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Application Note 574
Conclusion
Thermo Scientific ActivX Desthiobiotin-ATP and -ADP
probe technology combined with Q Exactive LC-MS
analysis creates a powerful workflow enabling global
kinase identification and drug-inhibition profiling. These
novel active-site probes specifically captured ATPase
subfamily members and improved kinase MS detection for
inhibitor selectivity profiling and binding affinity assessment.
Unique data acquisition methods incorporating HR/AM
full-scan MS and multiplexed SIM events were used to
build spectral libraries and target kinase active-site peptides.
Multiplexed SIM events were also used to maximize cycle
times for co-eluting active-site peptides which increased
signals 7-fold over background. This increased dynamic
range enabled low-level peptide quantitation necessary for
IC50 determination. Integrated software further facilitated
automated method building and data processing to reduce
analysis time. This workflow was successfully used to
identify and quantify inhibition of over 126 kinases in a
60 minute gradient using the same instrument platform.
Acknowledgment
The authors would like to thank Dr. M. Patricelli of ActivX
Biosciences for his valuable insights and discussions.
References
1. Patricelli, M.P., et al. Chem Biol, 2011, 18(6),
699-710.
2. Patricelli, M.P., et al. Biochemistry, 2007, 46, 350-358.
3. Bomgarden, R., et al. Thermo Scientific Application
Note 515, 2011.
4. Michalski, A., et al. Mol Cell Proteomics, 2011, 10(9),
M111.011015
5. Karaman, M.W., et al. Nature Biotech, 2008 , 26,
127-132.