Advertisement
MCP
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/mcp.M200048-MCP200 on September 26, 2002.
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
M200048-MCP200v1
1/10/791    most recent
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Glossary
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wells, L.
Right arrow Articles by Hart, G. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wells, L.
Right arrow Articles by Hart, G. W.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Molecular & Cellular Proteomics 1:791-804, 2002.
© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Mapping Sites of O-GlcNAc Modification Using Affinity Tags for Serine and Threonine Post-translational Modifications*

Lance Wells{ddagger},§, Keith Vosseller{ddagger},§, Robert N. Cole{ddagger}, Janet M. Cronshaw, Michael J. Matunis and Gerald W. Hart{ddagger},||

{ddagger} Department of Biological Chemistry, Johns Hopkins University School of Medicine
Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 21218

Identifying sites of post-translational modifications on proteins is a major challenge in proteomics. O-Linked ß-N-acetylglucosamine (O-GlcNAc) is a dynamic nucleocytoplasmic modification more analogous to phosphorylation than to classical complex O-glycosylation. We describe a mass spectrometry-based method for the identification of sites modified by O-GlcNAc that relies on mild ß-elimination followed by Michael addition with dithiothreitol (BEMAD). Using synthetic peptides, we also show that biotin pentylamine can replace dithiothreitol as the nucleophile. The modified peptides can be efficiently enriched by affinity chromatography, and the sites can be mapped using tandem mass spectrometry. This same methodology can be applied to mapping sites of serine and threonine phosphorylation, and we provide a strategy that uses modification-specific antibodies and enzymes to discriminate between the two post-translational modifications. The BEMAD methodology was validated by mapping three previously identified O-GlcNAc sites, as well as three novel sites, on Synapsin I purified from rat brain. BEMAD was then used on a purified nuclear pore complex preparation to map novel sites of O-GlcNAc modification on the Lamin B receptor and the nucleoporin Nup155. This method is amenable for performing quantitative mass spectrometry and can also be adapted to quantify cysteine residues. In addition, our studies emphasize the importance of distinguishing between O-phosphate versus O-GlcNAc when mapping sites of serine and threonine post-translational modification using ß-elimination/Michael addition methods.

|| To whom reprint requests should be addressed. E-mail: gwhart{at}jhmi.edu


Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 GlycobiologyHome page
N. Nakamura, S. H Stalnaker, D. Lyalin, O. Lavrova, L. Wells, and V. M Panin
Drosophila Dystroglycan is a target of O-mannosyltransferase activity of two protein O-mannosyltransferases, Rotated Abdomen and Twisted
Glycobiology, March 1, 2010; 20(3): 381 - 394.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. Lee, K.-Y. Kim, J. Lee, and Y.-K. Paik
Regulation of Dauer Formation by O-GlcNAcylation in Caenorhabditis elegans
J. Biol. Chem., January 29, 2010; 285(5): 2930 - 2939.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
Q. Zeidan and G. W. Hart
The intersections between O-GlcNAcylation and phosphorylation: implications for multiple signaling pathways
J. Cell Sci., January 1, 2010; 123(1): 13 - 22.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. G. Kang, S. Y. Park, S. Ji, I. Jang, S. Park, H. S. Kim, S.-M. Kim, J. I. Yook, Y.-I. Park, J. Roth, et al.
O-GlcNAc Protein Modification in Cancer Cells Increases in Response to Glucose Deprivation through Glycogen Degradation
J. Biol. Chem., December 11, 2009; 284(50): 34777 - 34784.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
W. B. Dias, W. D. Cheung, Z. Wang, and G. W. Hart
Regulation of Calcium/Calmodulin-dependent Kinase IV by O-GlcNAc Modification
J. Biol. Chem., August 7, 2009; 284(32): 21327 - 21337.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
R. J. Chalkley, A. Thalhammer, R. Schoepfer, and A. L. Burlingame
Identification of protein O-GlcNAcylation sites using electron transfer dissociation mass spectrometry on native peptides
PNAS, June 2, 2009; 106(22): 8894 - 8899.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. ProteomicsHome page
M. Gharib, M. Marcantonio, S. G. Lehmann, M. Courcelles, S. Meloche, A. Verreault, and P. Thibault
Artifactual Sulfation of Silver-stained Proteins: Implications for the Assignment of Phosphorylation and Sulfation Sites
Mol. Cell. Proteomics, March 1, 2009; 8(3): 506 - 518.
[Abstract] [Full Text] [PDF]

