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 Mg(2+) and ATP. We found that Abl kinase thiophosphorylation rates can be "rescued" using Mn(2+) in the presence of Mg(2+). Under our ideal conditions, titration of Mn(2+) and ATPgammaS in the presence of Mg(2+) allowed relatively rapid, highly specific thiophosphorylation by Abl tyrosine kinase, both as purified enzyme and in complex cell extracts.

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phosphorylation analogue abl kinase thiophosphorylation malditofms based assay thiophosphopeptide production

Laurie L, Parker Alexander B, Schilling Stephen J, Kron Stephen B H, Kent,.Optimizing thiophosphorylation in the presence of competing phosphorylation with MALDI-TOF-MS detection.. 4 (5),1863-6.

Laurie L, Parker Alexander B, Schilling Stephen J, Kron Stephen B H, Kent,"Optimizing thiophosphorylation in the presence of competing phosphorylation with MALDI-TOF-MS detection." 4

Laurie L, Parker Alexander B, Schilling Stephen J, Kron Stephen B H, Kent,"Optimizing thiophosphorylation in the presence of competing phosphorylation with MALDI-TOF-MS detection."