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

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• 作者：Reta Birhanu Kitata ; Baby Rorielyn T. Dimayacyac-Esleta ; Wai-Kok Choong ; Chia-Feng Tsai ; Tai-Du Lin ; Chih-Chiang Tsou ; Shao-Hsing Weng ; Yi-Ju Chen ; Pan-Chyr Yang ; Susan D. Arco ; Alexey I. Nesvizhskii ; Ting-Yi Sung ; Yu-Ju Chen
• 刊名：Journal of Proteome Research
• 出版年：2015
• 出版时间：September 4, 2015
• 年：2015
• 卷：14
• 期：9
• 页码：3658-3669
• 全文大小：552K
• ISSN：1535-3907

文摘

Despite significant efforts in the past decade toward complete mapping of the human proteome, 3564 proteins (neXtProt, 09-2014) are still 鈥渕issing proteins鈥? 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.