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 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.
Parker
Laurie L
LL
Department of Biochemistry and Molecular Biology, University of Chicago, CIS 201, 929 E. 57 Street, Chicago, IL 60637, USA. lparker@uchicago.edu
Schilling
Alexander B
AB
Kron
Stephen J
SJ
Kent
Stephen B H
SB
D000255:Adenosine Triphosphate / Q000737:chemistry; D019921:Biotinylation; D002138:Calibration; D003035:Cobalt / Q000737:chemistry; D006801:Humans; D020014:K562 Cells; D008274:Magnesium / Q000737:chemistry*; D008345:Manganese / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020543:Proteome; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D010455:Peptides; D010748:Phosphopeptides; D020543:Proteome; D003035:Cobalt; D008345:Manganese; D000255:Adenosine Triphosphate; D008274:Magnesium