Deep Coverage of Global Protein Expression and Phosphorylation in Breast Tumor Cell Lines Using TMT 10-plex Isobaric Labeling

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.