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Identification of O-Linked N-Acetylglucosamine (O-GlcNAc)-modified Osteoblast Proteins by Electron Transfer Dissociation Tandem Mass Spectrometry Reveals Proteins Critical for Bone Formation

2013; Elsevier BV; Volume: 12; Issue: 4 Linguagem: Inglês

10.1074/mcp.m112.026633

1535-9484

Alexis K. Nagel, Michael Schilling, Susana Comte‐Walters, Mary N. Berkaw, Lauren E. Ball,

Carbohydrate Chemistry and Synthesis

The nutrient-responsive β-O-linked N-acetylglucosamine (O-GlcNAc) modification of critical effector proteins modulates signaling and transcriptional pathways contributing to cellular development and survival. An elevation in global protein O-GlcNAc modification occurs during the early stages of osteoblast differentiation and correlates with enhanced transcriptional activity of RUNX2, a key regulator of osteogenesis. To identify other substrates of O-GlcNAc transferase in differentiating MC3T3E1 osteoblasts, O-GlcNAc-modified peptides were enriched by wheat germ agglutinin lectin weak affinity chromatography and identified by tandem mass spectrometry using electron transfer dissociation. This peptide fragmentation approach leaves the labile O-linkage intact permitting direct identification of O-GlcNAc-modified peptides. O-GlcNAc modification was observed on enzymes involved in post-translational regulation, including MAST4 and WNK1 kinases, a ubiquitin-associated protein (UBAP2l), and the histone acetyltransferase CREB-binding protein. CREB-binding protein, a transcriptional co-activator that associates with CREB and RUNX2, is O-GlcNAcylated at Ser-147 and Ser-2360, the latter of which is a known site of phosphorylation. Additionally, O-GlcNAcylation of components of the TGFβ-activated kinase 1 (TAK1) signaling complex, TAB1 and TAB2, occurred in close proximity to known sites of Ser/Thr phosphorylation and a putative nuclear localization sequence within TAB2. These findings demonstrate the presence of O-GlcNAc modification on proteins critical to bone formation, remodeling, and fracture healing and will enable evaluation of this modification on protein function and regulation. The nutrient-responsive β-O-linked N-acetylglucosamine (O-GlcNAc) modification of critical effector proteins modulates signaling and transcriptional pathways contributing to cellular development and survival. An elevation in global protein O-GlcNAc modification occurs during the early stages of osteoblast differentiation and correlates with enhanced transcriptional activity of RUNX2, a key regulator of osteogenesis. To identify other substrates of O-GlcNAc transferase in differentiating MC3T3E1 osteoblasts, O-GlcNAc-modified peptides were enriched by wheat germ agglutinin lectin weak affinity chromatography and identified by tandem mass spectrometry using electron transfer dissociation. This peptide fragmentation approach leaves the labile O-linkage intact permitting direct identification of O-GlcNAc-modified peptides. O-GlcNAc modification was observed on enzymes involved in post-translational regulation, including MAST4 and WNK1 kinases, a ubiquitin-associated protein (UBAP2l), and the histone acetyltransferase CREB-binding protein. CREB-binding protein, a transcriptional co-activator that associates with CREB and RUNX2, is O-GlcNAcylated at Ser-147 and Ser-2360, the latter of which is a known site of phosphorylation. Additionally, O-GlcNAcylation of components of the TGFβ-activated kinase 1 (TAK1) signaling complex, TAB1 and TAB2, occurred in close proximity to known sites of Ser/Thr phosphorylation and a putative nuclear localization sequence within TAB2. These findings demonstrate the presence of O-GlcNAc modification on proteins critical to bone formation, remodeling, and