This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We previously completed a large-scale analysis of physical protein interactions in yeast resulting in the identification of over 500 protein complexes (Nature 440, 637). More recently, we completed a screen for epistatically interacting genes in collaboration with Prof. Kevan Shokat, and have found that pairs of genes interacting positively correlate strongly with physical interactions, in particular between pairs of kinases/phosphatases and their substrate proteins. In this project, affinity tagged potential substrate proteins will be purified from yeast, digested into peptides, and enriched for phosphopeptides using the method of Beltrao et al., (PLoS Biol. 7(6):e1000134). Enriched fractions will be analyzed by LCMS.