33: A mass spectrometry-based approach for studying kinase activity

Abstract

Protein kinases play a key role in numerous cell signaling pathways. They have emerged as an important class of enzymes since aberrant kinase activity is associated with human diseases such as cancer and inflammatory disorders such as rheumatoid arthritis. While the human kinome is comprised of over 500 members, only a fraction have been exploited as attractive drug targets for therapeutic intervention. Thus, many members of the kinase superfamily and their role in human disease remain poorly understood. This can largely be attributed to the fact they are in low cellular abundance in complex protein samples and their detection is often reliant on the quality of available phosphoantibodies. In recent years, efforts have been made to develop immobilized small-molecule kinase inhibitors as kinase-capturing reagents. This kinase enrichment step coupled with highly sensitive mass spectrometry analysis allows for the comprehensive analysis of the kinome [1] . In this study, we have used two kinase-capturing reagents to profile the kinome from various cellular sources including HEK293Ts, human G-CSF mobilized neutrophils and murine bone marrow-derived macrophages (BMDMs). Kinome enrichment was achieved using CTx-0294885 resin (KiNET-1 beads, Synkinase) and a pan-JAK1/2 inhibitor Cyt387 (Cytopia) coupled to NHS-Sepharose beads. In this way we have identified a number of novel kinases within specific cell types, some of which may play important roles in the context of disease. This kinome profiing strategy was performed alongside a phosphopeptide enrichment strategy for quantitative mass spectrometry analysis. Using this approach we identified a number of novel phosphopeptide sites within clinically-relevant kinases including the JAK protein tyrosine kinases. We identified 40 phosphorylation sites in murine JAK2 (23 novel and 17 known sites), while 43 phosphorylation sites were identified in murine JAK1 (32 novel and 11 known sites). Mutational studies are currently underway to screen a selection of these evolutionarily-conserved candidate phosphorylation sites for functional relevance. The authors would like to acknowledge Leanne Daly from Synkinase for supplying the KiNET-1 beads used in this study.