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• W3039492798 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. 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 inhibitor treatment. 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 inhibitor treatment. Parkinson's disease (PD) is the second most common neurodegenerative condition, and no disease-modifying therapies exist to date (1Alessi D.R. Sammler E.J.S. LRRK2 kinase in Parkinson's disease.Science. 2018; 360: 36-37Crossref PubMed Scopus (153) Google Scholar, 2Eyers P.A. Back to the future: new target-validated Rab antibodies for evaluating LRRK2 signalling in cell biology and Parkinson's disease.Biochem. J. 2018; 475: 185-189Crossref PubMed Scopus (4) Google Scholar). Although most PD cases are idiopathic, mutations in several genes have been linked to familial forms of the disease (3Chang D. Nalls M.A. Hallgrímsdóttir I.B. Hunkapiller J. van der Brug M. Cai F. Kerchner G.A. Ayalon G. Bingol B. Sheng M. Hinds D. Behrens T.W. Singleton A.B. Bhangale T.R. Graham R.R. A meta-analysis of genome-wide association studies identifies 17 new Parkinson's disease risk loci.Nat. Genet. 2017; 49: 1511-1516Crossref PubMed Scopus (622) Google Scholar). Among those, mutations in the Leucine-rich repeat kinase 2 (LRRK2) comprise the predominant genetic cause of PD and account for 1% of sporadic and 4% of familial cases worldwide, and much higher in some populations (4Healy D.G. Falchi M. O'Sullivan S.S. Bonifati V. Durr A. Bressman S. Brice A. Aasly J. Zabetian C.P. Goldwurm S. Ferreira J.J. Tolosa E. Kay D.M. Klein C. Williams D.R. Marras C. Lang A.E. Wszolek Z.K. Berciano J. Schapira A.H.V. Lynch T. Bhatia K.P. Gasser T. Lees A.J. Wood N.W. Phenotype, genotype, and worldwide genetic penetrance of LRRK2-associated Parkinson's disease: a case-control study.Lancet. Neurol. 2008; 7: 583-590Abstract Full Text Full Text PDF PubMed Scopus (1066) Google Scholar). At least six pathogenic missense mutations in LRRK2, including the most frequent G2019S substitution, have been identified (5Paisán‐Ruiz C.J.H.M. LRRK2 gene variation and its contribution to Parkinson disease.Hum. Mutat. 2009; 30: 1153-1160Crossref PubMed Scopus (60) Google Scholar) and several studies confirmed that these mutations increase its kinase activity (5Paisán‐Ruiz C.J.H.M. LRRK2 gene variation and its contribution to Parkinson disease.Hum. Mutat. 2009; 30: 1153-1160Crossref PubMed Scopus (60) Google Scholar, 6Sheng Z. Zhang S. Bustos D. Kleinheinz T. Le Pichon C.E. Dominguez S.L. Solanoy H.O. Drummond J. Zhang X. Ding X. Cai F. Song Q. Li X. Yue Z. van der Brug M.P. Burdick D.J. Gunzner-Toste J. Chen H. Liu X. Estrada A.A. Sweeney Z.K. Scearce-Levie K. Moffat J.G. Kirkpatrick D.S. Zhu H. Ser1292 autophosphorylation is an indicator of LRRK2 kinase activity and contributes to the cellular effects of PD mutations.Sci. Transl. Med. 2012; 4: 164ra161Crossref PubMed Scopus (256) Google Scholar, 7West A.B. Moore D.J. Biskup S. Bugayenko A. Smith W.W. Ross C.A. Dawson V.L. Dawson T.M. Parkinson's disease-associated mutations in leucine-rich repeat kinase 2 augment kinase activity.Proc. Natl. Acad. Sci. U S A. 2005; 102: 16842-16847Crossref PubMed Scopus (932) Google Scholar, 8Steger M. Tonelli F. Ito G. Davies P. Trost M. Vetter M. Wachter S. Lorentzen E. Duddy G. Wilson S. Baptista M.A. Fiske B.K. Fell M.J. Morrow J.A. Reith A.D. Alessi D.R. Mann M. Phosphoproteomics reveals that Parkinson's disease kinase LRRK2 regulates a subset of Rab GTPases.Elife. 