Detection of in vitro kinase generated protein phosphorylation sites using gamma[18O4]-ATP and mass spectrometry.
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.
Zhou
Ming
M
SAIC-Frederick, Inc., National Cancer Institute at Frederick, Frederick, Maryland 21702-1201, USA.
Meng
Zhaojing
Z
Jobson
Andrew G
AG
Pommier
Yves
Y
Veenstra
Timothy D
TD
D000255:Adenosine Triphosphate / Q000032:analysis* / Q000737:chemistry*; D000595:Amino Acid Sequence; D002250:Carbon Radioisotopes / Q000737:chemistry; D008969:Molecular Sequence Data; D010104:Oxygen Radioisotopes / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011494:Protein Kinases / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013379:Substrate Specificity
D002250:Carbon Radioisotopes; D010104:Oxygen Radioisotopes; D010455:Peptides; D000255:Adenosine Triphosphate; D011494:Protein Kinases