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

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.