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

J Proteome Res. 2016 Nov 4;15(11):4020-4029. doi: 10.1021/acs.jproteome.6b00389. Epub 2016 Aug 12.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Databases, Protein*
  • Detergents / chemistry
  • Detergents / metabolism
  • Filtration
  • Humans
  • MCF-7 Cells
  • Membrane Proteins / analysis
  • Membrane Proteins / isolation & purification*
  • Membrane Proteins / metabolism
  • Peptides
  • Proteomics / methods*


  • Detergents
  • Membrane Proteins
  • Peptides