Mining Missing Membrane Proteins by High-pH Reverse-Phase StageTip Fractionation and Multiple Reaction Monitoring Mass Spectrometry

Despite
significant efforts in the past decade toward complete
mapping of the human proteome, 3564 proteins (neXtProt, 09-2014) are
still “missing proteins”. Over one-third of these missing
proteins are annotated as membrane proteins, owing to their relatively
challenging accessibility with standard shotgun proteomics. Using
nonsmall cell lung cancer (NSCLC) as a model study, we aim to mine
missing proteins from disease-associated membrane proteome, which
may be still largely under-represented. To increase identification
coverage, we employed Hp-RP StageTip prefractionation of membrane-enriched
samples from 11 NSCLC cell lines. Analysis of membrane samples from
20 pairs of tumor and adjacent normal lung tissue was incorporated
to include physiologically expressed membrane proteins. Using multiple
search engines (X!Tandem, Comet, and Mascot) and stringent evaluation
of FDR (MAYU and PeptideShaker), we identified 7702 proteins (66%
membrane proteins) and 178 missing proteins (74 membrane proteins)
with PSM-, peptide-, and protein-level FDR of 1%. Through multiple
reaction monitoring using synthetic peptides, we provided additional
evidence of eight missing proteins including seven with transmembrane
helix domains. This study demonstrates that mining missing proteins
focused on cancer membrane subproteome can greatly contribute to map
the whole human proteome. All data were deposited into ProteomeXchange
with the identifier PXD002224.