Sensitive and Accurate Quantitation of Phosphopeptides Using TMT Isobaric Labeling Technique

Phosphorylation
is an essential post-translational modification
for regulating protein function and cellular signal transduction.
Mass spectrometry (MS) combined with isobaric tandem mass tags (TMTs)
has become a powerful platform for simultaneous, large-scale phospho-proteome
site identification and quantitation. To improve the accuracy of isobaric
tag-based quantitation in complex proteomic samples, MS3-based acquisition
methods such as Synchronous Precursor Selection (SPS) have been used.
However, the method suffers from lower peptide identification rates
when applied to enriched phosphopeptide samples compared with unmodified
samples due to differences in phosphopeptide fragmentation patterns
during tandem MS. We developed and optimized two new acquisition methods
for analysis of TMT-labeled multiplexed phosphoproteome samples, which
resulted in more phosphopeptide identifications with less ratio distortion
when compared with previous methods. We also applied these improved
methods to a large-scale study of phosphorylation levels in A549 cell
lines treated with insulin or insulin growth factor 1 (IGF-1). Overall,
3378 protein groups and 12 465 phosphopeptides were identified,
of which 10 436 were quantified across 10 samples without prefractionation.
The accurate measurement enabled us to map to numerous signaling pathways
including mechanistic target of rapamycin (mTOR), epidermal growth
factor receptor (EGFR, ErbB), and insulin signaling pathways.