Ultradeep O- GlcNAc proteomics reveals widespread O- GlcNAcylation on tyrosine residues of proteins

• 成果类型: Article
• 署名作者: Hou, Chunyan; Deng, Jingtao; Wu, Ci; Zhang, Jing; Byers, Stephen; Moremen, Kelley W.; Pei, Huadong; Ma, Junfeng
• 署名单位: Georgetown University; University System of Georgia; Georgia State University; University System of Georgia; Georgia State University; University System of Georgia; University of Georgia
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-11804
• DOI: 10.1073/pnas.2409501121
• 发表日期: 2024-11-19
• 关键词: electron-transfer dissociation; linked n-acetylglucosamine; mass-spectrometry; cytoplasmic proteins; substrate recognition; sequence-analysis; identification; phosphorylation; glycosylation; transferase

摘要

As a unique type of glycosylation, O- linked beta-N- acetylglucosamine (O- GlcNAc) modification (O- GlcNAcylation) on Ser/Thr residues of proteins was discovered 40 y ago. O- GlcNAcylation is catalyzed by two enzymes: O- GlcNAc transferase (OGT) and O- GlcNAcase (OGA), which add and remove O- GlcNAc, respectively. O- GlcNAcylation is an essential glycosylation that regulates the functions of many proteins in virtually all cellular processes. However, deep and site- specific characterization of O- GlcNAcylated proteins remains a challenge. We developed an ultradeep O- GlcNAc proteomics workflow by integrating digestion with multiple proteases, two mass spectrometric approaches (i.e., electron- transfer/higher- energy collision dissociation [EThcD] and HCD product- dependent electron- transfer/higher- energy collision dissociation [HCD- pd- EThcD]), and two data analysis tools (i.e., MaxQuant and Proteome Discoverer). The performance of this strategy was benchmarked by the analysis of whole lysates from PANC-1 (a pancreatic cancer cell line). In total, 2,831 O- GlcNAc sites were unambiguously identified, representing the largest O- GlcNAc dataset of an individual study reported so far. Unexpectedly, in addition to confirming known sites and identifying many other sites of Ser/Thr modification, O- GlcNAcylation was found on 121 tyrosine (Tyr) residues of 93 proteins. In vitro enzymatic assays showed that OGT catalyzes the transfer of O- GlcNAc onto Tyr residues of peptides and OGA catalyzes its removal. Taken together, our work reveals widespread O- GlcNAcylation on Tyr residues of proteins and that Tyr O- GlcNAcylation is mediated by OGT and OGA. As another form of glycosylation, Tyr O- GlcNAcylation is likely to have important regulatory roles.