A strategy for identification and quantitation of phosphopeptides by liquid chromatography/tandem mass spectrometry.

Liquid chromatography/tandem mass spectrometry (LC/MS/MS) is a state-of-the-art method of structural analysis of peptides/proteins. Here, using activating transcription factor-2 (ATF2) as an example, we report how LC/MS/MS data were processed to generate selected ion tracings for identification of phosphorylated peptides based on their parallel elution behavior with their nonphosphorylated analogs. Via this approach, we verified that amino acid residues Thr-69, Thr-71, and Ser-90 of ATF2 were the in vitro targets for c-Jun kinase. Selected ion tracing method was also used to quantitatively determine phosphorylation states of peptides. We demonstrated that the phosphorylation of Thr-69/Thr-71 was increased in response to ultraviolet irradiation specifically in subconfluent but not in confluent cultures. About 24% of Thr-69/Thr-71-containing segment were singly phosphorylated in subconfluent cultures, while minimal phosphorylation occurred in confluent cultures. In contrast, Ser-112 phosphorylation remained unaffected by cell densities. This strategy could be applied to the studies of a variety of modifications seen in various regulated cellular processes.