Mining missing membrane proteins by high-ph reverse-phase stagetip fractionation and multiple reaction monitoring mass spectrometry
Journal
Journal of Proteome Research
Journal Volume
14
Journal Issue
9
Pages
3658-3669
Date Issued
2015
Author(s)
Kitata R.B.
Choong W.-K.
Tsai C.-F.
Lin T.-D.
Tsou C.-C.
Weng S.-H.
Chen Y.-J.
Arco S.-D.
Nesvizhskii A.-I.
Sung T.-Y.
Chen Y.-J.
Abstract
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. ? 2015 American Chemical Society.
Subjects
Hp-RP StageTip; lung cancer; membrane proteins; missing proteins; MRM
SDGs
Other Subjects
epidermal growth factor receptor; heat shock protein 90; membrane protein; messenger RNA; proteome; synthetic peptide; membrane protein; proteome; amino acid sequence; Article; cellular distribution; clinical article; controlled study; exon; female; genetic transcription; human; human cell; human tissue; in vivo study; lung cancer cell line; mass spectrometry; non small cell lung cancer; pH; priority journal; protein analysis; protein expression; protein family; protein localization; proteomics; signal noise ratio; tandem mass spectrometry; chemistry; liquid chromatography; molecular genetics; pH; procedures; tumor cell line; Amino Acid Sequence; Cell Line, Tumor; Chromatography, Liquid; Humans; Hydrogen-Ion Concentration; Membrane Proteins; Molecular Sequence Data; Proteome; Tandem Mass Spectrometry
Publisher
American Chemical Society
Type
journal article