Even though protein phosphatases are key regulators of signal transduction, 
their cellular mechanisms of action are poorly understood. Here, we undertook a 
large-scale proteomics survey to identify cellular protein targets of a 
serine/threonine phosphatase. We used SILAC-based quantitative MS to measure 
differences in protein expression and phosphorylation upon ablation of the 
serine/threonine phosphatase Ppt1 in Saccharomyces cerevisiae. Phosphopeptide 
fractionation by strong cation exchange chromatography combined with immobilized 
metal affinity chromatography (IMAC) enrichment enabled quantification of more 
than 8000 distinct phosphorylation sites in Ppt1 wild-type versus Ppt1-deficient 
yeast cells. We further quantified the relative expression of 1897 yeast 
proteins and detected no major protein changes accompanying Ppt1 deficiency. 
Notably, we found 33 phosphorylation sites to be significantly and reproducibly 
up-regulated while no phosphorylation events were repressed in cells lacking 
Ppt1. Ppt1 acted on its cellular target proteins in a sequence- and 
site-specific fashion. Several of the regulated phosphoproteins were involved in 
the response to heat stress in agreement with known Ppt1 functions. 
Additionally, biosynthetic enzymes were particularly prominent among 
Ppt1-regulated phosphoproteins, pointing to unappreciated roles of Ppt1 in the 
control of various metabolic functions. These results demonstrate the utility of 
large-scale and quantitative phosphoproteomics to identify cellular sites of 
serine/threonine phosphatase action in an unbiased manner.