fracture healing and will enable evaluation of this modification on protein function and regulation. Advances in proteomic approaches are revealing the abundance of β-O-linked N-acetylglucosamine (O-GlcNAc) 1The abbreviations used are:O-GlcNAcO-linked N-acetylglucosamineLWAClectin weak affinity chromatographyETDelectron transfer dissociationCBPCREB-binding proteinRUNX2runt-related transcription factor 2OGTO-GlcNAc transferaseOGAO-GlcNAcaseWGAwheat germ agglutininALPalkaline phosphataseOCNosteocalcinPTHparathyroid hormoneIGF-1insulin-like growth factor 1BMPbone morphogenetic proteinCREBcAMP-response element-binding proteinHCDhigher energy collision-induced dissociationPUGNAcO-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino N-phenyl carbamateCTDC-terminal domainNHOstnormal human osteoblast. 1The abbreviations used are:O-GlcNAcO-linked N-acetylglucosamineLWAClectin weak affinity chromatographyETDelectron transfer dissociationCBPCREB-binding proteinRUNX2runt-related transcription factor 2OGTO-GlcNAc transferaseOGAO-GlcNAcaseWGAwheat germ agglutininALPalkaline phosphataseOCNosteocalcinPTHparathyroid hormoneIGF-1insulin-like growth factor 1BMPbone morphogenetic proteinCREBcAMP-response element-binding proteinHCDhigher energy collision-induced dissociationPUGNAcO-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino N-phenyl carbamateCTDC-terminal domainNHOstnormal human osteoblast. modification on highly phosphorylated proteins and regulatory enzymes involved in post-translational processing (1Alfaro J.F. Gong C.X. Monroe M.E. Aldrich J.T. Clauss T.R. Purvine S.O. Wang Z. Camp 2nd, D.G. Shabanowitz J. Stanley P. Hart G.W. Hunt D.F. Yang F. Smith R.D. Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins, including EGF domain-specific O-GlcNAc transferase targets.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 7280-7285Crossref PubMed Scopus (231) Google Scholar, 2Trinidad J.C. Barkan D.T. Gulledge B.F. Thalhammer A. Sali A. Schoepfer R. Burlingame A.L. Global identification and characterization of both O-GlcNAcylation and phosphorylation at the murine synapse.Mol. Cell. Proteomics. 2012; 11: 215-229Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). First reported in 1984 in B-lymphocytes (3Torres C.R. Hart G.W. Topography and polypeptide distribution of terminal N-acetylglucosamine residues on the surfaces of intact lymphocytes. Evidence for O-linked GlcNAc.J. Biol. Chem. 1984; 259: 3308-3317Abstract Full Text PDF PubMed Google Scholar), O-GlcNAc modification is now appreciated as a nutrient-responsive mechanism for modulating signal transduction (4Wells L. Vosseller K. Hart G.W. Glycosylation of nucleocytoplasmic proteins: signal transduction and O-GlcNAc.Science. 2001; 291: 2376-2378Crossref PubMed Scopus (803) Google Scholar, 5Hart G.W. Slawson C. Ramirez-Correa G. Lagerlof O. Cross-talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease.Annu. Rev. Biochem. 2011; 80: 825-858Crossref PubMed Scopus (915) Google Scholar) and transcriptional regulation (6Love D.C. Krause M.W. Hanover J.A. O-GlcNAc cycling: emerging roles in development and epigenetics.Semin. Cell Dev. Biol. 2010; 21: 646-654Crossref PubMed Scopus (87) Google Scholar, 7Ozcan S. Andrali S.S. Cantrell J.E. Modulation of transcription factor function by O-GlcNAc modification.Biochim. Biophys. Acta. 2010; 1799: 353-364Crossref PubMed Scopus (181) Google Scholar). In contrast to N- and O-linked glycosylation taking place in the endoplasmic reticulum and Golgi apparatus, reversible O-GlcNAcylation occurs in the cytoplasm and nucleus and is catalyzed by O-GlcNAc transferase (OGT), which transfers GlcNAc from UDP-GlcNAc to Ser/Thr residues, and O-GlcNAcase (OGA), which removes it. These highly conserved enzymes are expressed in all mammalian tissues (8Lubas W.A. Frank D.W. Krause M. Hanover J.A. O-Linked GlcNAc transferase is a conserved nucleocytoplasmic protein containing tetratricopeptide repeats.J. Biol. Chem. 1997; 272: 9316-9324Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar, 9Heckel D. Comtesse N. Brass N. Blin N. Zang K.D. Meese E. Novel immunogenic antigen homologous to hyaluronidase in meningioma.Hum. Mol. Genet. 