Global Identification of O-GlcNAc-Modified Proteins - Analytical

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Anal. Chem. 2006, 78, 452-458

Global Identification of O-GlcNAc-Modified Proteins Animesh Nandi,† Robert Sprung,† Deb K. Barma,† Yingxin Zhao,† Sung Chan Kim,† John R. Falck,‡ and Yingming Zhao†,‡,*

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

The O-linked N-acetylglucosamine (O-GlcNAc) modification of serine/threonine residues is an abundant posttranslational modification present in cytosolic and nuclear proteins. The functions and subproteome of O-GlcNAc modification remain largely undefined. Here we report the application of the tagging-via-substrate (TAS) approach for global identification of O-GlcNAc-modified proteins. The TAS method utilizes an O-GlcNAc azide analogue for metabolic labeling of O-GlcNAc-modified proteins, which can be chemoselectively conjugated for detection and enrichment of the proteins for proteomics studies. Our study led to the identification of 199 putative O-GlcNAcmodified proteins from HeLa cells, among which 23 were confirmed using reciprocal immunoprecipitation. Functional classification shows that proteins with diverse functions are modified by O-GlcNAc, implying that OGlcNAc might be involved in the regulation of multiple cellular pathways. The modification of nuclear and cytoplasmic proteins at serine and threonine residues with O-linked N-acetylglucosamine (OGlcNAc) was first described over two decades ago.1 The modification was found in various classes of proteins including enzymes, transcription factors, cytoskeletal proteins, signaling proteins, receptors, nuclear pore complex proteins, and kinases.2 Similar to phosphorylation, the O-GlcNAc modification is dynamic with a turnover rate faster than that of the proteins it modifies.3 The O-GlcNAc modification has been shown to affect protein-protein interactions, protein-DNA interactions, protein stability and activity, and cell signaling cascades.4 Disregulation of the OGlcNAc modification has been implicated in the development of disease states including diabetes, cancer, and Alzheimer’s.2,4 Given the potentially broad regulatory influence of the O-GlcNAc modification, a more comprehensive understanding of the targets of O-GlcNAc transferase is needed to elucidate its functional consequences. * Corresponding author. E-mail: [email protected]. Fax: (214) 6482797. 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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In this report, we describe the global detection and proteomic analysis of O-GlcNAc-modified proteins in HeLa cells. An affinitytagged version of the O-GlcNAc modification is metabolically incorporated onto proteins using an azide-tagged analogue of N-acetylglucosamine. The azido-GlcNAc-modified proteins thus contain an azide handle for chemoselective conjugation using a biotinylated phosphine reagent. The resulting conjugates were affinity-purified with streptavidin beads and subsequently digested with trypsin and analyzed by nano-HPLC-MS/MS. Using this strategy, we identified 199 azido-GlcNAc-modified proteins in HeLa cells. We subsequently validated the presence of this modification among 10 previously reported and 13 newly identified O-GlcNAcmodified proteins using specific antibodies. Our results reveal that proteins with a wide range of functions are modified by O-GlcNAc, implying its diverse cellular functions. EXPERIMENTAL PROCEDURES Materials. DMEM and glucose-free DMEM were purchased from Life Technologies (Gaithesburg, MD). Bovine serum albumin, 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 Biotechnology (Rockford, IL). Biotinylated phosphine capture reagent 3 (Figure 1) and peracetylated N-(2-azidoacetyl)glucosamine 1 (Figure 1) were synthesized in-house. Primary antibodies and Protein A/G agarose beads were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Western Lighting plus chemiluminescence detection kit was from Perkin-Elmer Life Sciences (Boston, MA). Bradford protein estimation reagent and Bio-gel P6 DG desalting column were from Bio-Rad (Hercules, CA). Streptavidin HRP was from Amersham (Piscataway, NJ). Protease inhibitor cocktail was from Calbiochem (San Diego, CA). Methods. Cell Culture and Metabolic Labeling. Hela cells were cultured in DMEM (4.5 g of glucose/L) supplemented with 10% FBS and antibiotics at 37 °C with 5% CO2. For labeling, culture medium was replaced at 70% cell confluency with DMEM (1 g of glucose/L) containing 250 µM peracetylated GlcNAc or peracetylated azido-GlcNAc in DMSO. The cells were labeled for 24 h. Three hours before harvest, glucosamine was added to the cultures to a final concentration of 4 mM as an inhibitor of O-GlcNAcase during the harvest procedure. Isolation of Nucleocytoplasmic Proteins. Fifty dishes (15 cm) of labeled cells were harvested by scraping in chilled PBS containing 4 mM glucosamine. The cells were collected by centrifugation at 10.1021/ac051207j CCC: $33.50

