A Multiple Reaction Monitoring (MRM) Method to Detect Bcr-Abl Kinase Activity in CML Using a Peptide Biosensor

Abstract

The protein kinase Bcr-Abl plays a major role in the pathogenesis of chronic myelogenous leukemia (CML), and is the target of the breakthrough drug imatinib (Gleevec™). While most patients respond well to imatinib, approximately 30% never achieve remission or develop resistance within 1–5 years of starting imatinib treatment. Evidence from clinical studies suggests that achieving at least 50% inhibition of a patient’s Bcr-Abl kinase activity (relative to their level at diagnosis) is associated with improved patient outcomes, including reduced occurrence of resistance and longer maintenance of remission. Accordingly, sensitive assays for detecting Bcr-Abl kinase activity compatible with small amounts of patient material are desirable as potential companion diagnostics for imatinib. Here we report the detection of Bcr-Abl activity and inhibition by imatinib in the human CML cell line K562 using a cell-penetrating peptide biosensor and multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer. MRM enabled reproducible, selective detection of the peptide biosensor at fmol levels from aliquots of cell lysate equivalent to ∼15,000 cells. This degree of sensitivity will facilitate the miniaturization of the entire assay procedure down to cell numbers approaching 15,000, making it practical for translational applications in patient cells in which the limited amount of available patient material often presents a major challenge.

A Cell-Based Assay for Measuring Endogenous BcrAbl Kinase Activity and Inhibitor Resistance

“…The adjacent sequence (APTYSPPPPP) selectively binds the Abl SH3 domain and increases the affinity of the peptide for Abl kinase. The SH3-probe interaction is hypothesized to enhance specific phosphorylation of the substrate by BcrAbl, as implicated in our previous work [ 27 – 29 ] Finally the TAT cell penetrating sequence (RKKRRQRRRLL) facilitates uptake by the cell. We assess BcrAbl activity by measuring phosphorylation of the substrate sequence [ 27 ].…”

“…The multifunctional peptide substrate’s unique ability to enter many cell types and measure endogenous kinase activity could overcome many of the limitations described above that are associated with the current techniques. Our previous demonstrations of the BcrAbl assay have used mass spectrometry, immunoblot, and ELISA for detection of substrate phosphorylation [ 21 , 27 – 29 ]. Here we adapt our assay for measuring cell-based BcrAbl kinase activity in a multi-well plate format and improve assay performance.…”

Assays for tyrosine phosphorylation in human cells

“…As an alternative, substrate peptides can be conjugated with cell penetrating peptides and delivered to cells without the need for expression of a genetically engineered sensor construct. Phosphorylation of the substrate peptide can then be measured using many types of read-outs, including single cell electrophoresis (Soughayer et al, 2004;Turner et al, 2016), mass spectrometry (Placzek, Plebanek, Lipchik, Kidd, & Parker, 2010;Yang, Eissler, Hall, & Parker, 2013), fluorescence lifetime imaging (Damayanti, Parker, & Irudayaraj, 2013), or generic antiphosphotyrosine antibodies (Lipchik, Killins, Geahlen, & Parker, 2012;Ouellette, Noel, & Parker, 2016), depending on the particular substrate design and application. A unique advantage of these kinds of synthetic substrates is the ability to incorporate non-natural amino acids and other chemical moieties that would not be compatible with genetic encoding into sensor proteins, such as D amino acids to improve peptide stability (Proctor, Wang, Lawrence, & Allbritton, 2012a) or constrained phosphotyrosine analogs that are resistant to dephosphorylation (Turner et al, 2016).…”

NCI investment in nanotechnology: achievements and challenges for the future

“…21,22 A more recent example is from Dr. Laurie Parker and colleagues to develop a silver nanorod-based kinase protein biosensor for monitoring the efficacy of leukemia treatments. 23 This biosensor platform has surpassed critical early stage milestones and has received follow-up support for advanced development and validation. A different solicitation to support Bioengineering Research Partnerships (BRP), led by another institute at NIH, the National Institute for Biomedical Imaging and Bioengineering (NIBIB) with the participation of several NIH institutes, also provided early support for such projects.…”