1998; 7: 1859-1872Crossref PubMed Scopus (104) Google Scholar, 10Shafi R. Iyer S.P. Ellies L.G. O'Donnell N. Marek K.W. Chui D. Hart G.W. Marth J.D. The O-GlcNAc transferase gene resides on the X chromosome and is essential for embryonic stem cell viability and mouse ontogeny.Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 5735-5739Crossref PubMed Scopus (593) Google Scholar, 11Comtesse N. Maldener E. Meese E. Identification of a nuclear variant of MGEA5, a cytoplasmic hyaluronidase and a β-N-acetylglucosaminidase.Biochem. Biophys. Res. Commun. 2001; 283: 634-640Crossref PubMed Scopus (138) Google Scholar). O-linked N-acetylglucosamine lectin weak affinity chromatography electron transfer dissociation CREB-binding protein runt-related transcription factor 2 O-GlcNAc transferase O-GlcNAcase wheat germ agglutinin alkaline phosphatase osteocalcin parathyroid hormone insulin-like growth factor 1 bone morphogenetic protein cAMP-response element-binding protein higher energy collision-induced dissociation O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino N-phenyl carbamate C-terminal domain normal human osteoblast. O-linked N-acetylglucosamine lectin weak affinity chromatography electron transfer dissociation CREB-binding protein runt-related transcription factor 2 O-GlcNAc transferase O-GlcNAcase wheat germ agglutinin alkaline phosphatase osteocalcin parathyroid hormone insulin-like growth factor 1 bone morphogenetic protein cAMP-response element-binding protein higher energy collision-induced dissociation O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino N-phenyl carbamate C-terminal domain normal human osteoblast. The OGT gene encodes three splice variants differing in the number of N-terminal tetratricopeptide repeats that mediate protein-protein interactions and subcellular localization (8Lubas W.A. Frank D.W. Krause M. Hanover J.A. O-Linked GlcNAc transferase is a conserved nucleocytoplasmic protein containing tetratricopeptide repeats.J. Biol. Chem. 1997; 272: 9316-9324Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar, 12Kreppel L.K. Blomberg M.A. Hart G.W. Dynamic glycosylation of nuclear and cytosolic proteins. Cloning and characterization of a unique O-GlcNAc transferase with multiple tetratricopeptide repeats.J. Biol. Chem. 1997; 272: 9308-9315Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar). The full-length nucleocytoplasmic form of OGT is O-GlcNAc-modified, tyrosine-phosphorylated, and dynamically redistributed within the cell upon insulin stimulation (2Trinidad J.C. Barkan D.T. Gulledge B.F. Thalhammer A. Sali A. Schoepfer R. Burlingame A.L. Global identification and characterization of both O-GlcNAcylation and phosphorylation at the murine synapse.Mol. Cell. Proteomics. 2012; 11: 215-229Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 13Tai H.C. Khidekel N. Ficarro S.B. Peters E.C. Hsieh-Wilson L.C. Parallel identification of O-GlcNAc-modified proteins from cell lysates.J. Am. Chem. Soc. 2004; 126: 10500-10501Crossref PubMed Scopus (99) Google Scholar, 14Olsen J.V. Vermeulen M. Santamaria A. Kumar C. Miller M.L. Jensen L.J. Gnad F. Cox J. Jensen T.S. Nigg E.A. Brunak S. Mann M. Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis.Sci. Signal. 