title: Searching Missing Proteins Based on the Optimization of Membrane Protein Enrichment and Digestion Process.
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.
authors.affiliation: 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.
authors.forename: Mingzhi
authors.initials: M
authors.lastname: Zhao
authors.affiliation: 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.
authors.forename: Wei
authors.initials: W
authors.lastname: Wei
authors.affiliation: Department of Medical Molecular Biology, Beijing Institute of Biotechnology , 27 Tai-Ping Lu Road, Beijing 100850, China.
authors.forename: Long
authors.initials: L
authors.lastname: Cheng
authors.affiliation: 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.|Institute of Microbiology, Chinese Academy of Science , Beijing 100101, China.
authors.forename: Yao
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: 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.|Life Science College, Southwest Forestry University , Kunming 650224, P. R. China.
authors.forename: Feilin
authors.initials: F
authors.lastname: Wu
authors.affiliation: 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.
authors.forename: Fuchu
authors.initials: F
authors.lastname: He
authors.affiliation: 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.|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.|Anhui Medical University , Hefei 230032, Anhui, P. R. China.
authors.forename: Ping
authors.initials: P
authors.lastname: Xu
mesh_terms: D030562:Databases, Protein; D003902:Detergents / Q000737:chemistry / Q000378:metabolism; D005374:Filtration; D006801:Humans; D061986:MCF-7 Cells; D008565:Membrane Proteins / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D010455:Peptides; D040901:Proteomics / Q000379:methods*
chemical_list: D003902:Detergents; D008565:Membrane Proteins; D010455:Peptides
keywords: detergent; digestion; membrane proteomics; missing proteins; ultracentrifugation