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

Laurie L. Parker, Alexander B. Schilling, Stephen J. Kron, Stephen B.H. Kent

Research output: Contribution to journalArticlepeer-review

9 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1863-1866
Number of pages4
JournalJournal of Proteome Research
Volume4
Issue number5
DOIs
StatePublished - Sep 2005

Keywords

  • Bcr-Abl
  • Kinase activity
  • MALDI-TOF-MS
  • Phosphoproteomics
  • Thiophosphorylation

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