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 Abl kinase thiophosphorylation rates can be 'rescued' using Mn ²⁺ 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.