Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets

Joshua F. Alfaro, Cheng Xin Gong, Matthew E. Monroe, Joshua T. Aldrich, Therese R W Clauss, Samuel O. Purvine, Zihao Wang, David G. Camp, Jeffrey Shabanowitz, Pamela Stanley, Gerald W. Hart, Donald F. Hunt, Feng Yang, Richard D. Smith

Research output: Contribution to journalArticle

159 Citations (Scopus)

Abstract

O-linked N-acetylglucosamine (O-GlcNAc) is a reversible posttranslational modification of Ser and Thr residues on cytosolic and nuclear proteins of higher eukaryotes catalyzed by O-GlcNAc transferase (OGT). O-GlcNAc has recently been found on Notch1 extracellular domain catalyzed by EGF domain-specific OGT. Aberrant O-GlcNAc modification of brain proteins has been linked to Alzheimer's disease (AD). However, understanding specific functions of O-GlcNAcylation in AD has been impeded by the difficulty in characterization of O-GlcNAc sites on proteins. In this study, we modified a chemical/enzymatic photochemical cleavage approach for enriching O-GlcNAcylated peptides in samples containing ∼100 μg of tryptic peptides from mouse cerebrocortical brain tissue. A total of 274 O-GlcNAcylated proteins were identified. Of these, 168 were not previously known to be modified by O-GlcNAc. Overall, 458 O-GlcNAc sites in 195 proteins were identified. Many of the modified residues are either known phosphorylation sites or located proximal to known phosphorylation sites. These findings support the proposed regulatory cross-talk between O-GlcNAcylation and phosphorylation. This study produced the most comprehensive O-GlcNAc proteome of mammalian brain tissue with both protein identification and O-GlcNAc site assignment. Interestingly, we observed O-β-GlcNAc on EGF-like repeats in the extracellular domains of five membrane proteins, expanding the evidence for extracellular O-GlcNAcylation by the EGF domain-specific OGT. We also report a GlcNAc-β-1,3-Fuc-α-1-O-Thr modification on the EGF-like repeat of the versican core protein, a proposed substrate of Fringe β-1,3-N- acetylglucosaminyltransferases.

Original languageEnglish (US)
Pages (from-to)7280-7285
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume109
Issue number19
DOIs
StatePublished - May 8 2012

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Tandem Mass Spectrometry
Epidermal Growth Factor
Brain
Proteins
Phosphorylation
N-Acetylglucosaminyltransferases
Alzheimer Disease
Versicans
Peptides
Acetylglucosamine
Proteome
Post Translational Protein Processing
Nuclear Proteins
Eukaryota
O-GlcNAc transferase
Membrane Proteins

Keywords

  • Chemical/enzymatic photochemical cleavage enrichment
  • Glycosylation
  • Mouse cerebral cortex

ASJC Scopus subject areas

  • General

Cite this

Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets. / Alfaro, Joshua F.; Gong, Cheng Xin; Monroe, Matthew E.; Aldrich, Joshua T.; Clauss, Therese R W; Purvine, Samuel O.; Wang, Zihao; Camp, David G.; Shabanowitz, Jeffrey; Stanley, Pamela; Hart, Gerald W.; Hunt, Donald F.; Yang, Feng; Smith, Richard D.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 109, No. 19, 08.05.2012, p. 7280-7285.

Research output: Contribution to journalArticle

Alfaro, JF, Gong, CX, Monroe, ME, Aldrich, JT, Clauss, TRW, Purvine, SO, Wang, Z, Camp, DG, Shabanowitz, J, Stanley, P, Hart, GW, Hunt, DF, Yang, F & Smith, RD 2012, 'Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets', Proceedings of the National Academy of Sciences of the United States of America, vol. 109, no. 19, pp. 7280-7285. https://doi.org/10.1073/pnas.1200425109
Alfaro, Joshua F. ; Gong, Cheng Xin ; Monroe, Matthew E. ; Aldrich, Joshua T. ; Clauss, Therese R W ; Purvine, Samuel O. ; Wang, Zihao ; Camp, David G. ; Shabanowitz, Jeffrey ; Stanley, Pamela ; Hart, Gerald W. ; Hunt, Donald F. ; Yang, Feng ; Smith, Richard D. / Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets. In: Proceedings of the National Academy of Sciences of the United States of America. 2012 ; Vol. 109, No. 19. pp. 7280-7285.
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abstract = "O-linked N-acetylglucosamine (O-GlcNAc) is a reversible posttranslational modification of Ser and Thr residues on cytosolic and nuclear proteins of higher eukaryotes catalyzed by O-GlcNAc transferase (OGT). O-GlcNAc has recently been found on Notch1 extracellular domain catalyzed by EGF domain-specific OGT. Aberrant O-GlcNAc modification of brain proteins has been linked to Alzheimer's disease (AD). However, understanding specific functions of O-GlcNAcylation in AD has been impeded by the difficulty in characterization of O-GlcNAc sites on proteins. In this study, we modified a chemical/enzymatic photochemical cleavage approach for enriching O-GlcNAcylated peptides in samples containing ∼100 μg of tryptic peptides from mouse cerebrocortical brain tissue. A total of 274 O-GlcNAcylated proteins were identified. Of these, 168 were not previously known to be modified by O-GlcNAc. Overall, 458 O-GlcNAc sites in 195 proteins were identified. Many of the modified residues are either known phosphorylation sites or located proximal to known phosphorylation sites. These findings support the proposed regulatory cross-talk between O-GlcNAcylation and phosphorylation. This study produced the most comprehensive O-GlcNAc proteome of mammalian brain tissue with both protein identification and O-GlcNAc site assignment. Interestingly, we observed O-β-GlcNAc on EGF-like repeats in the extracellular domains of five membrane proteins, expanding the evidence for extracellular O-GlcNAcylation by the EGF domain-specific OGT. We also report a GlcNAc-β-1,3-Fuc-α-1-O-Thr modification on the EGF-like repeat of the versican core protein, a proposed substrate of Fringe β-1,3-N- acetylglucosaminyltransferases.",
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AU - Alfaro, Joshua F.

AU - Gong, Cheng Xin

AU - Monroe, Matthew E.

AU - Aldrich, Joshua T.

AU - Clauss, Therese R W

AU - Purvine, Samuel O.

AU - Wang, Zihao

AU - Camp, David G.

AU - Shabanowitz, Jeffrey

AU - Stanley, Pamela

AU - Hart, Gerald W.

AU - Hunt, Donald F.

AU - Yang, Feng

AU - Smith, Richard D.

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