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

糖肽 聚糖 N-糖酰胺酶F 糖组学 质谱法 色谱法 生物化学 分子生物学 串联质谱法
作者
Christina M. Woo,Peder Lund,Andrew C. Huang,Mark M. Davis,Carolyn R. Bertozzi,Sharon J. Pitteri
出处
期刊:Molecular & Cellular Proteomics [Elsevier BV]
卷期号:17 (4): 764-775 被引量:96
标识
DOI:10.1074/mcp.ra117.000261
摘要

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.
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