Journal
NATURE PROTOCOLS
Volume 13, Issue 7, Pages 1632-1661Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/s41596-018-0006-9
Keywords
-
Categories
Funding
- National Cancer Institute (NCI) Clinical Proteomic Tumor Analysis Consortium (CPTAC) [1U24CA210986, U24CA210955, U24CA210985, U24CA160035]
- DOE [DE-AC05-76RL01830]
- Washington University Institute of Clinical and Translational Sciences grant from the National Center for Advancing Translational Sciences (NCATS) of the National Institutes of Health (NIH) [UL1 TR000448]
Ask authors/readers for more resources
Here we present an optimized workflow for global proteome and phosphoproteome analysis of tissues or cell lines that uses isobaric tags (TMT (tandem mass tags)-10) for multiplexed analysis and relative quantification, and provides 3x higher throughput than iTRAQ (isobaric tags for absolute and relative quantification)-4-based methods with high intra- and inter-laboratory reproducibility. The workflow was systematically characterized and benchmarked across three independent laboratories using two distinct breast cancer subtypes from patient-derived xenograft models to enable assessment of proteome and phosphoproteome depth and quantitative reproducibility. Each plex consisted of ten samples, each being 300 mu g of peptide derived from < 50 mg of wet-weight tissue. Of the 10,000 proteins quantified per sample, we could distinguish 7,700 human proteins derived from tumor cells and 3100 mouse proteins derived from the surrounding stroma and blood. The maximum deviation across replicates and laboratories was < 7%, and the inter-laboratory correlation for TMT ratio-based comparison of the two breast cancer subtypes was r > 0.88. The maximum deviation for the phosphoproteome coverage was < 24% across laboratories, with an average of > 37,000 quantified phosphosites per sample and differential quantification correlations of r > 0.72. The full procedure, including sample processing and data generation, can be completed within 10 d for ten tissue samples, and 100 samples can be analyzed in similar to 4 months using a single LC-MS/MS instrument. The high quality, depth, and reproducibility of the data obtained both within and across laboratories should enable new biological insights to be obtained from mass spectrometry-based proteomics analyses of cells and tissues together with proteogenomic data integration.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available