Quantification of Protein Kinase Activities by LC-MS - LC-MS/MS in Proteomics: Methods and Applications - page 322

Chapter 20

Quantification of Protein Kinase Activities by LC-MS

Maria P. Alcolea and Pedro R. Cutillas

Abstract

Measuring the enzymatic activity of protein kinases in cell and tissue extracts represents a difficult task
owing to the complex regulation and dynamics of such enzymes. Here we describe a sensitive and specific
approach for the quantitative analysis of PI3K-dependent protein kinase activity based on the mass spec-
trometry measurement of reaction products. The principle of this method can be applied to develop other
kinase assays and thus should contribute to the understanding of processes controlled by protein kinases.
Because of the enhanced sensitivity of this technique, it may be applied to the multiplex measurement of
pathway activities when sample amounts are limiting.

Key words: PI3K signalling pathway, AKT/PKB, kinase enzymatic assay, strong cation exchange
(SCX), kinase activity quantification, mass spectrometry, cancer.

1. Introduction

Protein kinases are enzymes of significant importance in biochem-
istry since they control the regulation of pathways involved in
many different biological processes, including energy metabolism,
cellular proliferation, differentiation, migration and cell death ( 1 ) .
All of these processes are known to be deregulated in cancer, mak-
ing protein kinases drug targets of increasing interest in oncol-
ogy ( 2 ) .
Accurate and sensitive methods to quantify the activation
of protein kinases are an essential requirement in preclinical
research to evaluate the potential of developing drugs against spe-
cific kinases or pathways. These approaches are invaluable dur-
ing the drug developmental process to monitor the extent to
which lead compounds inhibit their target in cells and tissues (i.e.