Cellular processes such as proliferation, differentiation, and adaptation to 
environmental changes are regulated by protein phosphorylation. Development of 
sensitive and comprehensive analytical methods for determination of protein 
phosphorylation is therefore a necessity in the pursuit of a detailed molecular 
view of complex biological processes. We present a quantitative 
modification-specific proteomic approach that combines stable isotope labeling 
by amino acids in cell culture (SILAC) for quantitation with IMAC for 
phosphopeptide enrichment and three stages of mass spectrometry (MS/MS/MS) for 
identification. This integrated phosphoproteomic technology identified and 
quantified phosphorylation in key regulator and effector proteins of a 
prototypical G-protein-coupled receptor signaling pathway, the yeast pheromone 
response. SILAC encoding of yeast proteomes was achieved by incorporation of 
[(13)C(6)]arginine and [(13)C(6)]lysine in a double auxotroph yeast strain. 
Pheromone-treated yeast cells were mixed with SILAC-encoded cells as the control 
and lysed, and extracted proteins were digested with trypsin. Phosphopeptides 
were enriched by a combination of strong cation exchange chromatography and 
IMAC. Phosphopeptide fractions were analyzed by LC-MS using a linear ion 
trap-Fourier transform ion cyclotron resonance mass spectrometer. MS/MS and 
neutral loss-directed MS/MS/MS analysis allowed detection and sequencing of 
phosphopeptides with exceptional accuracy and specificity. Of more than 700 
identified phosphopeptides, 139 were differentially regulated at least 2-fold in 
response to mating pheromone. Among these regulated proteins were components 
belonging to the mitogen-activated protein kinase signaling pathway and to 
downstream processes including transcriptional regulation, the establishment of 
polarized growth, and the regulation of the cell cycle.