Journal
JOURNAL OF PROTEOME RESEARCH
Volume 4, Issue 5, Pages 1863-1866Publisher
AMER CHEMICAL SOC
DOI: 10.1021/pr050150e
Keywords
thiophosphorylation; MALDI-TOF-MS; kinase activity; Bcr-Abl; phosphoproteomics
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Funding
- NCI NIH HHS [R21 CA103235, R33 CA 103235, R33 CA103235, F32 CA117672-01, F32 CA117672] Funding Source: Medline
- NHLBI NIH HHS [T32 HL 9723] Funding Source: Medline
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Thiophosphorylation provides a metabolically stable, chemically reactive phosphorylation analogue for analyzing the phosphoproteome in vitro and in vivo. We developed a MALDI-TOF-MS based assay for optimizing thiophosphopeptide production by a kinase even in the presence of Mg2+ and ATP. We found that Ab1 kinase thiophosphorylation rates can be 'rescued' using Mn2+ in the presence of Mg2+. Under our ideal conditions, titration of Mn2+ and ATP gamma S in the presence of Mg2+ allowed relatively rapid, highly specific thiophosphorylation by Ab1 tyrosine kinase, both as purified enzyme and in complex cell extracts.
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