© 2006 American Chemical Society Published on Web 12/15/2005

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

1000g. Four milliliters of hypotonic lysis buffer (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, and protease inhibitor cocktail) was added to the pellet and the resultant mixture incubated on ice for 30 min. The sample was Dounce homogenized using a “B”-rated pestle, and NaCl was added drop by drop to a final concentration of 400 mM to lyse nuclei as previously described.5 The sample was ultracentrifuged at 100000g for 1 h at 4 °C. The supernatant (nucleocytosolic fraction) was carefully removed, and the protein was estimated by the Bradford method. Conjugation with Biotinylated Capture Reagent and Affinity Purification. The nucleocytosolic protein extract was precipitated with eight volumes of ice-cold acetone and one volume of TCA for 2 h at -20 °C. The protein pellet was obtained by centrifugation at 20000g, washed with ice-cold acetone, and resuspended in PBS containing 2% SDS at a concentration of 4-8 µg/µL. For conjugation, biotinylated phosphine capture reagent was added to a final concentration of 50 µM. The samples were kept agitating for 1012 h in the dark at room temperature. Unconjugated capture reagent was removed using a Bio-gel P6 DG desalting column. The samples were diluted with PBS to a final SDS concentration of 0.2% and mixed with streptavidin beads (for protein affinity purification and subsequent identification) or avidin monomer beads (for verification of azido-GlcNAc modification by Western blot analysis) for 1 h at room temperature. For protein identification, the streptavidin beads were washed with PBS containing 2% SDS three times followed by 8 M urea three times and finally with 1 M KCl three times. The beads were then washed with 50 mM NH4HCO3 (pH 8.0) and digested overnight at 37 °C with 0.5 µg of trypsin. The peptides were collected, and the beads were washed with buffer (ACN/HOAc/water, 40:1:59, v/v/v). The eluates were pooled and dried in a Speed-Vac for protein (5) Comer, F. I.; Vosseller, K.; Wells, L.; Accavitti, M. A.; Hart, G. W. Anal. Biochem. 2001, 293, 169-177.

identification. For Western blotting analysis, avidin monomer beads were collected by centrifugation and washed with PBS containing 0.5% NP40 five times. The beads were then boiled in 1× SDS sample buffer prior to SDS-PAGE. Protein Identification by Nano-HPLC-MS/MS. Tryptic peptides obtained above were cleaned with ZipTip C18 (Millipore, Bedford, MA) prior to nano-HPLC/tandem mass spectrometry analysis. Nano-HPLC/tandem mass spectrometry analysis was performed in an LCQ DECA XP ion trap mass spectrometer (ThermoFinnigan, San Jose, CA) equipped with a nano-ESI source (ThermoFinnigan). The electrospray source was coupled online with an 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 manually loaded into a capillary HPLC column (50 mm length × 75 µm i.d., 5-µm particle size, 300-Å pore diameter) packed in-house with Luna C18 resin (Phenomenex, St. Torrance, CA). The peptides were 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 A over 30 min. The eluted peptides were electrosprayed directly into the LCQ mass spectrometer. The MS/MS spectra were acquired in a data-dependent mode that determined the masses of the parent ions and fragments of the three strongest ions. Protein Sequence Database Search. Tandem mass spectra were searched against NCBI-nr database with MASCOT search engine (Matrix Science, London, U.K.). Enzyme was specified as trypsin with one or two missing cleavages. Mass error for parent ion mass was 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 one spectrum were assigned to one peptide, each spectrum was given a Mascot score and only the spectrum with the highest score was used for fragmentation analysis. Peptides identified with a Mascot score higher than 30 were considered as potential positive Analytical Chemistry, Vol. 78, No. 2, January 15, 2006

