Mapping and Quantification of Over 2000 O-linked Glycopeptides in Activated Human T Cells with Isotope-Targeted Glycoproteomics (Isotag)

Mol Cell Proteomics. 2018 Apr;17(4):764-775. doi: 10.1074/mcp.RA117.000261. Epub 2018 Jan 19.


Post-translational modifications (PTMs) on proteins often function to regulate signaling cascades, with the activation of T cells during an adaptive immune response being a classic example. Mounting evidence indicates that the modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc), the only mammalian glycan found on nuclear and cytoplasmic proteins, helps regulate T cell activation. Yet, a mechanistic understanding of how O-GlcNAc functions in T cell activation remains elusive, partly because of the difficulties in mapping and quantifying O-GlcNAc sites. Thus, to advance insight into the role of O-GlcNAc in T cell activation, we performed glycosite mapping studies via direct glycopeptide measurement on resting and activated primary human T cells with a technique termed Isotope Targeted Glycoproteomics. This approach led to the identification of 2219 intact O-linked glycopeptides across 1045 glycoproteins. A significant proportion (>45%) of the identified O-GlcNAc sites lie near or coincide with a known phosphorylation site, supporting the potential for PTM crosstalk. Consistent with other studies, we find that O-GlcNAc sites in T cells lack a strict consensus sequence. To validate our results, we employed gel shift assays based on conjugating mass tags to O-GlcNAc groups. Notably, we observed that the transcription factors c-JUN and JUNB show higher levels of O-GlcNAc glycosylation and higher levels of expression in activated T cells. Overall, our findings provide a quantitative characterization of O-GlcNAc glycoproteins and their corresponding modification sites in primary human T cells, which will facilitate mechanistic studies into the function of O-GlcNAc in T cell activation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Acetylglucosamine / metabolism*
  • Cells, Cultured
  • Deuterium
  • Glycopeptides / metabolism*
  • Humans
  • Lymphocyte Activation
  • Protein Processing, Post-Translational
  • Proteomics
  • Proto-Oncogene Proteins c-jun / metabolism
  • T-Lymphocytes / metabolism*


  • Glycopeptides
  • Proto-Oncogene Proteins c-jun
  • Deuterium
  • Acetylglucosamine