Correlation profiling for determining kinase-substrate relationships.

ABSTRACT

Arguably the most important issue in phosphorylation studies is the determination of kinase-substrate relationships. Kinase Activity-Abundance Correlation (KAAC) profiling is a technique that can be used to suggest the kinase responsible for a particular phosphorylation event. The method involves the separation of a lysate at the protein level, after which fractions are analyzed to produce an activity profile for each substrate peptide. This activity profile is correlated with the kinase abundance profiles (obtained using shotgun proteomics) to obtain candidate kinases responsible for phosphorylation of the substrate peptide. We demonstrate this approach by determining potential kinase-substrate pairs for six peptides, selected from the literature, that were shown to be upregulated during mitosis. Finally, for a subset of fractions we evaluated the use of stable isotope labeling to improve quantification.