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Optimizing Thiophosphorylation in the Presence of Competing Phosphorylation with MALDI-TOF−MS Detection
Ist Teil von
Journal of proteome research, 2005-09, Vol.4 (5), p.1863-1866
Ort / Verlag
United States: American Chemical Society
Erscheinungsjahr
2005
Link zum Volltext
Quelle
Alma/SFX Local Collection
Beschreibungen/Notizen
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 Abl kinase thiophosphorylation rates can be ‘rescued' using Mn2+ in the presence of Mg2+. Under our ideal conditions, titration of Mn2+ and ATPγS in the presence of Mg2+ allowed relatively rapid, highly specific thiophosphorylation by Abl tyrosine kinase, both as purified enzyme and in complex cell extracts. Keywords: thiophosphorylation • MALDI-TOF-MS • kinase activity • Bcr-Abl • phosphoproteomics