Pharmaceutical Sciences Faculty Publications
Detection of In Vitro Kinase Generated Protein Phosphorylation Sites Using Gamma[18O4]-ATP and Mass Spectrometry
Document Type
Article
Publication Date
10-15-2007
Journal Title
Analytical Chemistry
ISSN
0003-2700
Volume
79
Issue
20
First Page
7603
Last Page
7610
DOI
10.1021/ac071584r
PubMed ID
17877366
Abstract
A novel stable-isotope labeling approach for identification of phosphopeptides that utilizes adenosine triphosphate, in which four oxygen-16 atoms attached to the terminal phosphate group are substituted with oxygen-18 [gamma((18)O4)-ATP], has been developed. The ability to use gamma((18)O4)-ATP to monitor phosphorylation modification within various proteins was conducted by performing in vitro kinase reactions in the presence of a 1:1 mixture of gamma((18)O4)-ATP and normal isotopic abundance ATP (ATP). After tryptic digestion, the peptides were analyzed using mass spectrometry (MS). Phosphorylated peptides are easily recognized within the MS spectrum owing to the presence of doublets separated by 6.01 Da; representing versions of the peptide modified by ATP and gamma((18)O4)-ATP. Standard peptides phosphorylated using gamma((18)O4)-ATP via in vitro kinase reactions showed no exchange loss of (18)O with (16)O. The identity of these doublets as phosphorylated peptides could be readily confirmed using tandem MS. The method described here provides the first direct stable-isotope labeling method to definitely detect phosphorylation sites within proteins.
Keywords
Adenosine triphosphate, amino acid sequence, carbon radioisotopes, oxygen radioisotopes, peptides, phosphorylation, protein kinases, spectrometry, mass, substrate specificity
Recommended Citation
Zhou, Ming; Meng, Zhaojing; Jobson, Andrew G.; Pommier, Yves; and Veenstra, Timothy D., "Detection of In Vitro Kinase Generated Protein Phosphorylation Sites Using Gamma[18O4]-ATP and Mass Spectrometry" (2007). Pharmaceutical Sciences Faculty Publications. 362.
https://digitalcommons.cedarville.edu/pharmaceutical_sciences_publications/362