2010; 3: ra3Crossref PubMed Scopus (1144) Google Scholar, 15Yang X. Ongusaha P.P. Miles P.D. Havstad J.C. Zhang F. So W.V. Kudlow J.E. Michell R.H. Olefsky J.M. Field S.J. Evans R.M. Phosphoinositide signalling links O-GlcNAc transferase to insulin resistance.Nature. 2008; 451: 964-969Crossref PubMed Scopus (446) Google Scholar). Ablation of OGT is embryonically lethal or developmentally limiting in animal models (10Shafi R. Iyer S.P. Ellies L.G. O'Donnell N. Marek K.W. Chui D. Hart G.W. Marth J.D. The O-GlcNAc transferase gene resides on the X chromosome and is essential for embryonic stem cell viability and mouse ontogeny.Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 5735-5739Crossref PubMed Scopus (593) Google Scholar, 16O'Donnell N. Zachara N.E. Hart G.W. Marth J.D. Ogt-dependent X-chromosome-linked protein glycosylation is a requisite modification in somatic cell function and embryo viability.Mol. Cell. Biol. 2004; 24: 1680-1690Crossref PubMed Scopus (339) Google Scholar, 17Webster D.M. Teo C.F. Sun Y. Wloga D. Gay S. Klonowski K.D. Wells L. Dougan S.T. O-GlcNAc modifications regulate cell survival and epiboly during zebrafish development.BMC Dev. Biol. 2009; 9: 28Crossref PubMed Scopus (77) Google Scholar) with the noted exception of Caenorhabditis elegans. In C. elegans, the phenotypes of oga-1 and ogt-1 null mutants suggest the enzymes are involved in macronutrient storage and life span (18Hanover J.A. Forsythe M.E. Hennessey P.T. Brodigan T.M. Love D.C. Ashwell G. Krause M. A Caenorhabditis elegans model of insulin resistance: altered macronutrient storage and dauer formation in an OGT-1 knockout.Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 11266-11271Crossref PubMed Scopus (176) Google Scholar, 19Forsythe M.E. Love D.C. Lazarus B.D. Kim E.J. Prinz W.A. Ashwell G. Krause M.W. Hanover J.A. Caenorhabditis elegans ortholog of a diabetes susceptibility locus: oga-1 (O-GlcNAcase) knockout impacts O-GlcNAc cycling, metabolism, and dauer.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 11952-11957Crossref PubMed Scopus (129) Google Scholar, 20Rahman M.M. Stuchlick O. El-Karim E.G. Stuart R. Kipreos E.T. Wells L. Intracellular protein glycosylation modulates insulin mediated life span in C. elegans.Aging. 2010; 2: 678-690Crossref PubMed Scopus (63) Google Scholar), and subsequent studies revealed the presence of O-GlcNAc modification at over 800 promoters associated with transcriptional programs influencing carbohydrate metabolism, stress responses, and longevity (21Love D.C. Ghosh S. Mondoux M.A. Fukushige T. Wang P. Wilson M.A. Iser W.B. Wolkow C.A. Krause M.W. Hanover J.A. Dynamic O-GlcNAc cycling at promoters of Caenorhabditis elegans genes regulating longevity, stress, and immunity.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 7413-7418Crossref PubMed Scopus (115) Google Scholar). The presence of OGT in polycomb group repressor complexes further implicates OGT in the regulation of myriad genes involved in tissue differentiation and spatial patterning during embryogenesis (22Gambetta M.C. Oktaba K. Müller J. Essential role of the glycosyltransferase sxc/Ogt in polycomb repression.Science. 2009; 325: 93-96Crossref PubMed Scopus (246) Google Scholar, 23Sinclair D.A. Syrzycka M. Macauley M.S. Rastgardani T. Komljenovic I. Vocadlo D.J. Brock H.W. Honda B.M. Drosophila O-GlcNAc transferase (OGT) is encoded by the Polycomb group (PcG) gene, super sex combs (sxc).Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 13427-13432Crossref PubMed Scopus (184) Google Scholar). In cellular models of differentiation, modulation of OGT and OGA impacts the expression and activity of markers characteristic of tissue specification (24Li X. Molina H. Huang H. Zhang Y.Y. Liu M. Qian S.W. Slawson C. Dias W.B. Pandey A. Hart G.W. Lane M.D. Tang Q.Q. O-Linked N-acetylglucosamine modification on CCAAT enhancer-binding protein β: role during adipocyte differentiation.J. Biol. Chem. 2009; 284: 19248-19254Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 25Ji S. Park S.Y. Roth J. Kim H.S. Cho J.W. O-GlcNAc modification of PPARγ reduces its transcriptional activity.Biochem. Biophys. Res. Commun. 