Deep coverage of global protein expression and phosphorylation in breast tumor cell lines using TMT 10-plex isobaric labeling Journal Article


Authors: Huang, F. K.; Zhang, G.; Lawlor, K.; Nazarian, A.; Philip, J.; Tempst, P.; Dephoure, N.; Neubert, T. A.
Article Title: Deep coverage of global protein expression and phosphorylation in breast tumor cell lines using TMT 10-plex isobaric labeling
Abstract: Labeling peptides with isobaric tags is a popular strategy in quantitative bottom-up proteomics. In this study, we labeled six breast tumor cell lysates (1.34 mg proteins per channel) using 10-plex tandem mass tag reagents and analyzed the samples on a Q Exactive HF Quadrupole-Orbitrap mass spectrometer. We identified a total of 8,706 proteins and 28,186 phosphopeptides, including 7,394 proteins and 23,739 phosphosites common to all channels. The majority of technical replicates correlated with a R2 ≥ 0.98, indicating minimum variability was introduced after labeling. Unsupervised hierarchical clustering of phosphopeptide data sets successfully classified the breast tumor samples into Her2 (epidermal growth factor receptor 2) positive and Her2 negative groups, whereas mRNA abundance did not. The tyrosine phosphorylation levels of receptor tyrosine kinases, phosphoinositide-3-kinase, protein kinase C delta, and Src homology 2, among others, were significantly higher in the Her2 positive than the Her2 negative group. Despite ratio compression in MS2-based experiments, we demonstrated the ratios calculated using an MS2 method are highly correlated (R2 > 0.65) with ratios obtained using MS3-based quantitation (using a Thermo Orbitrap Fusion mass spectrometer) with reduced ratio suppression. Given the deep coverage of global and phosphoproteomes, our data show that MS2-based quantitation using TMT can be successfully used for large-scale multiplexed quantitative proteomics. © 2017 American Chemical Society.
Keywords: mass spectrometry; titanium dioxide; tyrosine phosphorylation; quantitative proteomics; phosphoproteomics; hierarchical clustering; tandem mass tag
Journal Title: Journal of Proteome Research
Volume: 16
Issue: 3
ISSN: 1535-3893
Publisher: American Chemical Society  
Date Published: 2017-03-03
Start Page: 1121
End Page: 1132
Language: English
DOI: 10.1021/acs.jproteome.6b00374
PROVIDER: scopus
PMCID: PMC5336479
PUBMED: 28102081
DOI/URL:
Notes: Article -- Export Date: 3 April 2017 -- Source: Scopus
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MSK Authors
  1. Paul J Tempst
    307 Tempst
  2. Kevin S Lawlor
    9 Lawlor
  3. John Philip
    20 Philip