2016; 5Crossref PubMed Google Scholar). LRRK2-associated PD is clinically largely indistinguishable from idiopathic PD, suggesting that LRRK2 inhibition may be a useful a therapy for a larger group of patients (4Healy D.G. Falchi M. O'Sullivan S.S. Bonifati V. Durr A. Bressman S. Brice A. Aasly J. Zabetian C.P. Goldwurm S. Ferreira J.J. Tolosa E. Kay D.M. Klein C. Williams D.R. Marras C. Lang A.E. Wszolek Z.K. Berciano J. Schapira A.H.V. Lynch T. Bhatia K.P. Gasser T. Lees A.J. Wood N.W. Phenotype, genotype, and worldwide genetic penetrance of LRRK2-associated Parkinson's disease: a case-control study.Lancet. Neurol. 2008; 7: 583-590Abstract Full Text Full Text PDF PubMed Scopus (1066) Google Scholar). Clinical trials with selective LRRK2 kinase inhibitors are ongoing and have already passed phase 1. Downstream targets of the LRRK2 kinase have long been enigmatic and controversial, but using genetic mouse models, specific inhibitors and a novel phosphoproteomics workflow, we have recently identified and verified a subset of Rab GTPases (Rab3A/B/C/D, Rab5A/B/C, Rab8A/B, Rab10, Rab12, Rab29, Rab35, and Rab43) as bona fide substrates (8Steger M. Tonelli F. Ito G. Davies P. Trost M. Vetter M. Wachter S. Lorentzen E. Duddy G. Wilson S. Baptista M.A. Fiske B.K. Fell M.J. Morrow J.A. Reith A.D. Alessi D.R. Mann M. Phosphoproteomics reveals that Parkinson's disease kinase LRRK2 regulates a subset of Rab GTPases.Elife. 2016; 5Crossref PubMed Google Scholar, 9Steger M. Diez F. Dhekne H.S. Lis P. Nirujogi R.S. Karayel O. Tonelli F. Martinez T.N. Lorentzen E. Pfeffer S.R. Alessi D.R. Mann M. Systematic proteomic analysis of LRRK2-mediated Rab GTPase phosphorylation establishes a connection to ciliogenesis.Elife. 2017; 6Crossref PubMed Scopus (216) Google Scholar). Among these, Rab10 appears to be a key physiological kinase substrate (8Steger M. Tonelli F. Ito G. Davies P. Trost M. Vetter M. Wachter S. Lorentzen E. Duddy G. Wilson S. Baptista M.A. Fiske B.K. Fell M.J. Morrow J.A. Reith A.D. Alessi D.R. Mann M. Phosphoproteomics reveals that Parkinson's disease kinase LRRK2 regulates a subset of Rab GTPases.Elife. 2016; 5Crossref PubMed Google Scholar, 9Steger M. Diez F. Dhekne H.S. Lis P. Nirujogi R.S. Karayel O. Tonelli F. Martinez T.N. Lorentzen E. Pfeffer S.R. Alessi D.R. Mann M. Systematic proteomic analysis of LRRK2-mediated Rab GTPase phosphorylation establishes a connection to ciliogenesis.Elife. 2017; 6Crossref PubMed Scopus (216) Google Scholar, 10Atashrazm F. Hammond D. Perera G. Bolliger M.F. Matar E. Halliday G.M. Schule B. Lewis S.J. Nichols R.J. Dzamko N LRRK2‐mediated Rab10 phosphorylation in immune cells from Parkinson's disease patients.Mov. Disord. 2018; 34: 406-415Crossref PubMed Scopus (50) Google Scholar, 11Fan Y. Howden A.J.M. Sarhan A.R. Lis P. Ito G. Martinez T.N. Brockmann K. Gasser T. Alessi D.R. Sammler E.M. Interrogating Parkinson's disease LRRK2 kinase pathway activity by assessing Rab10 phosphorylation in human neutrophils.Biochem. J. 2018; 475: 23-44Crossref PubMed Scopus (81) Google Scholar, 12Ito G. et al.Phos-tag analysis of Rab10 phosphorylation by LRRK2: a powerful assay for assessing kinase function and inhibitors.Biochem. J. 2016; 473: 2671-2685Crossref PubMed Scopus (99) Google Scholar, 13Thirstrup K. Dächsel J.C. Oppermann F.S. Williamson D.S. Smith G.P. Fog K. Christensen K.V. Selective LRRK2 kinase inhibition reduces phosphorylation of endogenous Rab10 and Rab12 in human peripheral mononuclear blood cells.Sci. Rep. 2017; 7: 10300Crossref PubMed Scopus (61) Google Scholar, 14Lis P. Burel S. Steger M. Mann M. Brown F. Diez F. Tonelli F. Holton J.L. Ho P.W. Ho S.