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

• DOI: 10.1074/mcp.ra120.002055
• Publication Date: 2020-06-29T19:55:11Z
• Authors: Özge Karayel; Francesca Tonelli; Sebastian Virreir...; Phillip E. Geyer; Ying Fan; Esther M. Sammler; Dario R. Alessi; Martin Steger; Matthias Mann
• Keywords: LRRK2; Phosphopeptide

Pathogenic mutations in the Leucine-rich repeat kinase 2 (LRRK2) are predominant genetic cause of Parkinson's disease (PD). They increase its activity, resulting augmented Rab10-Thr73 phosphorylation and conversely, LRRK2 inhibition decreases pRab10 levels. Currently, there is no assay to quantify levels for drug target engagement or patient stratification. To meet this challenge, we developed an high accuracy sensitivity targeted mass spectrometry (MS)-based determining stoichiometry human samples. It uses synthetic stable isotope-labeled (SIL) analogues both phosphorylated nonphosphorylated tryptic peptides surrounding directly derive percentage Rab10 from attomole amounts endogenous phosphopeptide. The SIL phosphopeptides separately admitted into Orbitrap analyzer with appropriate injection times. We test reproducibility our by neutrophils mutation carriers before after inhibition. 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Stoichiometry based on heavy-to-light ratios analogs proteolytic counterparts can overcome problem allow precise Alternatively, recombinant chemically enzymatically spike-in (28Hanke Besir Oesterhelt SILAC accurate mixtures down level.J. Proteome Res. 1118-1130Crossref (184) 29Zeiler al.A Protein Epitope Signature Tag (PrEST) library SILAC-based multiplexed copy numbers lines.Mol. Cell. Proteomics. 11 (O111–009613)Abstract (118) combined tailored especially suited given analyte (30Domon Gallien Recent advances clinical applications.Proteomics. Clin. Appl. 9: 423-431Crossref (46) Instruments operated scan modes such high-resolution selected ion (SIM) parallel (PRM). precursor ions isolated narrow m/z range quadrupole filter introduction analyzer, thereby providing signal noise ratio interest allowing attomole-level limits (31Michalski al.Mass spectrometry-based Q Exactive, high-performance benchtop spectrometer.Mol. (M111.011015)Abstract (626) 32Gallien Duriez Crone Kellmann Moehring Domon Targeted quadrupole-orbitrap 11: 1709-1723Abstract (364) As fragmentation involved SIM, relies resolution alone. PRM, fragment peptide. approach specific, overall PRM lower, distributed across fragments (32Gallien Here describe highly-sensitive MS-assay how disease. evaluate patients, defined cause. subtle phosphorylation, beyond what detectable typical immunoassays. show measured precisely serve robust stratification studies. experimental design statistical rationale experiments conducted study described detail subsection figure legend. Briefly, neutrophil samples individuals technical replicates (n = 3). LOQ experiment IP For CV experiments, repeated measurements (analytical), workflow same gel (intra-assay) different gels (inter-assay) phosphoprotein 6). was applied collected 14 three four setting up validating assay, recruited seven volunteers Department Proteomics Signal Transduction Max Planck Institute Biochemistry who kindly donated study. data shown Fig. 1, 2, 3 supplemental Figs. S1–S3 derived donors, provided written informed consent, prior approval ethics committee Society.Fig. 2mxSIM accurately determines stoichiometry. A, Heavy-to-light formulas protein- peptide-centric approaches. B, HA-Rab10-pThr73 LRRK2-Y1699C-expressing HEK293 determined versus approaches HA-IP. C, either enrichment anti-Rab10-IP SDS-PAGE followed in-gel digestion expressing LRRK2-Y1699C (−/+ 200 nm MLi-2, 60 min). D, Endogenous Rab10-pThr73 standards mock Samples (B), (C), (D) analyzed triplicates mxSIM occupancies presented means ± SEM. E, Benchmarking Thr73-phosphorylated unlabeled (1-175 aa) standards. Correlation median intact two independent experiments. F, mixed mimic 10, 30, 50, 70, 90% spiked digested triplicates. solid line represents (75.4 1.5%) whereas dashed shows estimated (70%) analysis. G, CVs calculated repeating 6).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3Reliable lysates. Workflow assay. Immunoblotting WT R1441G knock-in MEFs treated indicated concentrations MLi-2 intervals monoclonal MJFF-pRAB10 (pThr73) GAPDH antibodies. Dose–response curve generate based-IC50 values MLi-2. Each point triplicate 90 min. (DMSO 30 100 treated) Normalized Rab10- subtracting red. H, Individual-specific dose–response curves Error bars represent SEM.View (PPT) 4, carrying heterozygous nonPD had previously publication ((11Fan Scholar), respectively, Table S4). All procedures performed compliance local review boards participants consent. These subjected blinded set-up, identity only being revealed completion purchased Tocris Bioscience (cat# 5756). DIFP, HA-agarose trypsin Sigma LysC Wako. Microcystin-LR Enzo Life Sciences. Complete protease tablets Roche. quantities (AQUA) Sigma-Aldrich. Anti-GAPDH (#5174), anti-HA (#3724), anti-Rab10 (#8127) Cell Signaling Technologies. Rabbit