Optimizing thiophosphorylation in the presence of competing phosphorylation with MALDI-TOF-MS detection.
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ABSTRACT:
SUBMITTER: Parker LL
PROVIDER: S-EPMC4568822 | biostudies-literature | 2005 Sep-Oct
REPOSITORIES: biostudies-literature
Publications
Optimizing thiophosphorylation in the presence of competing phosphorylation with MALDI-TOF-MS detection.
Parker Laurie L LL Schilling Alexander B AB Kron Stephen J SJ Kent Stephen B H SB
Journal of proteome research 20050901 5
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 relat ...[more]
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