2012; 417: 1158-1163Crossref PubMed Scopus (56) Google Scholar, 26Ogawa M. Mizofuchi H. Kobayashi Y. Tsuzuki G. Yamamoto M. Wada S. Kamemura K. Terminal differentiation program of skeletal myogenesis is negatively regulated by O-GlcNAc glycosylation.Biochim. Biophys. Acta. 2012; 1820: 24-32Crossref PubMed Scopus (36) Google Scholar, 27Andrés-Bergós J. Tardio L. Larranaga-Vera A. Gómez R. Herrero-Beaumont G. Largo R. The increase in O-linked N-acetylglucosamine protein modification stimulates chondrogenic differentiation both in vitro and in vivo.J. Biol. Chem. 2012; 287: 33615-33628Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 28Kim S.H. Kim Y.H. Song M. An S.H. Byun H.Y. Heo K. Lim S. Oh Y.S. Ryu S.H. Suh P.G. O-GlcNAc modification modulates the expression of osteocalcin via OSE2 and Runx2.Biochem. Biophys. Res. Commun. 2007; 362: 325-329Crossref PubMed Scopus (28) Google Scholar). This has been observed in differentiating osteoblasts whereby inhibition of OGA enhanced the expression of osteocalcin, a marker indicative of progression to the later stages of osteoblastogenesis (28Kim S.H. Kim Y.H. Song M. An S.H. Byun H.Y. Heo K. Lim S. Oh Y.S. Ryu S.H. Suh P.G. O-GlcNAc modification modulates the expression of osteocalcin via OSE2 and Runx2.Biochem. Biophys. Res. Commun. 2007; 362: 325-329Crossref PubMed Scopus (28) Google Scholar). The increased rate of differentiation in osteoblasts was attributed to the potentiation of the transcriptional activity of runt-related transcription factor 2 (RUNX2), a master regulator of osteogenic programs, by O-GlcNAc modification of RUNX2 (28Kim S.H. Kim Y.H. Song M. An S.H. Byun H.Y. Heo K. Lim S. Oh Y.S. Ryu S.H. Suh P.G. O-GlcNAc modification modulates the expression of osteocalcin via OSE2 and Runx2.Biochem. Biophys. Res. Commun. 2007; 362: 325-329Crossref PubMed Scopus (28) Google Scholar). Accumulating evidence continues to establish the adult skeleton as a nutrient- and insulin-sensitive tissue that communicates with other organs via bone-specific endocrine signals (e.g. osteocalcin (OCN) and fibroblast growth factor (FGF23)) thereby coupling bone metabolism to whole-body nutrient and mineral homeostasis (29Lee N.K. Sowa H. Hinoi E. Ferron M. Ahn J.D. Confavreux C. Dacquin R. Mee P.J. McKee M.D. Jung D.Y. Zhang Z. Kim J.K. Mauvais-Jarvis F. Ducy P. Karsenty G. Endocrine regulation of energy metabolism by the skeleton.Cell. 2007; 130: 456-469Abstract Full Text Full Text PDF PubMed Scopus (1944) Google Scholar, 30Ferron M. Wei J. Yoshizawa T. Del Fattore A. DePinho R.A. Teti A. Ducy P. Karsenty G. Insulin signaling in osteoblasts integrates bone remodeling and energy metabolism.Cell. 2010; 142: 296-308Abstract Full Text Full Text PDF PubMed Scopus (854) Google Scholar, 31Fulzele K. Riddle R.C. DiGirolamo D.J. Cao X. Wan C. Chen D. Faugere M.C. Aja S. Hussain M.A. Brüning J.C. Clemens T.L. Insulin receptor signaling in osteoblasts regulates postnatal bone acquisition and body composition.Cell. 2010; 142: 309-319Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar, 32Yoshikawa Y. Kode A. Xu L. Mosialou I. Silva B.C. Ferron M. Clemens T.L. Economides A.N. Kousteni S. Genetic evidence points to an osteocalcin-independent influence of osteoblasts on energy metabolism.J. Bone Miner. Res. 2011; 26: 2012-2025Crossref PubMed Scopus (117) Google Scholar, 33Fukumoto S. Martin T.J. Bone as an endocrine organ.Trends Endocrinol. Metab. 