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J Proteome Res. 2016 Nov 4;15(11):4020-4029. Epub 2016 Aug 12.

Searching Missing Proteins Based on the Optimization of Membrane Protein Enrichment and Digestion Process.

Zhao M1, Wei W1, Cheng L2, Zhang Y1,3, Wu F1,4, He F1, Xu P1,5,6.

Author information

1
State Key Laboratory of Proteomics, National Engineering Research Center for Protein Drugs, Beijing Proteome Research Center, National Center for Protein Sciences Beijing, Beijing Institute of Radiation Medicine , Beijing 102206, P. R. China.
2
Department of Medical Molecular Biology, Beijing Institute of Biotechnology , 27 Tai-Ping Lu Road, Beijing 100850, China.
3
Institute of Microbiology, Chinese Academy of Science , Beijing 100101, China.
4
Life Science College, Southwest Forestry University , Kunming 650224, P. R. China.
5
Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, and Wuhan University School of Pharmaceutical Sciences , Wuhan 430071, P. R. China.
6
Anhui Medical University , Hefei 230032, Anhui, P. R. China.

Abstract

A membrane protein enrichment method composed of ultracentrifugation and detergent-based extraction was first developed based on MCF7 cell line. Then, in-solution digestion with detergents and eFASP (enhanced filter-aided sample preparation) with detergents were compared with the time-consuming in-gel digestion method. Among the in-solution digestion strategies, the eFASP combined with RapiGest identified 1125 membrane proteins. Similarly, the eFASP combined with sodium deoxycholate identified 1069 membrane proteins; however, the in-gel digestion characterized 1091 membrane proteins. Totally, with the five digestion methods, 1390 membrane proteins were identified with ≥1 unique peptides, among which 1345 membrane proteins contain unique peptides ≥2. This is the biggest membrane protein data set for MCF7 cell line and even breast cancer tissue samples. Interestingly, we identified 13 unique peptides belonging to 8 missing proteins (MPs). Finally, eight unique peptides were validated by synthesized peptides. Two proteins were confirmed as MPs, and another two proteins were candidate detections.

KEYWORDS:

detergent; digestion; membrane proteomics; missing proteins; ultracentrifugation

PMID:
27485413
DOI:
10.1021/acs.jproteome.6b00389
[Indexed for MEDLINE]

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