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Technical Note

Optimizing Thiophosphorylation in the Presence of Competing Phosphorylation with MALDI-TOF−MS Detection

Supporting Info

Laurie L. Parker,*^† Alexander B. Schilling,^† Stephen J. Kron,^‡ and Stephen B. H. Kent^†

Department of Biochemistry and Molecular Biology, University of Chicago, CIS 201, 929 E. 57^th Street, Chicago, Illinois 60637, and Center for Molecular Oncology, University of Chicago, Knapp R322, 924 E. 57^th Street, Chicago, Illinois 60637

J. Proteome Res., 2005, 4 (5), pp 1863–1866

DOI: 10.1021/pr050150e

Publication Date (Web): September 15, 2005

To whom correspondence should be addressed. Tel: (773) 834-9989. Fax: (773) 702-0439. E-mail: lparker@uchicago.edu.

†

Department of Biochemistry and Molecular Biology.

‡

Center for Molecular Oncology.

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 Mn²⁺ 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.

Keywords: thiophosphorylation • MALDI-TOF-MS • kinase activity • Bcr-Abl • phosphoproteomics

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