-L. Chou M.-Y. Polinski N.K. Martinez T.N. Davies P. Alessi D.R. Development of phospho-specific Rab protein antibodies to monitor in vivo activity of the LRRK2 Parkinson's disease kinase.Biochem. J. 2018; 475: 1-22Crossref PubMed Scopus (73) Google Scholar). We found that LRRK2 directly phosphorylates Rab10 at Thr73 and all known pathogenic forms of LRRK2 enhance this phosphorylation (8Steger M. Tonelli F. Ito G. Davies P. Trost M. Vetter M. Wachter S. Lorentzen E. Duddy G. Wilson S. Baptista M.A. Fiske B.K. Fell M.J. Morrow J.A. Reith A.D. Alessi D.R. Mann M. Phosphoproteomics reveals that Parkinson's disease kinase LRRK2 regulates a subset of Rab GTPases.Elife. 2016; 5Crossref PubMed Google Scholar, 9Steger M. Diez F. Dhekne H.S. Lis P. Nirujogi R.S. Karayel O. Tonelli F. Martinez T.N. Lorentzen E. Pfeffer S.R. Alessi D.R. Mann M. Systematic proteomic analysis of LRRK2-mediated Rab GTPase phosphorylation establishes a connection to ciliogenesis.Elife. 2017; 6Crossref PubMed Scopus (216) Google Scholar). Intriguingly, the PD-associated D620N mutation of the retromer complex protein VPS35 also activates LRRK2 kinase activity, which in turn results in augmented Rab10 phosphorylation (15Mir R. Tonelli F. Lis P. Macartney T. Polinski N.K. Martinez T.N. Chou M.-Y. Howden A.J.M. König T. Hotzy C. Milenkovic I. Brücke T. Zimprich A. Sammler E. Alessi D.R. The Parkinson's disease VPS35 [D620N] mutation enhances LRRK2-mediated Rab protein phosphorylation in mouse and human.Biochem. J. 2018; 475: 1861-1883Crossref PubMed Scopus (100) Google Scholar). Thus, multiple PD-associated factors are interconnected and dysregulation of a common LRRK2-Rab signaling pathway appears to be an underlying cause of PD. The LRRK2 autophosphorylation site Ser1292 has been widely used for assessing LRRK2 kinase activity (16Deng X. Dzamko N. Prescott A. Davies P. Liu Q. Yang Q. Lee J.-D. Patricelli M.P. Nomanbhoy T.K. Alessi D.R. Gray N.S. Characterization of a selective inhibitor of the Parkinson's disease kinase LRRK2.Nat. Chem. Biol. 2011; 7: 203-205Crossref PubMed Scopus (325) Google Scholar, 17Dzamko N. Chua G. Ranola M. Rowe D.B. Halliday G.M. Measurement of LRRK2 and Ser910/935 phosphorylated LRRK2 in peripheral blood mononuclear cells from idiopathic Parkinson's disease patients.J. Parkinsons. Dis. 2013; 3: 145-152Crossref PubMed Scopus (36) Google Scholar, 18Dzamko N. Deak M. Hentati F. Reith A.D. Prescott A.R. Alessi D.R. Nichols R.J. Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser910/Ser935, disruption of 14-3-3 binding and altered cytoplasmic localization.Biochem. J. 2010; 430: 405-413Crossref PubMed Scopus (285) Google Scholar, 19Nichols R.J. Dzamko N. Morrice N.A. Campbell D.G. Deak M. Ordureau A. Macartney T. Tong Y. Shen J. Prescott A.R. Alessi D.R. 14-3-3 binding to LRRK2 is disrupted by multiple Parkinson's disease-associated mutations and regulates cytoplasmic localization.Biochem. J. 2010; 430: 393-404Crossref PubMed Scopus (282) Google Scholar). However, its stoichiometry (percentage of molecules phosphorylated) appears to be extremely low and there is no sensitive phospho-specific antibody available to reliably detect and quantify phosphorylation at this site (6Sheng Z. Zhang S. Bustos D. Kleinheinz T. Le Pichon C.E. Dominguez S.L. Solanoy H.O. Drummond J. Zhang X. Ding X. Cai F. Song Q. Li X. Yue Z. van der Brug M.P. Burdick D.J. Gunzner-Toste