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 activa ting transcription factor-2 (ATF2) as an example, we report how LC/MS/MS da ta were processed to generate selected ion tracings for identification of p hosphorylated peptides based on their parallel elution behavior with their nonphosphorylated analogs. Via this approach, we verified that amino acid r esidues Thr-69, Thr-71, and Ser-90 of ATFS were the in vitro targets for c- Jun kinase. Selected ion tracing method was also used to quantitatively det ermine phosphorylation states of peptides. We demonstrated that the phospho rylation of Thr-69/Thr-71 was increased in response to ultraviolet irradiat ion specifically in subconfluent but not in confluent cultures. About 24% o f Thr-69/Thr-71-containing segment were singly phosphorylated in subconflue nt 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 modification s seen in various regulated cellular processes. (C) 2000 Academic Press.