期刊
TALANTA
卷 241, 期 -, 页码 -出版社
ELSEVIER
DOI: 10.1016/j.talanta.2021.123167
关键词
O-GlcNAc; Derivatization; Enrichment; Chromatin-associated proteins; Genomic loci
资金
- National Key R&D Program of China [2017YFA0505002]
- National Natural Science Foundation of China [22077027, 21778015, 32088101, 22074158]
- Doctoral Initiation Fund [5101039170344]
- National Key Laboratory of Proteomics [SKLP-K201706, 2021-NCPSB-003]
The study developed a novel chemical enrichment method (AFT-OCAP) for simultaneous analysis of O-GlcNAc chromatin-associated proteins (OCAPs) and mapping their binding DNA, providing valuable tools for understanding the functions of OCAPs and the regulatory machinery of O-GlcNAcylation on gene transcription.
O-Linked beta-N-acetylglucosamine (O-GlcNAc), a versatile posttranslational modification (PTM), is found on many chromatin-associated proteins (CAPs), such as transcription factors and their cofactors (TFCs). O-GlcNAc turnover influences the dynamic interactions of CAPs with chromatin and thereby regulates gene expression. Therefore, both global profiling of O-GlcNAc chromatin-associated proteins (OCAPs) and genome-wide mapping of their DNA binding sites are invaluable for understanding the functions of OCAPs and the regulatory machinery of O-GlcNAcylation on gene transcription. However, it is difficult to conduct genome-and proteome-wide OCAP studies using the widely adopted chromatin immunoprecipitation (ChIP) method due to the lack of highly OGlcNAc-specific panantibodies. Therefore, we developed a chemical enrichment method (AFT-OCAP) for simultaneously profiling OCAPs and mapping their binding DNA via mass spectrometry (MS) analysis and DNA sequencing. In our method, we developed an alkynyl-functionalized trimethylpiperidine (AFT) reagent to perform highly efficient chemical derivatizations of azide-labeled OCAP-DNA complexes. The reversible affinity between the immobilized anti-trimethylpiperidine antibody resin and AFT reagent leads to specific enrichment and efficient elution of the OCAP-DNA complexes for both MS identification and sequencing. Deep coverage of OCAPs was achieved from HeLa cells, including 1951 O-GlcNAc peptides from 1136 O-GlcNAc chromatin associated transcription factors and cofactors (TFCs) using HCD fragmentation and 669 O-GlcNAc sites using EThcD fragmentation. In addition, the distributions of O-GlcNAcylation across the genome and the dynamic interactions of OCAPs upon O-GlcNAc regulation were obtained.
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