Mapping and quantification of over 2,000 O-linked glycopeptides in activated human T cells with isotope-targeted glycoproteomics

ABSTRACT: 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 Nacetylglucosamine (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 extensive 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 over 2,000 intact O-GlcNAccontaining glycopeptides across 1,046 glycoproteins. A significant proportion (>45%) of the identified O-GlcNAc sites lie in close proximity to or coincide with known phosphorylation sites, 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.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): T Cell, Blood

SUBMITTER: Christina Woo  

LAB HEAD: Christina Woo

PROVIDER: PXD004559 | Pride | 2018-01-25

REPOSITORIES: Pride