Optimizing thiophosphorylation in the presence of competing phosphorylation with MALDI-TOF-MS detection

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.