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Generic Plug-and-Play Strategy for High-Throughput Analysis of PTM-Mediated Protein Complexes.
Qin, Yunqiu; Zheng, Zhendong; Chu, Bizhu; Kong, Qian; Ke, Mi; Voss, Courtney; Li, Shawn S C; Tian, Ruijun.
Afiliação
  • Qin Y; School of Chemistry and Chemical Engineering, Harbin Institute of Technology, Harbin 150001, China.
  • Zheng Z; Department of Chemistry, College of Science, Southern University of Science and Technology, Shenzhen 518055, China.
  • Chu B; School of Chemistry and Chemical Engineering, Harbin Institute of Technology, Harbin 150001, China.
  • Kong Q; Department of Chemistry, College of Science, Southern University of Science and Technology, Shenzhen 518055, China.
  • Ke M; School of Pharmaceutical Sciences, Health Science Center, Shenzhen University, Shenzhen 518060, China.
  • Voss C; Department of Chemistry, College of Science, Southern University of Science and Technology, Shenzhen 518055, China.
  • Li SSC; State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR 999077, China.
  • Tian R; Department of Chemistry, College of Science, Southern University of Science and Technology, Shenzhen 518055, China.
Anal Chem ; 94(18): 6799-6808, 2022 05 10.
Article em En | MEDLINE | ID: mdl-35471023
Protein complexes mediated by various post-translational modifications (PTMs) play important roles in almost every aspect of biological processes. PTM-mediated protein complexes often have weak and transient binding properties, which limit their unbiased profiling especially in complex biological samples. Here, we developed a plug-and-play chemical proteomic approach for high-throughput analyis of PTM-mediated protein complexes. Taking advantage of the glutathione-S-transferase (GST) tag, which is the gold standard for protein purification and has wide access to a variety of proteins of interest (POIs), a glutathione (GSH) group- and photo-cross-linking group-containing trifunctional chemical probe was developed to tag POIs and assembled onto a streptavidin-coated 96-well plate for affinity purification, photo-cross-linking, and proteomics sample preparation in a fully integrated manner. Compared with the previously developed photo-pTyr-scaffold strategy, by assembling the tyrosine phosphorylation (pTyr) binding domain through covalent NHS chemistry, the new plug-and-play strategy using a noncovalent GST-GSH interaction has comparable enrichment efficiency for EGF stimulation-dependent pTyr protein complexes. To further prove its feasibility, we additionally assembled four pTyr-binding domains in the 96-well plate and selectively identified their pTyr-dependent interacting proteins. Importantly, we systematically optimized and applied the plug-and-play approach for exploring protein methylation-mediated protein complexes, which are difficult to be characterized due to their weak binding affinity and the lack of efficient enrichment strategies. We explored a comprehensive protein methylation-mediated interaction network assembled by five protein methylation binding domains including the chromo domain of MPP8, tandem tudor domain of KDM4A, full-length CBX1, PHD domain of RAG2, and tandem tudor domain of TP53BP1 and validated the chromo domain- and tudor domain-mediated interaction with histone H3. Collectively, this plug-and-play approach provides a convenient and generic strategy for exploring PTM-dependent protein complexes for any POIs with the GST tag.
Assuntos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Processamento de Proteína Pós-Traducional / Proteômica Idioma: En Revista: Anal Chem Ano de publicação: 2022 Tipo de documento: Article País de afiliação: China

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Processamento de Proteína Pós-Traducional / Proteômica Idioma: En Revista: Anal Chem Ano de publicação: 2022 Tipo de documento: Article País de afiliação: China