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identifications, and each of them was manually verified by the method specified as following. Manual Verification of Protein Identification. Strict manual analysis was applied to validate protein identification results using a procedure previously described.6 The following criteria were used for manual verification. y, b, and a ions as well as their water loss or amine loss peaks are considered. All the major isotoperesolved peaks should match fragment masses of the identified peptide. The isotope-resolved peaks were emphasized because a single peak could come from an electronic spark and are less likely to be relevant to peptide fragments. The major isotope-resolved peaks are defined as (1) those isotope-resolved daughter ions with m/z higher than parent m/z and intensity higher than 5% of the maximum intensity or (2) those isotopically resolved peaks with intensities higher than 20% of the maximum intensity and m/z values between one-third of the parent m/z and the parent m/z. Typically >7 isotope-resolved peaks were matched to theoretical masses of the peptide fragments. Identified proteins were functionally classified based on their annotation in the NCBI protein database (www.ncbi.nlm.nih.gov) and NIAIDs DAVID.7,8 Verification of Azido-GlcNAc Modification. Cells were harvested in PBS containing 0.5% NP40. The cell lysate was centrifuged at 20000g for 1 h at 4 °C, and the supernatant was carefully removed. For immunoprecipitation, 5 mg of whole cell lysate was mixed with 4 µg of specific antibody and 20 µL of protein A/G agarose beads for 2 h at room temperature. The beads were collected by centrifugation and washed with PBS containing 0.5% NP40 five times. The proteins were eluted by boiling for 5 min in 2% SDS containing PBS. The supernatant was removed and then mixed with biotinylated capture reagent 3 (final concentration ∼200 µM). The conjugation reaction was carried out at room temperature for 12 h. The unconjugated capture reagent was removed using a Bio-gel P6 DG desalting column. SDS sample buffer was added to the eluted biotin-conjugated protein sample and analyzed by Western blot using streptavidin-HRP. Alternatively, protein OGlcNAc modification was confirmed by avidin/Western blotting analysis. In this experiment, the azido-O-GlcNAc-modified proteins were conjugated using the same method as described above and isolated by avidin-conjugated agarose beads. The isolated proteins were eluted by boiling in SDS sample buffer and analyzed by Western blotting analysis using an antibody of interest. RESULTS Selective Metabolic Labeling of O-GlcNAc-Modified Proteins. The effective proteomic analysis of proteins bearing specific posttranslational modifications requires selective enrichment of the proteins of interest from a complex protein mixture. With respect to the O-GlcNAc modification, the TAS strategy involves metabolic labeling of cells with an azide-derivatized analogue of peracetylated N-acetylglucosamine 1 (Figure 1). This compound is modified by the cell’s endogenous metabolic machinery into UDP-azido-GlcNAc 2, which is appended onto proteins in place (6) Chen, Y.; Kwon, S. W.; Kim, S. C.; Zhao, Y. J. Proteome Res. 2005, 4, 9981005. (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.

