A Novel Quantitative Proteomics Strategy To Study Phosphorylation-Dependent Peptide−Protein Interactions

Phosphorylation-dependent protein−protein interactions provide the mechanism for a large number
of intracellular signal transduction pathways. One of the goals of signal transduction research is to
understand more precisely the nature of these phosphorylation-dependent interactions. Here, we report
a novel strategy based on quantitative proteomics that allows for the rapid analysis of peptide−protein
interactions with more than one phosphorylation site involved. The phosphorylation of two tyrosine
residues, Y342 and Y346, within the linker B region of the protein-tyrosine kinase Syk is important for
optimal signaling from the B cell receptor for antigen. We employed four amino-specific, isobaric
reagents to differentially label proteins interacting in vitro with four Syk peptides containing none,
one, or two phosphates on tyrosine residues Y342 and Y346, respectively. In total, 76 proteins were
identified and quantified, 11 of which were dependent on the phosphorylation of individual tyrosine
residues. One of the proteins, peroxiredoxin 1, preferably bound to phosphorylated Y346, which was
further verified by Western blotting results. Thus, we demonstrate that the use of 4-fold multiplexing
allows for relative protein measurements simultaneously for the identification of interacting proteins
dependent on the phosphorylation of specific residues.
Keywords: Quantitative proteomics • Mass spectrometry • Isotope labeling • Phosphorylation • Tyrosine kinases