Accurate MS-based Rab10 phosphorylation stoichiometry determination as readout for LRRK2 activity in Parkinson’s disease

ABSTRACT: 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. Monitoring pRab10 can thus serve as a readout for LRRK2 activity; however, no sufficiently accurate assay to quantify pRab10 levels for drug target engagement or patient stratification exists. Here, we developed an ultra-sensitive 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 non-phosphorylated tryptic peptides surrounding Rab10-Thr73 to directly derive the percentage of Rab10 phosphorylation from attomole amounts of the endogenous phosphopeptide. We test the reproducibility of our assay by determining Rab10-Thr73 phosphorylation stoichiometry in human neutrophils before and after LRRK2 inhibition. Compared to healthy controls, neutrophils of LRRK2 G2019S and VPS35 D620N carriers robustly display 1.4-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 inhibitor treatment.

INSTRUMENT(S): LTQ Orbitrap, Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Neutrophil, Blood

DISEASE(S): Parkinson's Disease

SUBMITTER: Mario Oroshi  

LAB HEAD: Matthias Mann

PROVIDER: PXD015219 | Pride | 2020-07-03

REPOSITORIES: Pride