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

of the O-GlcNAc modification. Incorporation of the azide-tagged analogue of O-GlcNAc onto proteins would then allow the selective isolation, detection, and characterization of O-GlcNAc-modified proteins via the enhanced Staudinger ligation between the azide and a phosphine probe 3 engineered with an affinity tag, such as biotin.9 In this study, we applied the previously characterized TAS technology toward the proteomic analysis of O-GlcNAc-modified proteins in HeLa cells. This strategy takes advantage of the promiscuity of metabolic enzymes in tolerating small perturbations in the structure of the modification substrate.9-11 Detection of Azido-GlcNAc Modification in Nucleocytoplasmic Proteins. Metabolic incorporation of an azide-tagged GlcNAc molecule onto O-GlcNAc-modified proteins would allow the efficient detection, isolation, and characterization of O-GlcNAcmodified proteins. To demonstrate our ability to label and detect O-GlcNAc-modified proteins, HeLa cells were grown to 70% confluency and labeled for 24 h with either peracetylated GlcNAc or peracetylated azido-GlcNAc. Nucleocytoplasmic proteins were extracted from the labeled cells and conjugated with biotinylated phosphine capture reagent 3. As shown in Figure 2, O-GlcNAcmodified proteins can be detected in samples prepared from azidoGlcNAc-labeled HeLa cells, only after conjugation with 3 (lane 3, Figure 2). The Western blotting signal can be competitively inhibited by performing the capture reaction in the presence of 0.1 mM concentration of an exogenous azide-containing substrate, azido-farnesyl diphosphate (FPP-N3) (lane 2). The signal can also be competitively inhibited by probing the nitrocellulose membrane in the presence of 0.1 mM D-biotin (lanes 4-6). In addition, there (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 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 spectrum of peptide AHSSMVGVNLPQK, unique to phosphoglycerate kinase. The analysis led to the identification of 199 proteins.

is little cross-reactivity for proteins from those cells labeled with peracetylated O-GlcNAc (lanes 7-12). These results demonstrate that the endogenous cellular machinery can incorporate azidoGlcNAc into proteins, which can then be selectively conjugated with biotinylated phosphine capture reagent. The resulting biotinylated, azido-O-GlcNAc-modified proteins can be specifically detected using streptavidin-HRP. Isolation and Identification of O-GlcNAc-Modified Proteins. The selective enrichment and identification of proteins bearing the O-GlcNAc modification can give insight into the regulatory roles of the modification. Having shown our ability to selectively detect azido-GlcNAc-modified proteins, we wanted to apply the TAS strategy toward the proteomic profiling of azidoGlcNAc-modified proteins using an affinity purification strategy and subsequent protein identification. Nucleocytoplasmic proteins of HeLa cells labeled with peracetylated GlcNAc or peracetylated azido-GlcNAc were isolated. The proteins were conjugated with biotinylated phosphine capture reagent 3. The biotinylated, azido-

GlcNAc-modified proteins were then selectively affinity-purified using streptavidin beads. The strong interaction between biotin and streptavidin permitted the use of harsh washing conditions to facilitate removal of any nonspecifically bound proteins. The remaining proteins bearing the azido-GlcNAc modification were identified by nano-HPLC-MS/MS analysis of tryptic digests from the affinity-purified samples. A representative chromatogram and mass fingerprint spectrum are presented in Figure 3. In a parallel experiment, we also labeled the cells with peracetylated GlcNAc and performed the conjugation reaction and affinity purification using the same conditions as described above in order to identify potential contaminant proteins. Subtractive analysis of proteins identified from peracetylated azido-GlcNAc- and peracetylated GlcNAc-treated cells led to identification of 199 azido-GlcNAc-modified proteins (Figure 4 and Supporting Information). Significantly, 21 of the identified proteins were previously reported to be O-GlcNAc-modified.12 Analytical Chemistry, Vol. 78, No. 2, January 15, 2006

