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J Proteome Res. 2015 Sep 4;14(9):3658-69. doi: 10.1021/acs.jproteome.5b00477. Epub 2015 Aug 6.

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

Author information

1
Institute of Chemistry, Academia Sinica , No. 128, Academia Road Sec. 2, Taipei 115, Taiwan.
2
Department of Chemistry, National Tsing Hua University , 101, Sec 2, Kuang-Fu Road, Hsinchu 30013, Taiwan.
3
Molecular Science and Technology Program, Taiwan International Graduate Program, Academia Sinica , No. 1, Roosevelt Road, Sec. 4, Taipei 10617, Taiwan.
4
Institute of Chemistry, University of the Philippines , Diliman Quezon City, Philippines.
5
Institute of Information Science, Academia Sinica , 128 Academia Road, Section 2, Taipei 115, Taiwan.
6
Department of Biochemical Sciences, National Taiwan University , 1 Roosevelt Road, Sec. 4, Taipei 106, Taiwan.
7
Department of Computational Medicine and Bioinformatics and Department of Pathology, University of Michigan Medical School , 1301 Catherine, Ann Arbor, Michigan 48109, United States.
8
Genome and Systems Biology Degree Program, National Taiwan University , 1, Roosevelt Road, Section 4, Taipei 10617, Taiwan.
9
Department of Internal Medicine, National Taiwan University Hospital , 1 Jen-Ai Road, Section 1, Taipei 10051, Taiwan.
10
National Taiwan University College of Medicine , No. 1, Section 1, Ren'ai Road, Taipei 100, Taiwan.
11
Institute of Biomedical Science, Academia Sinica , 128 Academia Road, Section 2, Taipei 115, Taiwan.

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.

KEYWORDS:

Hp-RP StageTip; MRM; lung cancer; membrane proteins; missing proteins

PMID:
26202522
PMCID:
PMC4761227
DOI:
10.1021/acs.jproteome.5b00477
[Indexed for MEDLINE]
Free PMC Article

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