Elaboration of a MALDI-TOF Mass Spectrometry-based Assay of Parkin Activity and High-Throughput screening platform for Parkin Activators

Parkinson’s disease (PD) is a progressive neurological disorder that manifests clinically as alterations in movement (bradykinesia, postural instability, loss of balance, and resting tremors) as well as multiple non-motor symptoms including but not limited to cognitive and autonomic abnormalities. Mitochondrial dysfunction has been linked to sporadic PD and loss-of-function mutations in genes encoding the ubiquitin E3 ligase Parkin and protein kinase, PTEN-induced kinase 1 (PINK1), that regulate mitophagy, are causal for familial and juvenile PD 1-3 . Among several therapeutic approaches being explored to treat or improve PD patient’s prognosis, the use of small molecules able to reinstate or boost Parkin activity represents a potential pharmacological treatment strategy 4 . A major barrier is the lack of high throughput platforms based on robust and accurate quantification of Parkin activity in vitro . Here we present two different and complementary Matrix Assisted Laser Desorption/Ionization-Time of Flight mass spectrometry (MALDI-TOF MS) based approaches for the quantification of Parkin E3 ligase activity in vitro . These methods recapitulate distinct aspects of ubiquitin conjugation: Parkin auto-ubiquitylation and Parkin-catalysed discharge on lysine residues. Both approaches are scalable for high-throughput primary screening to facilitate the identification of Parkin modulators.