Home page
 GlycobiologyHome page
J. F Valliere-Douglass, L. J Brady, C. Farnsworth, D. Pace, A. Balland, A. Wallace, W. Wang, M. J Treuheit, and B. Yan
O-Fucosylation of an antibody light chain: Characterization of a modification occurring on an IgG1 molecule
Glycobiology, February 1, 2009; 19(2): 144 - 152.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
Z. Wang, K. Park, F. Comer, L. C. Hsieh-Wilson, C. D. Saudek, and G. W. Hart
Site-Specific GlcNAcylation of Human Erythrocyte Proteins: Potential Biomarker(s) for Diabetes
Diabetes, February 1, 2009; 58(2): 309 - 317.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y. Hu, J. Suarez, E. Fricovsky, H. Wang, B. T. Scott, S. A. Trauger, W. Han, Y. Hu, M. O. Oyeleye, and W. H. Dillmann
Increased Enzymatic O-GlcNAcylation of Mitochondrial Proteins Impairs Mitochondrial Function in Cardiac Myocytes Exposed to High Glucose
J. Biol. Chem., January 2, 2009; 284(1): 547 - 555.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
B. Laczy, B. G. Hill, K. Wang, A. J. Paterson, C. R. White, D. Xing, Y.-F. Chen, V. Darley-Usmar, S. Oparil, and J. C. Chatham
Protein O-GlcNAcylation: a new signaling paradigm for the cardiovascular system
Am J Physiol Heart Circ Physiol, January 1, 2009; 296(1): H13 - H28.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
G. A. Ngoh and S. P. Jones
New Insights into Metabolic Signaling and Cell Survival: The Role of {beta}-O-Linkage of N-Acetylglucosamine
J. Pharmacol. Exp. Ther., December 1, 2008; 327(3): 602 - 609.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. ProteomicsHome page
V. Dehennaut, M.-C. Slomianny, A. Page, A.-S. Vercoutter-Edouart, C. Jessus, J.-C. Michalski, J.-P. Vilain, J.-F. Bodart, and T. Lefebvre
Identification of Structural and Functional O-Linked N-Acetylglucosamine-bearing Proteins in Xenopus laevis Oocyte
Mol. Cell. Proteomics, November 1, 2008; 7(11): 2229 - 2245.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
R. J. Copeland, J. W. Bullen, and G. W. Hart
Cross-talk between GlcNAcylation and phosphorylation: roles in insulin resistance and glucose toxicity
Am J Physiol Endocrinol Metab, July 1, 2008; 295(1): E17 - E28.
[Abstract] [Full Text] [PDF]

Home page
 Plant Cell PhysiolHome page
M. Abe, M. Fujiwara, K.-i. Kurotani, S. Yokoi, and K. Shimamoto
Identification of Dynamin as an Interactor of Rice GIGANTEA by Tandem Affinity Purification (TAP)
Plant Cell Physiol., March 1, 2008; 49(3): 420 - 432.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. ProteomicsHome page
Z. Wang, A. Pandey, and G. W. Hart
Dynamic Interplay between O-Linked N-Acetylglucosaminylation and Glycogen Synthase Kinase-3-dependent Phosphorylation
Mol. Cell. Proteomics, August 1, 2007; 6(8): 1365 - 1379.
[Abstract] [Full Text] [PDF]

Home page
 JCBHome page
S. K. Olson, J. R. Bishop, J. R. Yates, K. Oegema, and J. D. Esko
Identification of novel chondroitin proteoglycans in Caenorhabditis elegans: embryonic cell division depends on CPG-1 and CPG-2
J. Cell Biol., June 19, 2006; 173(6): 985 - 994.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. ProteomicsHome page
K. Vosseller, J. C. Trinidad, R. J. Chalkley, C. G. Specht, A. Thalhammer, A. J. Lynn, J. O. Snedecor, S. Guan, K. F. Medzihradszky, D. A. Maltby, et al.
O-Linked N-Acetylglucosamine Proteomics of Postsynaptic Density Preparations Using Lectin Weak Affinity Chromatography and Mass Spectrometry
Mol. Cell. Proteomics, May 1, 2006; 5(5): 923 - 934.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. ProteomicsHome page
L. E. Ball, M. N. Berkaw, and M. G. Buse
Identification of the Major Site of O-Linked {beta}-N-Acetylglucosamine Modification in the C Terminus of Insulin Receptor Substrate-1
Mol. Cell. Proteomics, February 1, 2006; 5(2): 313 - 323.
[Abstract] [Full Text] [PDF]