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

Summary of Identified Azido-GlcNAc-Modified Proteins. Identified proteins were functionally classified based on their annotation 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 regulatory mechanism connected to energy availability due to its position in the hexosamine biosynthetic pathway.13 It is estimated that 25% of the energy generated by a cell is used for protein synthesis and processing.14 Thus, one might expect these processes to be tightly regulated by energy availability. Significantly, more than a quarter of the proteins identified in this study are annotated as being involved in protein synthesis and processing, representing the largest functional group among the identified proteins. Numerous factors involved in transcription and RNA processing were also identified. The prevalence of O-GlcNAc on these proteins provides a mechanism through which energy availability may be coupled directly to protein metabolism and gene expression patterns. The next largest functional group is cellular housekeeping and maintenance proteins. This group is composed of structural and signal transduction proteins involved in the regulation of cellular growth, motility, and division. Fifteen proteins among the various categories were annotated as being involved in the regulation of the cell cycle. Thirty proteins were identified with signal transduction activity. These include mediators of G-protein signaling, the MAP kinase pathway, tyrosine kinases, IkB/NFkB signaling, calmodulin-binding signal transducers, and components of the caspase cascade as well as Wnt and Jak/Stat signaling proteins. These processes may be regulated in part by O-GlcNAc modifications. Proteins involved in intracellular transport were also well represented among identified proteins. This group includes nuclear transport proteins as well as proteins associated with the transport of membrane-bound vesicles. Such trafficking is essential for many metabolic and cell housekeeping functions and is regulated through various cell-signaling cascades. The O-GlcNAc modification may influence trafficking by mediating proteinprotein interactions or through modulation of the signaling cascades leading to the transport process. (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.

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O-GlcNAc was also found on many enzymes involved in amino acid, carbohydrate, lipid, cofactor, and nucleotide metabolism. Such enzymes mark a primary interface between energy generation and energy use and represent ideal targets for a regulatory modification associated with energy status. Five proteins involved in the cellular response to DNA damage were identified, suggesting that the O-GlcNAc modification may play a role in mediating cell survival pathways. It is already known that O-GlcNAcase is a target for caspase cleavage. Although the in vivo consequences of this cleavage are unknown, it does not affect the catalytic activities of the enzyme in vitro.15 Cleavage may impact the enzyme’s regulation or substrate recognition. The O-GlcNAc modification has been implicated as a regulatory modification related to the stress response in cells.12 In addition to the DNA-damage responsive proteins identified, 16 proteins were annotated as playing a role in redox homeostasis. A number of chaperone proteins important in the unfolded protein response were also identified. These results are in agreement with the hypothesis that the O-GlcNAc modification is involved in the regulation of stress-induced pathways. An additional 17 proteins were identified for which a functional annotation was not available. The identification of 21 previously known O-GlcNAc-modified proteins represents almost 20% coverage of previously reported O-GlcNAc-modified proteins. This serves as a good positive control and helps confirm that the TAS technology can be successfully applied to studies of the O-GlcNAc modification. We are unaware of any single study to yield identification of so many previously reported O-GlcNAc-modified proteins. In addition, we have nearly tripled the list of putative O-GlcNAc-modified proteins, extending the list to >200 (Figure 4 and Supporting Information). Verification of the O-GlcNAc Modification in Proteins. To further validate the presence of the azido-GlcNAc modification in the identified proteins, we used reciprocal immunoprecipitation to confirm some previously known and putative O-GlcNAcmodified proteins from the list of identified proteins. For those proteins whose only antibodies for Western blotting analysis (but not immunoprecipitation antibody) are available, nucleocytoplasmic proteins were isolated from cells labeled with peracetylated GlcNAc (lanes 1 and 2, Figure 5) or peracetylated (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 5. Verification of azido-O-GlcNAc modification. Avidin/WB: proteins were conjugated with 3, affinity-purified with avidin-monomer agarose, and detected by Western blot analysis using specific antibody. Reciprocal IP: protein was immunoprecipitated with specific antibody, conjugated with 3, and detected by Western blot analysis using streptavidin-HRP. aIndicates novel O-GlcNAc-modified proteins identified in this study.

