Mass Spectrometry Based Method to Increase Throughput for Kinome Analyses Using ATP Probes
1 Department of Cell Biology, Harvard Medical School, Boston, MA; 2 HMS LINCS Center, Harvard Medical School, Boston, MA; 3 These authors contributed equally to this work.
SynopsisKinome-wide profiling aims to provide a systematic, unbiased look at kinase levels and modification states across biological and pathological processes. Current kinase analysis methods, however, are low in throughput and often poor at detecting low-abundance kinases unless time-consuming cellular fractionation steps are undertaken. We describe here a robust method that combines ActivX ATP probe (AAP) affinity reagents (ATP analogues) with 6-plex tandem mass tags (TMT) isotopic labeling of cell lysates and enables multiplexed analysis of ~90 kinases across six conditions in a single LC-MS run.
- Combination of ActivX ATP probe (AAP) affinity reagents with multiplexed isotopic labeling (TMT) enables systematic kinome analysis across up to six samples in a single LC-MS run.
- AAPs crosslink to both inactive and active kinases. Inclusion of a phosphopeptide enrichment step provides additional information on kinase activation state.
Protein kinases play critical roles in many biological and pathological processes, making them important targets for therapeutic drugs. Here, we desired to increase the throughput for kinome-wide profiling. A new workflow coupling ActivX ATP probe (AAP) affinity reagents with isotopic labeling to quantify the relative levels and modification states of kinases in cell lysates is described. We compared the new workflow to a classical proteomics approach in which fractionation was used to identify low-abundance kinases. We find that AAPs enriched approximately 90 kinases in a single analysis involving six cell lines or states in a single run, an 8-fold improvement in throughput relative to the classical approach. In general, AAPs crosslinked to both the active and inactive states of kinases, but performing phosphopeptide enrichment made it possible to measure the phospho sites of regulatory residues lying in the kinase activation loops, providing information on activation state. When we compared the kinome across the six cell lines, representative of different breast cancer clinical subtypes, we observed that many kinases, particularly receptor tyrosine kinases, varied widely in abundance, perhaps explaining the differential sensitivities to kinase inhibitor drugs. The improved kinome profiling methods described here represent an effective means to perform systematic analysis of kinases involved in cell signaling and oncogenic transformation and for analyzing the effect of different inhibitory drugs.
NIH grant HG3456 and NIH LINCS grant U54 HG006097.