Phospho-iTRAQ: Assessing Isobaric Labels for the Large-Scale Study Of Phosphopeptide Stoichiometry

The
ability to distinguish between phosphopeptides of high and
low stoichiometry is essential to discover the true extent of protein
phosphorylation. We here extend the strategy whereby a peptide sample
is briefly split in two identical parts and differentially labeled
preceding the phosphatase treatment of one part. Our use of isobaric
tags for relative and absolute quantitation (iTRAQ) marks the first
time that isobaric tags have been applied for the large-scale analysis
of phosphopeptides. Our Phospho-iTRAQ method focuses on the unmodified
counterparts of phosphorylated peptides, which thus circumvents the
ionization, fragmentation, and phospho-enrichment difficulties that
hamper quantitation of stoichiometry in most common phosphoproteomics
methods. Since iTRAQ enables multiplexing, simultaneous (phospho)­proteome
comparison between internal replicates and multiple samples is possible.
The technique was validated on multiple instrument platforms by adding
internal standards of high stoichiometry to a complex lysate of control
and EGF-stimulated HeLa cells. To demonstrate the flexibility of Phospho-iTRAQ
with regards to the experimental setup, the proteome coverage was
extended through gel fractionation, while an internal replicate measurement
created more stringent data analysis opportunities. The latest developments
in MS instrumentation promise to further increase the resolution of
the stoichiometric measurement of Phospho-iTRAQ in the future. The
data have been deposited to the ProteomeXchange with identifier PXD001574.