Yg. Tsay et al., A strategy for identification and quantitation of phosphopeptides by liquid chromatography/tandem mass spectrometry, ANALYT BIOC, 287(1), 2000, pp. 55-64
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.