Reproducible workflow for multiplexed deep-scale proteome and phosphoproteome analysis of tumor tissues by liquid chromatography-mass spectrometry.

Nat Protoc
Authors
Keywords
Abstract

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 3× 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 μg of peptide derived from 0.88. The maximum deviation for the phosphoproteome coverage was 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 ~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.

Year of Publication
2018
Journal
Nat Protoc
Volume
13
Issue
7
Pages
1632-1661
Date Published
2018 07
ISSN
1750-2799
DOI
10.1038/s41596-018-0006-9
PubMed ID
29988108
PubMed Central ID
PMC6211289
Links
Grant list
U24 CA210985 / CA / NCI NIH HHS / United States
U24 CA210986 / CA / NCI NIH HHS / United States
U01 CA114722 / CA / NCI NIH HHS / United States
U24 CA210955 / CA / NCI NIH HHS / United States
P30 CA091842 / CA / NCI NIH HHS / United States
UL1 TR000448 / TR / NCATS NIH HHS / United States
U24 CA160035 / CA / NCI NIH HHS / United States
UL1 TR002345 / TR / NCATS NIH HHS / United States
P41 RR000954 / RR / NCRR NIH HHS / United States
R01 CA095614 / CA / NCI NIH HHS / United States