A Strategy for Large-Scale Phosphoproteomics and SRM-Based Validation of Human Breast Cancer Tissue Samples

Protein phosphorylation is a key mechanism of cellular
signaling pathways and aberrant phosphorylation has been implicated
in a number of human diseases. Thus, approaches in phosphoproteomics
can contribute to the identification of key biomarkers to assess disease
pathogenesis and drug targets. Moreover, careful validation of large-scale
phosphoproteome analysis, which is lacking in the current protein-based
biomarker discovery, significantly increases the value of identified
biomarkers. Here, we performed large-scale differential phosphoproteome
analysis using IMAC coupled with the isobaric tag for relative quantification
(iTRAQ) technique and subsequent validation by selected/multiple reaction
monitoring (SRM/MRM) of human breast cancer tissues in high- and low-risk
recurrence groups. We identified 8309 phosphorylation sites on 3401
proteins, of which 3766 phosphopeptides (1927 phosphoproteins) were
able to be quantified and 133 phosphopeptides (117 phosphoproteins)
were differentially expressed between the two groups. Among them,
19 phosphopeptides were selected for further verification and 15 were
successfully quantified by SRM using stable isotope peptides as a
reference. The ratio of phosphopeptides between high- and low-risk
groups quantified by SRM was well correlated with iTRAQ-based quantification
with a few exceptions. These results suggest that large-scale phosphoproteome
quantification coupled with SRM-based validation is a powerful tool
for biomarker discovery using clinical samples.