title: A strategy for identification and quantitation of phosphopeptides by liquid chromatography/tandem mass spectrometry.
abstract: 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.
authors.affiliation: Institute of Molecular Medicine, National Taiwan University, Taipei, Taiwan.
authors.forename: Y G
authors.initials: YG
authors.lastname: Tsay
authors.forename: Y H
authors.initials: YH
authors.lastname: Wang
authors.forename: C M
authors.initials: CM
authors.lastname: Chiu
authors.forename: B J
authors.initials: BJ
authors.lastname: Shen
authors.forename: S C
authors.initials: SC
authors.lastname: Lee
mesh_terms: D051698:Activating Transcription Factor 2; D000595:Amino Acid Sequence; D002478:Cells, Cultured / Q000528:radiation effects; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002853:Chromatography, Liquid; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D006868:Hydrolysis; D048031:JNK Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D013058:Mass Spectrometry / Q000379:methods*; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000032:analysis*; D010766:Phosphorylation; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013601:T-Lymphocytes / Q000187:drug effects / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014357:Trypsin / Q000494:pharmacology; D014466:Ultraviolet Rays
chemical_list: C497105:ATF2 protein, human; D051698:Activating Transcription Factor 2; D017362:Cyclic AMP Response Element-Binding Protein; D010748:Phosphopeptides; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D048031:JNK Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D020929:Mitogen-Activated Protein Kinase Kinases; D014357:Trypsin