Comprehensive identification of staurosporine-binding kinases in the hepatocyte cell line HepG2 using Capture Compound Mass Spectrometry (CCMS).
The central role of kinases in cell signaling has set them in the focus of biomedical research. In functional proteomics analyses, large- scale profiling of kinases has become feasible through the use of affinity pulldown beads that carry immobilized kinase inhibitors. As an alternative approach to solid phase beads, Capture Compound Mass Spectrometry (CCMS) enables the functional isolation of protein-classes on the basis of small molecule-protein interactions in solution. Capture Compounds are trifunctional probes: a selectivity function interacts with the native target proteins in equilibrium, upon irradiation a photoactivatable reactivity function forms an irreversible covalent bond to the target proteins, and a sorting function allows the captured proteins to be isolated from a complex protein mixture. We report the design and application of a novel, fully water-soluble Capture Compound that carries the broadband kinase inhibitor staurosporine as selectivity function. We used this Capture Compound to profile the kinome of the human liver-derived cell line HepG2 and identified one hundred kinases. HepG2 cells are a widely used model system for hepatocarcinoma, hepatitis, and for investigation of drug toxicity effects. CCMS experiments in membrane fractions of human placenta are given as example for the applicability to human tissue.
Fischer
Jenny J
JJ
caprotec bioanalytics GmbH, Volmerstrasse 5, 12489 Berlin, Germany.
Graebner Baessler
Olivia Y
OY
Dalhoff
Christian
C
Michaelis
Simon
S
Schrey
Anna K
AK
Ungewiss
Jan
J
Andrich
Kathrin
K
Jeske
Danny
D
Kroll
Friedrich
F
Glinski
Mirko
M
Sefkow
Michael
M
Dreger
Mathias
M
Koester
Hubert
H
D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D010770:Phosphotransferases / Q000378:metabolism*; D019311:Staurosporine / Q000378:metabolism / Q000494:pharmacology*
D010770:Phosphotransferases; D019311:Staurosporine