azido-GlcNAc (lanes 3 and 4, Figure 5) and conjugated with biotinylated phosphine capture reagent 3. Biotinylated, azidoGlcNAc proteins were affinity purified using avidin (monomer) agarose and resolved by SDS-PAGE. After transfer to a nitrocellulose membrane, proteins were probed with specific antibodies to verify their presence among the azido-GlcNAc-modified proteins. For those proteins where immunoprecipitating antibodies were available, the protein of interest was isolated from cells labeled with peracetylated GlcNAc or peracetylated azido-GlcNAc by immunoprecipitation. The immunoprecipitated samples were conjugated with biotinylated phosphine capture reagent 3 and the resulting conjugate was subjected to Western blot analysis using streptavidin-HRP. This analysis confirmed O-GlcNAc modification of all of the 23 proteins assayed including 10 known and 13 newly identified O-GlcNAc-modified proteins (Figure 5). This result offers further evidence 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 is challenging due to the large number and wide dynamic range of proteins expressed at any given time. There are over 200 known posttranslational modifications of proteins, which add a layer of complexity to the proteome with important functional conse-

quences.16 Unfortunately, there are few methods currently available that are able to detect and enrich samples for proteins bearing specific modifications. The strategy presented here attempts to address this problem by providing a global approach to the detection and proteomic analysis of O-GlcNAc-modified proteins in HeLa cells under steady-state resting conditions. Identification of novel O-GlcNAc-modified proteins will help elucidate the functional consequences of the modification. We believe that the TAS technology represents a useful strategy for the detection, enrichment, and identification of proteins bearing specific posttranslational modifications. For example, no enzymatic purification, mutation, or overexpression is necessary using TAS. Also, the TAS strategy is not contingent on the presence of a specific sequence motif. Rather, the cell’s endogenous enzymatic machinery is utilized in vivo. The chemistry used in the TAS strategy is mild; the reaction conditions do not perturb the peptide backbone structure. The chemistry is also highly selective; there is no detectable cross reactivity with unmodified proteins (lanes 7-12, Figure 2). One reason for this selectivity is that neither phosphines nor azides occur in any known biomolecules, making them truly bioorthogonal. As a result, the TAS strategy does not directly affect posttranslational modifications beyond the modification of interest. The resulting covalent linkage forms specifically between the phosphine and azide. This opens up the possibility of tandem analysis of protein glycosylation and phosphorylation on a single sample. Additionally, TAS can be employed in the detection of dynamic changes in patterns of modification in response to a stimulus. This advantage stems from the fact that only those proteins posttranslationally modified after introduction and conversion of the substrate analogue will be detected, isolated, and identified. Thus, stimulation of a signaling pathway after the metabolic labeling step would allow for resolution between those proteins that are constitutively modified and those that are specifically modified in response to the stimulus. Such resolution is difficult to accomplish using other proteomic strategies. TAS does suffer from some disadvantages. The method requires metabolic labeling in cell culture and is not amenable to analysis of unlabeled tissue samples. In addition, it is not inherently quantitative and not all posttranslational modifications will be accessible using this strategy. In this study, we first demonstrated that peracetylated azidoGlcNAc could be used for the selective detection, enrichment, and identification of O-GlcNAc-modified proteins, in agreement with previous observations.9,17 Affinity purification, tryptic digestion, and LC-MS/MS analysis of samples led to the identification of 199 O-GlcNAc-modified proteins, including 21 previously reported to be O-GlcNAc-modified, which helps to validate the authenticity of our identifications. Two additional methods, avidin affinity purification/Western blotting analysis and reciprocal immunoprecipitation/Western blotting analysis, were employed to further validate the identifications. The identified proteins confirm some of the previously known O-GlcNAc modifications while extending the list to >200 proteins. These results represent the 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.

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to date. The presence of this modification in proteins representing diverse regulatory pathways reinforces the idea that the O-GlcNAc modification plays a significant role in cellular regulation and survival. ACKNOWLEDGMENT Y.Z. is supported by The Robert A. Welch Foundation (I-1550) and NIH (CA 107943). We thank Mark Lehrman for helpful discussions.

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SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review July 7, 2005. Accepted November 15, 2005. AC051207J