2009; 20: 230-236Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). O-GlcNAc modification of the insulin receptor substrates, critical mediators of insulin/IGF-1 signaling, raises the possibility that O-GlcNAc cycling serves as a nutrient-responsive regulatory mechanism in bone tissue (34Klein A.L. Berkaw M.N. Buse M.G. Ball L.E. O-Linked N-acetylglucosamine modification of insulin receptor substrate-1 occurs in close proximity to multiple SH2 domain binding motifs.Mol. Cell. Proteomics. 2009; 8: 2733-2745Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). It is evident from immunoblot analysis that many proteins are O-GlcNAc-modified in osteoblasts, and the extent of protein O-GlcNAc modification changes during osteoblast differentiation (28Kim S.H. Kim Y.H. Song M. An S.H. Byun H.Y. Heo K. Lim S. Oh Y.S. Ryu S.H. Suh P.G. O-GlcNAc modification modulates the expression of osteocalcin via OSE2 and Runx2.Biochem. Biophys. Res. Commun. 2007; 362: 325-329Crossref PubMed Scopus (28) Google Scholar). The aim of this study was to identify O-GlcNAc-modified substrates of OGT in differentiating osteoblasts. Advances in methodology to isolate O-GlcNAcylated peptides and the recent availability of multiple modes of peptide fragmentation during tandem mass spectrometry are revealing the ubiquitous nature of this modification (1Alfaro J.F. Gong C.X. Monroe M.E. Aldrich J.T. Clauss T.R. Purvine S.O. Wang Z. Camp 2nd, D.G. Shabanowitz J. Stanley P. Hart G.W. Hunt D.F. Yang F. Smith R.D. Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins, including EGF domain-specific O-GlcNAc transferase targets.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 7280-7285Crossref PubMed Scopus (231) Google Scholar, 2Trinidad J.C. Barkan D.T. Gulledge B.F. Thalhammer A. Sali A. Schoepfer R. Burlingame A.L. Global identification and characterization of both O-GlcNAcylation and phosphorylation at the murine synapse.Mol. Cell. Proteomics. 2012; 11: 215-229Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 35Wang Z. Udeshi N.D. O'Malley M. Shabanowitz J. Hunt D.F. Hart G.W. Enrichment and site mapping of O-linked N-acetylglucosamine by a combination of chemical/enzymatic tagging, photochemical cleavage, and electron transfer dissociation mass spectrometry.Mol. Cell. Proteomics. 2010; 9: 153-160Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 36Zhao P. Viner R. Teo C.F. Boons G.J. Horn D. Wells L. Combining high-energy C-trap dissociation and electron transfer dissociation for protein O-GlcNAc modification site assignment.J. Proteome Res. 2011; 10: 4088-4104Crossref PubMed Scopus (120) Google Scholar). The enrichment of O-GlcNAc-modified peptides from complex mixtures of digested proteins has been achieved by enzymatic labeling of the N-acetylglucosamine with chemically reactive moieties amenable to affinity purification, immunoprecipitation with pan-specific anti-O-GlcNAc antibodies, or by wheat germ agglutinin (WGA) lectin affinity chromatography (1Alfaro J.F. Gong C.X. Monroe M.E. Aldrich J.T. Clauss T.R. Purvine S.O. Wang Z. Camp 2nd, D.G. Shabanowitz J. Stanley P. Hart G.W. Hunt D.F. Yang F. Smith R.D. Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins, including EGF domain-specific O-GlcNAc transferase targets.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 7280-7285Crossref PubMed Scopus (231) Google Scholar, 2Trinidad J.C. Barkan D.T. Gulledge B.F. Thalhammer A. Sali A. Schoepfer R. Burlingame A.L. Global identification and characterization of both O-GlcNAcylation and phosphorylation at the murine synapse.Mol. Cell. Proteomics. 