Relative quantification of peptide phosphorylation in a complex mixture using 18O labeling

1 Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee, Wisconsin 2 Human and Molecular Genetics Center, Medical College of Wisconsin, Milwaukee, Wisconsin 3 Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin We have developed a method to determine the degree of phosphorylation of a peptide in a complex mixture without enrichment or operation of the mass spectrometer in negative ion mode. Yeast lysate containing known amounts of synthetic peptides (VPQLEIVPNSAEERLHSMK and VPQLEIVPN[pS]AEERLHSMK) was labeled with 16 O and 18 O during hydrolysis. After treatment of one sample with a cocktail of phosphatases, the two samples were pooled. The intensity of the dephosphorylated peptide peaks was used to infer the degree of phosphorylation present before treatment. The linear dynamic range of this method is >10-fold before either of the peptide envelopes becomes indistinguishable from the surrounding noise. Since both the site of posttranslational modification and the proportion of the protein population that is modified are vital in protein function, the employment of this technique will provide a valuable tool for the analysis of the functional implications of protein phosphorylation. mass spectrometry; peptide ratio; isotopic labeling