Kinase fokussierte Chemoproteomik unter Verwendung gezielter Affinitätsaufreinigung und modernster, quantitativer Massenspektrometrie
Biologische und Biomimetische Chemie
Zusammenfassung der Projektergebnisse
Here I report the successful synthesis and application of a number of type I (compounds 1-3, 9) and type II (compounds 4-8, 10-13) kinase affinity reagents. All probes were tested for their ability to enrich protein and lipid kinases from cellular extracts using affinity enrichment and nanoLC-MS/MS analysis for protein identification and quantification. With the goal to increase the analytical throughput and to minimize the consumption of starting materials (e.g. affinity resin, cellular protein) we optimized the chemoproteomics workflow. By employing in-situ digest of kinases after a mild wash of affinity resins with TBS (to remove detergents) we were able to significantly downscale the process. With 10 µl of kinase affinity resin mixture (i.e. Kinobeads) and 600 µg of cellular protein we were able to quantify ca. 310 protein and lipid kinases in single nanoLC-MS/MS runs. We expect that the workflow presented here will greatly facilitate the applicability of KinoBead-based chemoproteomics for kinase inhibitor profiling and the measurement of kinase abundance in cellular extracts from cell lines and clinical cancer samples. Further, in a series of preliminary experiments we tested the ability of the type I affinity reagents 2 and 3 to differentially enrich activated or inactivated kinases from cellular extracts. To this end we designed a duplex SILAC experiment with HeLa cells either stimulated with 50 ng/ml EGF of treated with vehicle. Cellular extracts from stimulated and non-stimulated cells were subjected to affinity enrichment and kinases quantified as described to detect changes in the enrichment efficiency. We found that affinity reagents 2 and 3 were able to differentially enrich 9 protein kinases known to be downstream effectors of EGFR signaling. This is in good agreement with a very recently published study that concluded that activation state-dependent enrichment of kinases is possible using single kinase affinity reagents. We believe that a set of type I and type II reagents can be designed for activation state-dependent enrichment of a substantial fraction of the kinome and we anticipate that the affinity reagents presented here will be useful to achieve this goal. Further, we hope that the method could facilitate the profiling of kinase activation states in clinical samples.
Projektbezogene Publikationen (Auswahl)
- Comparing SILAC- and stable isotope dimethyl-labeling approaches for quantitative proteomics. J. Proteome Res. 2014 Sep 5; 13(9): 4164-74
Lau HT, Suh HW, Golkowski M, Ong SE
(Siehe online unter https://doi.org/10.1021/pr500630a) - Rapid profiling of protein kinase inhibitors by quantitative proteomics. MedChemComm. 2014 Mar; 5(3): 363-369
Golkowski M, Brigham JL, Perera GK, Romano GE, Maly DJ, Ong SE
(Siehe online unter https://doi.org/10.1039/c3md00315a)