2012; 11: 215-229Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 36Zhao P. Viner R. Teo C.F. Boons G.J. Horn D. Wells L. Combining high-energy C-trap dissociation and electron transfer dissociation for protein O-GlcNAc modification site assignment.J. Proteome Res. 2011; 10: 4088-4104Crossref PubMed Scopus (120) Google Scholar, 37Vosseller K. Wells L. Hart G.W. Nucleocytoplasmic O-glycosylation: O-GlcNAc and functional proteomics.Biochimie. 2001; 83: 575-581Crossref PubMed Scopus (72) Google Scholar, 38Vosseller K. Trinidad J.C. Chalkley R.J. Specht C.G. Thalhammer A. Lynn A.J. Snedecor J.O. Guan S. Medzihradszky K.F. Maltby D.A. Schoepfer R. Burlingame A.L. O-Linked N-acetylglucosamine proteomics of postsynaptic density preparations using lectin weak affinity chromatography and mass spectrometry.Mol. Cell. Proteomics. 2006; 5: 923-934Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 39Chalkley R.J. Thalhammer A. Schoepfer R. Burlingame A.L. Identification of protein O-GlcNAcylation sites using electron transfer dissociation mass spectrometry on native peptides.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 8894-8899Crossref PubMed Scopus (199) Google Scholar, 40Myers S.A. Panning B. Burlingame A.L. Polycomb repressive complex 2 is necessary for the normal site-specific O-GlcNAc distribution in mouse embryonic stem cells.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 9490-9495Crossref PubMed Scopus (106) Google Scholar). The identification of O-GlcNAcylated peptides by conventional collision-induced dissociation (CID) tandem mass spectrometry can be complicated by the labile nature of the O-linkage, which upon fragmentation results in neutral loss of GlcNAc from the Ser/Thr prior to fragmentation of the peptide backbone (36Zhao P. Viner R. Teo C.F. Boons G.J. Horn D. Wells L. Combining high-energy C-trap dissociation and electron transfer dissociation for protein O-GlcNAc modification site assignment.J. Proteome Res. 2011; 10: 4088-4104Crossref PubMed Scopus (120) Google Scholar, 41Chalkley R.J. Burlingame A.L. Identification of GlcNAcylation sites of peptides and α-crystallin using Q-TOF mass spectrometry.J. Am. Soc. Mass Spectrom. 2001; 12: 1106-1113Crossref PubMed Scopus (71) Google Scholar, 42Jebanathirajah J. Steen H. Roepstorff P. Using optimized collision energies and high resolution, high accuracy fragment ion selection to improve glycopeptide detection by precursor ion scanning.J. Am. Soc. Mass Spectrom. 2003; 14: 777-784Crossref PubMed Scopus (73) Google Scholar). This impedes detection of O-GlcNAc-modified peptides by database search algorithms and obscures identification of the sites of modification. Peptide fragmentation by electron transfer dissociation (ETD) provides an alternative approach that leaves the fragile O-linkage intact enabling the direct detection of O-GlcNAc-modified peptides and more confident assignment of the sites of modification (43Syka J.E. Coon J.J. Schroeder M.J. Shabanowitz J. Hunt D.F. Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 9528-9533Crossref PubMed Scopus (1999) Google Scholar, 44Mikesh L.M. Ueberheide B. Chi A. Coon J.J. Syka J.E. Shabanowitz J. Hunt D.F. The utility of ETD mass spectrometry in proteomic analysis.Biochim. Biophys. Acta. 2006; 1764: 1811-1822Crossref PubMed Scopus (462) Google Scholar). With the capability of multiple modes of fragmentation, the generation of N-acetylglucosamine (GlcNAc) oxonium ions or the neutral loss of GlcNAc from precursor ions during CID or higher energy collision-induced dissociation (HCD) (36Zhao P. Viner R. Teo C.F. Boons G.J. Horn D. Wells L. Combining high-energy C-trap dissociation and electron transfer dissociation for protein O-GlcNAc modification site assignment.J. Proteome Res. 2011; 10: 4088-4104Crossref PubMed Scopus (120) Google Scholar) can trigger the acquisition of an ETD spectrum yielding complementary tandem mass spectra of the peptide. The development of algorithms for searching ETD spectra against protein databases (45Baker P.R. Medzihradszky K.F. Chalkley R.J. Improving software performance for peptide electron transfer dissociation data analysis by implementation of charge state- and sequence-dependent scoring.Mol. Cell. Proteomics. 