Measuring Abl Kinase Activity Using Silver Nanoparticle-Based Biosensors
Authors:Andrew Lipchik , Jessica Malinchak
Mentor:Laurie Parker , Assistant Professor of Medicinal Chemistry and Molecular Pharmacology, Purdue University
Phosphorylation is the process of a protein gaining a phosphate group. This translational modification is catalyzed by a group of enzymes called kinases. These enzymes are responsible for using the gamma phosphate group from an energy molecule, adenine triphosphate (ATP), and attaching it to the amino acids serine and threonine, or tyrosine on a target protein. Phosphorylation by protein kinases is a tightly regulated process that is crucial for maintaining cellular functions. Cancer cells lose control of this signaling and gain capabilities to allow for uncontrolled proliferation. The importance of protein phosphorylation as a regulatory process has led to the development of kinase inhibitors as therapeutic drugs. Chronic myeloid leukemia (CML) is a clonal cancer with unregulated proliferation caused by an oncogenic fusion kinase, Bcr-Abl. Inhibition of this kinase is central to decreasing the development of CML. Imatinib (Gleevec, Glivec, formerly STI571) is the standard FDA approved inhibitor of Bcr-Abl that has been shown to decrease the amount of proliferating CML Bcr-Abl cells.
Sensitive and accurate methods of detecting Abl kinase activity are essential for prognosis and diagnosis of CML. The objective of this study is to detect Abl kinase activity using silver nanoparticle (AgNP)-based biosensors. AgNPs were synthesized and functionalized with an Abl specific peptide substrate. An in-vitro kinase assay was performed on the prepared biosensor and later analyzed with a Matrix-Assisted Laser Desorption/Ionization-Time Of Flight Mass Spectrometry (MALDI-TOF MS). Signal readouts from the MALDI-TOF MS could then be plotted on a calibrated curve to report the quantitative percent phosphorylation of the Abl substrate.