Abstract: A combination of affinity purification, 2D-PAGE and peptide mass fingerprinting was employed to study the phosphoprotein complement of Saccharomyces cerevisiae. Protein extracts were first passed through a phosphoprotein affinity column, and the phosphoprotein-enriched eluate fractions were then separated on 2D gels and visualized by staining with SYPRO Ruby. Proteins were excised from the gels and identified by peptide mass fingerprinting; 11/13 protein spots identified from a gel of the phosphoprotein-enriched fraction had prior published evidence indicating that they were phosphoproteins. Additional experiments using a specific stain for phosphoproteins, prior incubation of the protein extract with alkaline phosphatase and blotting with monoclonal antibodies to phosphothreonine, phosphoserine and phosphotyrosine demonstrated that the phosphoprotein affinity column was an effective method for enriching phosphoproteins. Further validating the method, growth of yeast in the presence of sorbic acid resulted in altered phosphorylation of 17 proteins, 13 of which had prior published evidence that they were phosphoproteins or had ATP binding activity. Copyright (c) 2005 John Wiley & Sons, Ltd.