2010; 9: 1795-1803Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), the incorporation of neutral loss information during searches of CID spectra (46Darula Z. Chalkley R.J. Baker P. Burlingame A.L. Medzihradszky K.F. Mass spectrometric analysis, automated identification and complete annotation of O-linked glycopeptides.Eur. J. Mass Spectrom. 2010; 16: 421-428Crossref PubMed Scopus (38) Google Scholar), and the association of a score with the assignment of the site of modification (47Chalkley R.J. Clauser K.R. Modification site localization scoring: strategies and performance.Mol. Cell. Proteomics. 2012; 11: 3-14Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) are further enabling this field. A recent study employing lectin weak affinity chromatography (LWAC) and CID/ETD permitted the identification of over 1,750 unique sites of O-GlcNAcylation on 2,434 synaptosome peptides (2Trinidad J.C. Barkan D.T. Gulledge B.F. Thalhammer A. Sali A. Schoepfer R. Burlingame A.L. Global identification and characterization of both O-GlcNAcylation and phosphorylation at the murine synapse.Mol. Cell. Proteomics. 2012; 11: 215-229Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). In this study, we employed lectin chromatography to identify O-GlcNAc-modified proteins by ETD MS/MS in MC3T3E1 preosteoblasts cultured for 7 days under osteogenic conditions. Although many of the proteins observed in this study were previously reported to be O-GlcNAc-modified in other cell types, in some cases different sites of O-GlcNAc were elucidated. Of particular interest with respect to osteoblast function were sites of O-GlcNAc modification on the TFGβ-activated kinase 1/MAP3K7-binding protein 1 and 2 (TAB1/TAB2), regulatory subunits associated with the TFGβ-activated kinase 1 (TAK1) signaling node (48Parker B.L. Gupta P. Cordwell S.J. Larsen M.R. Palmisano G. Purification and identification of O-GlcNAc-modified peptides using phosphate-based alkyne CLICK chemistry in combination with titanium dioxide chromatography and mass spectrometry.J. Proteome Res. 2011; 10: 1449-1458Crossref PubMed Scopus (43) Google Scholar). Recent findings demonstrate that TAK1 signaling is essential to osteoblast differentiation and skeletal ossification, and it modulates RUNX2 activation and its association with the CREB-binding protein (CBP) (49Greenblatt M.B. Shim J.H. Zou W. Sitara D. Schweitzer M. Hu D. Lotinun S. Sano Y. Baron R. Park J.M. Arthur S. Xie M. Schneider M.D. Zhai B. Gygi S. Davis R. Glimcher L.H. The p38 MAPK pathway is essential for skeletogenesis and bone homeostasis in mice.J. Clin. Invest. 2010; 120: 2457-2473Crossref PubMed Scopus (309) Google Scholar). CBP, a histone acetyltransferase and transcriptional co-activator, is critical for osteoblast differentiation and skeletal mineralization. A new site of O-GlcNAc modification was observed in the C-terminal domain of CBP at Ser-2360, a known site of phosphorylation (53Hornbeck P.V. Kornhauser J.M. Tkachev S. Zhang B. Skrzypek E. Murray B. Latham V. Sullivan M. PhosphoSitePlus: a comprehensive resource for investigating the structure and function of experimentally determined post-translational modifications in man and mouse.Nucleic Acids Res. 2012; 40: D261-D270Crossref PubMed Scopus (1138) Google Scholar, 54Huttlin E.L. Jedrychowski M.P. Elias J.E. Goswami T. Rad R. Beausoleil S.A. Villén J. Haas W. Sowa M.E. Gygi S.P. A tissue-specific atlas of mouse protein phosphorylation and expression.Cell. 2010