Home page
 Sci SignalHome page
D. C. Love and J. A. Hanover
The Hexosamine Signaling Pathway: Deciphering the "O-GlcNAc Code"
Sci. Signal., November 29, 2005; 2005(312): re13 - re13.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. ProteomicsHome page
M. D. M. Huq and L.-N. Wei
Post-translational Modification of Nuclear Co-repressor Receptor-interacting Protein 140 by Acetylation
Mol. Cell. Proteomics, July 1, 2005; 4(7): 975 - 983.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. ProteomicsHome page
K. M. Loyet, J. T. Stults, and D. Arnott
Mass Spectrometric Contributions to the Practice of Phosphorylation Site Mapping through 2003: A Literature Review
Mol. Cell. Proteomics, March 1, 2005; 4(3): 235 - 245.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
L. Wells, L. K. Kreppel, F. I. Comer, B. E. Wadzinski, and G. W. Hart
O-GlcNAc Transferase Is in a Functional Complex with Protein Phosphatase 1 Catalytic Subunits
J. Biol. Chem., September 10, 2004; 279(37): 38466 - 38470.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
N. Khidekel, S. B. Ficarro, E. C. Peters, and L. C. Hsieh-Wilson
Exploring the O-GlcNAc proteome: Direct identification of O-GlcNAc-modified proteins from the brain
PNAS, September 7, 2004; 101(36): 13132 - 13137.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
N. E. Zachara, N. O'Donnell, W. D. Cheung, J. J. Mercer, J. D. Marth, and G. W. Hart
Dynamic O-GlcNAc Modification of Nucleocytoplasmic Proteins in Response to Stress: A SURVIVAL RESPONSE OF MAMMALIAN CELLS
J. Biol. Chem., July 16, 2004; 279(29): 30133 - 30142.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
G. Parker, R. Taylor, D. Jones, and D. McClain
Hyperglycemia and Inhibition of Glycogen Synthase in Streptozotocin-treated Mice: ROLE OF O-LINKED N-ACETYLGLUCOSAMINE
J. Biol. Chem., May 14, 2004; 279(20): 20636 - 20642.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. ProteomicsHome page
K. F. Medzihradszky, Z. Darula, E. Perlson, M. Fainzilber, R. J. Chalkley, H. Ball, D. Greenbaum, M. Bogyo, D. R. Tyson, R. A. Bradshaw, et al.
O-Sulfonation of Serine and Threonine: Mass Spectrometric Detection and Characterization of a New Posttranslational Modification in Diverse Proteins Throughout the Eukaryotes
Mol. Cell. Proteomics, May 1, 2004; 3(5): 429 - 440.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
H. C. Hang, C. Yu, D. L. Kato, and C. R. Bertozzi
A metabolic labeling approach toward proteomic analysis of mucin-type O-linked glycosylation
PNAS, December 9, 2003; 100(25): 14846 - 14851.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
S. A. Whelan and G. W. Hart
Proteomic Approaches to Analyze the Dynamic Relationships Between Nucleocytoplasmic Protein Glycosylation and Phosphorylation
Circ. Res., November 28, 2003; 93(11): 1047 - 1058.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
D. J. Vocadlo, H. C. Hang, E.-J. Kim, J. A. Hanover, and C. R. Bertozzi
A chemical approach for identifying O-GlcNAc-modified proteins in cells
PNAS, August 5, 2003; 100(16): 9116 - 9121.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. ProteomicsHome page
R. J. Chalkley and A. L. Burlingame
Identification of Novel Sites of O-N-Acetylglucosamine Modification of Serum Response Factor Using Quadrupole Time-of-flight Mass Spectrometry
Mol. Cell. Proteomics, March 1, 2003; 2(3): 182 - 190.
[Abstract] [Full Text] [PDF]


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Journal of Biological Chemistry 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2002 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement