1. Robust label free targeted proteomic workflow to identify and validate biomarkers of
kinase inhibition: comparative evaluation of CK2 inhibitors for their inhibition of CK2 in
cells
Laszlo Gyenis1
, Sam R. Fess1
, Stephanie A. Zukowski1
, Jacob P. Turowec1
, Paula Pittock1
,
Gilles Lajoie1
and David W. Litchfield1,2
1
Department of Biochemistry and 2
Department of Oncology, Schulich School of Medicine &
Dentistry, Western University, London, Ontario, Canada. N6A 5C1
ABSTRACT (250 words max)
Protein kinases have emerged as promising therapeutic agents resulting from the discovery of
their role in numerous human diseases including cancer. While there has been considerable
interest in the therapeutic potential of kinase inhibitors, many have not been tested by unbiased
strategies to validate both inhibitor specificity and target engagement within living cells. To
overcome these limitations, we have developed a label free-targeted LC-MS workflow for
identification and validation of biomarkers for kinase inhibition that we have applied to
characterize inhibitors of protein kinase CK2. Although our methods are optimized for CK2,
they are easily adaptable for any substrate-kinase or inhibitor-kinase relation characterization.
This workflow is free of phospho-peptide enrichment, which provided a robust platform to
measure kinase inhibition through quantification of both phospho- and non-phospho- species of
the same peptide phosphorylated by the kinase. Since non-phosphorylated peptides are detected
easier in any MS instrument, our method provided an increased sensitivity to monitor CK2
inhibition with elongation factor 1-delta (EF1D) and demonstrated that eukaryotic translation
initiation factor 2 (IF2B) is superior biomarker of CK2 inhibition. Our comparison of eight
commercially available CK2 inhibitors (TBB, TBBz, DMAT, Ellagic Acid, Quinalirazin,
Resorufin, CX-4945 and inhibitor VIII) showed that CX-4945 and inhibitor VIII were most
effective at the inhibition of CK2 in human osteosarcoma U2OS and adenocarcinoma HeLa
cells. Overall, our methods have yielded implementation of systematic platforms for studying
CK2 inhibitors and further characterize the biological functions of CK2.