期刊
JOURNAL OF PROTEOME RESEARCH
卷 11, 期 8, 页码 4201-4210出版社
AMER CHEMICAL SOC
DOI: 10.1021/pr300322q
关键词
iTRAQ; MammaPrint; SRM/MRM; shotgun proteomics; PTS; biomarker; plasma membrane
资金
- Ministry of Health, Labour, and Welfare of Japan [H20-0005]
- Ministry of Education, Science, Sports, and Culture of Japan [21390354, 22800095]
- Grants-in-Aid for Scientific Research [24701015, 22800095, 21390354, 22590545, 23501303] Funding Source: KAKEN
Since LC-MS-based quantitative proteomics has become increasingly applied to a wide range of biological applications over the past decade, numerous studies have performed relative and/or absolute abundance determinations across large sets of proteins. In this study, we discovered prognostic biomarker candidates from limited breast cancer tissue samples using discovery-through-verification strategy combining iTRAQ method followed by selected reaction monitoring/multiple reaction monitoring analysis (SRM/MRM). We identified and quantified 5122 proteins with high confidence in 18 patient tissue samples (pooled high-risk (n = 9) or low-risk (n = 9)). A total of 2480 proteins (48.4%) of them were annotated as membrane proteins, 16.1% were plasma membrane and 6.6% were extracellular space proteins by Gene Ontology analysis. Forty-nine proteins with >2-fold differences in two groups were chosen for further analysis and verified in 16 individual tissue samples (high-risk (n = 9) or low-risk (n = 7)) using SRM/MRM. Twenty-three proteins were differentially expressed among two groups of which MFAP4 and GP2 were further confirmed by Western blotting in 17 tissue samples (high-risk (n = 9) or low-risk (n = 8)) and Immunohistochemistry (IHC) in 24 tissue samples (high-risk (n = 12) or low-risk (n = 12)). These results indicate that the combination of iTRAQ and SRM/MRM proteomics will be a powerful tool for identification and verification of candidate protein biomarkers.
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