J. Chen H. Liu X. Estrada A.A. Sweeney Z.K. Scearce-Levie K. Moffat J.G. Kirkpatrick D.S. Zhu H. Ser1292 autophosphorylation is an indicator of LRRK2 kinase activity and contributes to the cellular effects of PD mutations.Sci. Transl. Med. 2012; 4: 164ra161Crossref PubMed Scopus (256) Google Scholar, 20Kluss J.H. Conti M.M. Kaganovich A. Beilina A. Melrose H.L. Cookson M.R. Mamais A. Detection of endogenous S1292 LRRK2 autophosphorylation in mouse tissue as a readout for kinase activity.NPJ Parkinsons. Dis. 2018; 4: 13Crossref PubMed Scopus (35) Google Scholar, 21Reynolds A. Doggett E.A. Riddle S.M. Lebakken C.S. Nichols R.J.J. LRRK2 kinase activity and biology are not uniformly predicted by its autophosphorylation and cellular phosphorylation site status.Front. Mol. Neurosci. 2014; 7: 54Crossref PubMed Scopus (54) Google Scholar). We recently developed several high-affinity antibodies for detecting pRab proteins in cells and in tissues (14Lis P. Burel S. Steger M. Mann M. Brown F. Diez F. Tonelli F. Holton J.L. Ho P.W. Ho S.-L. Chou M.-Y. Polinski N.K. Martinez T.N. Davies P. Alessi D.R. Development of phospho-specific Rab protein antibodies to monitor in vivo activity of the LRRK2 Parkinson's disease kinase.Biochem. J. 2018; 475: 1-22Crossref PubMed Scopus (73) Google Scholar). Among those, a highly specific clone detects pThr73 levels in human peripheral blood cells of (mutant) LRRK2 G2019S and VPS35-D620N carriers with Parkinson's disease (11Fan Y. Howden A.J.M. Sarhan A.R. Lis P. Ito G. Martinez T.N. Brockmann K. Gasser T. Alessi D.R. Sammler E.M. Interrogating Parkinson's disease LRRK2 kinase pathway activity by assessing Rab10 phosphorylation in human neutrophils.Biochem. J. 2018; 475: 23-44Crossref PubMed Scopus (81) Google Scholar, 15Mir R. Tonelli F. Lis P. Macartney T. Polinski N.K. Martinez T.N. Chou M.-Y. Howden A.J.M. König T. Hotzy C. Milenkovic I. Brücke T. Zimprich A. Sammler E. Alessi D.R. The Parkinson's disease VPS35 [D620N] mutation enhances LRRK2-mediated Rab protein phosphorylation in mouse and human.Biochem. J. 2018; 475: 1861-1883Crossref PubMed Scopus (100) Google Scholar). Although pRab10 levels were markedly increased in VPS35 D620N carriers, no statistically significant differences in pRab10 levels were detected by immunoblotting analysis when comparing controls to LRRK2-G2019S carriers in peripheral blood mononuclear cells (PBMCs) and neutrophils (11Fan Y. Howden A.J.M. Sarhan A.R. Lis P. Ito G. Martinez T.N. Brockmann K. Gasser T. Alessi D.R. Sammler E.M. Interrogating Parkinson's disease LRRK2 kinase pathway activity by assessing Rab10 phosphorylation in human neutrophils.Biochem. J. 2018; 475: 23-44Crossref PubMed Scopus (81) Google Scholar, 15Mir R. Tonelli F. Lis P. Macartney T. Polinski N.K. Martinez T.N. Chou M.-Y. Howden A.J.M. König T. Hotzy C. Milenkovic I. Brücke T. Zimprich A. Sammler E. Alessi D.R. The Parkinson's disease VPS35 [D620N] mutation enhances LRRK2-mediated Rab protein phosphorylation in mouse and human.Biochem. J. 2018; 475: 1861-1883Crossref PubMed Scopus (100) Google Scholar). The reproducible quantification of immunoblots, particularly the detection of small (<2-fold) changes is challenging. In contrast, MS-based quantification has become a gold standard and has several advantages over traditional biochemical methods, as it is more specific and potentially more accurate. Importantly, MS allows simultaneous detection of both the phosphorylated peptide and the total protein pool and hence enables the direct calculation of the absolute fraction of the phosphorylated protein, also known as phosphorylation stoichiometry or occupancy (22Olsen J.V. Vermeulen M. Santamaria A. Kumar C. Miller M.L. Jensen L.J. Gnad F. Cox J. Jensen T.S. Nigg E.A. Brunak S. Mann M. Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis.Sci. Signal. 2010; 3: ra3Crossref PubMed Scopus (1145) Google Scholar). There are several strategies differing in their accuracy, throughput, and applicability for measuring phosphorylation stoichiometry by MS (22Olsen J.V. Vermeulen M. Santamaria A. Kumar C. Miller M.L. Jensen L.J. Gnad F. Cox J. Jensen T.S. Nigg E.A. Brunak S. Mann M. Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis.Sci. Signal. 2010; 3: ra3Crossref PubMed Scopus (1145) Google Scholar, 23Wu R. Haas W. Dephoure N. Huttlin E.L. Zhai B. Sowa M.E. Gygi S.P. A large-scale method to measure absolute protein phosphorylation stoichiometries.Nat. Methods. 2011; 8: 677-683Crossref PubMed Scopus (226) Google Scholar, 24Gerber S.A. Rush J. Stemman O. Kirschner M.W. Gygi S.P. Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS.Proc. Natl. Acad. Sci. U S A. 2003; 100: 6940-6945Crossref PubMed Scopus (1542) Google Scholar, 25Tsai C.-F. Wang Y.-T. Yen H.-Y. Tsou C.-C. Ku W.-C. Lin P.-Y. Chen H.-Y. Nesvizhskii A.I. Ishihama Y. Chen Y.-J. Large-scale determination of absolute phosphorylation stoichiometries in human cells by motif-targeting quantitative proteomics.Nat. Commun. 2015; 6: 6622Crossref PubMed Scopus (119) Google Scholar, 26Domanski D. Murphy L.C. Borchers C.H. Assay development for the determination of phosphorylation stoichiometry using multiple reaction monitoring methods with and without phosphatase treatment: application to breast cancer signaling pathways.Anal. Chem. 2010; 82: 5610-5620Crossref PubMed Scopus (64) Google Scholar, 27Steen H. Jebanathirajah J.A. Springer M. Kirschner M.W. Stable isotope-free relative and absolute quantitation of protein phosphorylation stoichiometry by MS.Proc. Natl. Acad. Sci. U S A. 2005; 102: 3948-3953Crossref PubMed Scopus (181) Google Scholar). One way is to compare the intensities of modified and unmodified peptides by label-free proteomics (27Steen H. Jebanathirajah J.A. Springer M. Kirschner M.W. Stable isotope-free relative and absolute quantitation of protein phosphorylation stoichiometry by MS.Proc. Natl. Acad. Sci. U S A. 2005; 102: 3948-3953Crossref PubMed Scopus (181) Google Scholar). However, because of potential differences in ionization efficiencies, the MS signals of individual peptides cannot be directly compared with each other. Stoichiometry determination based on heavy-to-light ratios of stable isotope-labeled (SIL) analogs of phosphorylated and nonphosphorylated proteolytic peptides and their endogenous counterparts can overcome this problem and allow a much more sensitive and precise readout for monitoring kinase activity (22Olsen J.V. Vermeulen M. Santamaria A. Kumar C. Miller M.L. Jensen L.J. Gnad F. Cox J. Jensen T.S. Nigg E.A. Brunak S. Mann M. Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis.Sci. Signal. 2010; 3: ra3Crossref PubMed Scopus (1145) Google Scholar, 23Wu R. Haas W. Dephoure N. Huttlin E.L. Zhai B. Sowa M.E. Gygi S.P. A large-scale method to measure absolute protein phosphorylation stoichiometries.Nat. Methods. 2011; 8: 677-683Crossref PubMed Scopus (226) Google Scholar). Alternatively, a SIL recombinant protein that is chemically or enzymatically phosphorylated can also be used as the spike-in standard (28Hanke S. