will be detected, isolated, and identified. Thus, stimula-tion of a signaling pathway after the metabolic labeling step wouldallow for resolution between those proteins that are constitutivelymodified and those that are specifically modified in response tothe stimulus. Such resolution is difficult to accomplish using otherproteomic strategies.

TAS does suffer from some disadvantages. The methodrequires metabolic labeling in cell culture and is not amenable toanalysis of unlabeled tissue samples. In addition, it is notinherently quantitative and not all posttranslational modificationswill be accessible using this strategy.

In this study, we first demonstrated that peracetylated azido-GlcNAc could be used for the selective detection, enrichment,and identification of O-GlcNAc-modified proteins, in agreementwith previous observations.9,17 Affinity purification, tryptic diges-tion, and LC-MS/MS analysis of samples led to the identificationof 199 O-GlcNAc-modified proteins, including 21 previouslyreported to be O-GlcNAc-modified, which helps to validate theauthenticity of our identifications. Two additional methods, avidinaffinity purification/Western blotting analysis and reciprocalimmunoprecipitation/Western blotting analysis, were employedto further validate the identifications. The identified proteinsconfirm some of the previously known O-GlcNAc modificationswhile extending the list to >200 proteins. These results representthe most O-GlcNAc-modified proteins identified in a single analysis

(16) Proteins: Analysis and Design; Gudepu, R. G.; Wold, F.; Angeletti, R. H.,Ed.; Academic: San Diego, 1998; pp 121-207.

(17) Vocadlo, D. J.; Hang, H. C.; Kim, E. J.; Hanover, J. A.; Bertozzi, C. R. Proc.Natl. Acad. Sci. U.S.A. 2003, 100, 9116-9121.

Figure 5. Verification of azido-O-GlcNAc modification. Avidin/WB:proteins were conjugated with 3, affinity-purified with avidin-monomeragarose, and detected by Western blot analysis using specificantibody. Reciprocal IP: protein was immunoprecipitated with specificantibody, conjugated with 3, and detected by Western blot analysisusing streptavidin-HRP. aIndicates novel O-GlcNAc-modified proteinsidentified in this study.

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to date. The presence of this modification in proteins representingdiverse regulatory pathways reinforces the idea that the O-GlcNAcmodification plays a significant role in cellular regulation andsurvival.

ACKNOWLEDGMENTY.Z. is supported by The Robert A. Welch Foundation (I-1550)

and NIH (CA 107943). We thank Mark Lehrman for helpfuldiscussions.

SUPPORTING INFORMATION AVAILABLE

Additional information as noted in text. This material isavailable free of charge via the Internet at http://pubs.acs.org.

Received for review July 7, 2005. Accepted November 15,2005.

AC051207J

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