Accurate MS-based Rab10 Phosphorylation Stoichiometry Determination as Readout for LRRK2 Activity in Parkinson's Disease

Pathogenic mutations in the Leucine-rich repeat kinase 2 (LRRK2) increase its activity leading to increased phosphorylation of Rab proteins. Here we introduce a sensitive and accurate assay to measure increased phospho Rab levels using synthetic stable isotope-labeled analogues for both phosphorylated and non-phosphorylated tryptic peptides surrounding Rab10-Thr73. Compared to healthy controls, carriers of mutated LRRK2 had 1.9-fold and those with VPS35 mutation 3.7-fold increased pRab10 levels in their neutrophils. Our generic MS-based assay helps to stratify PD patient and determine LRRK2 inhibitor efficiency in clinical trials. [Display omitted] Highlights •MS-based clinical assay that accurately determines phospho Rab10 occupancy.•Stable isotope labeled phosphopeptide injected as a standard with endogenous tryptic phospho Rab peptide for accurate ratio determination.•Determination of pRab levels in neutrophils of Parkinson disease patients.•Relevance of pRab levels as marker of PD. Pathogenic mutations in the Leucine-rich repeat kinase 2 (LRRK2) are the predominant genetic cause of Parkinson's disease (PD). They increase its activity, resulting in augmented Rab10-Thr73 phosphorylation and conversely, LRRK2 inhibition decreases pRab10 levels. Currently, there is no assay to quantify pRab10 levels for drug target engagement or patient stratification. To meet this challenge, we developed an high accuracy and sensitivity targeted mass spectrometry (MS)-based assay for determining Rab10-Thr73 phosphorylation stoichiometry in human samples. It uses synthetic stable isotope-labeled (SIL) analogues for both phosphorylated and nonphosphorylated tryptic peptides surrounding Rab10-Thr73 to directly derive the percentage of Rab10 phosphorylation from attomole amounts of the endogenous phosphopeptide. The SIL and the endogenous phosphopeptides are separately admitted into an Orbitrap analyzer with the appropriate injection times. We test the reproducibility of our assay by determining Rab10-Thr73 phosphorylation stoichiometry in neutrophils of LRRK2 mutation carriers before and after LRRK2 inhibition. Compared with healthy controls, the PD predisposing mutation carriers LRRK2 G2019S and VPS35 D620N display 1.9-fold and 3.7-fold increased pRab10 levels, respectively. Our generic MS-based assay further establishes the relevance of pRab10 as a prognostic PD marker and is a powerful tool for determining LRRK2 inhibitor efficacy and for stratifying PD patients for LRRK2