Phospho-iTRAQ: Assessing Isobaric Labels for the Large-Scale Study Of Phosphopeptide Stoichiometry
Pieter Glibert, Paulien Meert, Katleen Van Steendam, Filip Van Nieuwerburgh, Dieter De Coninck, Lennart Martens, Maarten Dhaenens, and Dieter Deforce
J. Proteome Res. (2015) 14: 839–849
With an estimated 30% of all proteins being phosphorylated, detailed analysis of the prevalence of this important modification is important for the elucidation of a range for cellular processes. While many workflows have focused on the enrichment of the phosphopeptides and subsequent quantitation by label-free or labeling approaches, the stoichiometry or occupancy of phosphorylation is not determined.
To measure the occupancy with the Phospho-iTRAQ method, a peptide sample is briefly split in two identical parts and differentially labeled preceding the phosphatase treatment of one part to remove the phosphate moieties. By focusing on the unmodified counterparts of phosphorylated peptides, the method circumvents the ionization, fragmentation, and enrichment difficulties that hamper quantitation of stoichiometry in most common phosphoproteomics methods. The technique was validated on multiple instrument platforms using a complex lysate of EGF-stimulated HeLa cells.