WEST LAFAYETTE, Ind.—Protein-protein interactions are typically mediated by post-translational modifications, but when one protein carries multiple modification sites, it can be difficult to determine which residues play what roles in the interaction. Recently, however, scientists at Purdue University developed a multiplex method to identify and quantitate interactions between proteins and selectively phosphorylated peptides.
As described in the Journal of Proteome Research, the researchers focused their attention on Syk, a protein-tyrosine kinase involved in multiple signal transduction pathways. The researchers synthesized four peptides carrying two tyrosine residues (Syk 338-353) without phosphorylation or phosphorylated at either or both tyrosines, and attached the peptides to resin beads.
The researchers incubated the beads with equal amounts of cell lysates to affinity purify peptide-binding proteins. After eluting the proteins and digesting them with trypsin, the researchers labeled the peptides associated with each peptide with a different iTRAQ tag. They then combined the labeled peptides for LC-MS/MS analysis.
Nonspecifically bound proteins will result in equally large iTRAQ peaks, whereas if a protein binds selectively to one peptide, the peak from that sample will be larger than for the other samples, and the ratio of peak size will be related to the degree of binding specificity. The researchers found 11 proteins with distinct binding preferences and were surprised to find some proteins never suspected to be involved with Syk.