Integrated and Quantitative Proteomic Approach for Charting Temporal and Endogenous Protein Complexes

Ke, Mi1; Liu, Jie2; Chen, Wendong1; Chen, Lan1; Gao, Weina1; Qin, Yunqiu1; He, An1; Chu, Bizhu1; Tang, Jun1,3; Xu, Ruilan3; Deng, Yi2,4; Tian, Ruijun1,4

2018-11-06

10.1021/acs.analchem.8b02667

ANALYTICAL CHEMISTRY   影响因子和分区

0003-2700

1520-6882

Proteins often assemble into multiprotein complexes for carrying out their biological functions. Affinity purification combined with mass spectrometry (AP-MS) is a method of choice for unbiasedly charting protein complexes. Typically, genetically tagged bait protein and associated proteins are immunoprecipitated from cell lysate and subjected to in-gel or on-bead digestion for MS analysis. However, the sample preparation procedures are often time-consuming and skipping reduction and alkylation steps results in incomplete digestion. Here, by seamlessly combining AP with the simple and integrated spintip-based proteomics technology (SISPROT), we developed an integrated AP-MS workflow for simultaneously processing more than 10 AP samples from cells cultured in six-well plates in 2 h. Moreover, we developed a quantitation-based data analysis workflow for differentiating potential interacting proteins from nonspecific interferences. The AP-SISPROT ensures high digestion efficiency especially for large transmembrane proteins such as EGFR and high quantification precision for profiling temporal interaction network of key EGFR signaling protein GRB2 across four time points of EGF treatment. More importantly, the integration feature allows minimum sample lose and helps the development of an ideal AP-MS workflow for studying endogenous protein complexes by the CRISPR Cas9 technology for the first time. By generating endogenously expressed bait protein fused with affinity tag, protein complexes associated with endogenous Integrin-linked kinase (ILK) was identified with much higher selectivity as compared with overexpressed and tagged ILK. The AP-SISPROT technology and its combination with CRISPR Cas9 technology should be generally applicable for studying protein complexes in a more efficient and physiologically relevant manner.

SCI ; EI

英语

NI期刊

第一 ; 通讯

Guangdong Provincial Grants[2017B030301018] ; Guangdong Provincial Grants[2016A030312016]

Chemistry

Chemistry, Analytical

WOS:000449722500036

AMER CHEMICAL SOC

20184305986364

Complex networks ; Mass spectrometry ; Molecular biology

Biology:461.9 ; Computer Systems and Equipment:722 ; Chemistry:801 ; Organic Compounds:804.1

CHEMISTRY

Web of Science

1.Southern Univ Sci & Technol, Dept Chem, Shenzhen 518055, Peoples R China
2.Southern Univ Sci & Technol, Dept Biol, Shenzhen 518055, Peoples R China
3.Jinan Univ, Clin Med Coll 2, Shenzhen Peoples Hosp, Shenzhen 518020, Peoples R China
4.Guangdong Prov Key Lab Cell Microenvironm & Dis R, Shenzhen 518055, Peoples R China

Ke, Mi,Liu, Jie,Chen, Wendong,et al. Integrated and Quantitative Proteomic Approach for Charting Temporal and Endogenous Protein Complexes[J]. ANALYTICAL CHEMISTRY,2018,90(21):12574-12583.

Ke, Mi.,Liu, Jie.,Chen, Wendong.,Chen, Lan.,Gao, Weina.,...&Tian, Ruijun.(2018).Integrated and Quantitative Proteomic Approach for Charting Temporal and Endogenous Protein Complexes.ANALYTICAL CHEMISTRY,90(21),12574-12583.

Ke, Mi,et al."Integrated and Quantitative Proteomic Approach for Charting Temporal and Endogenous Protein Complexes".ANALYTICAL CHEMISTRY 90.21(2018):12574-12583.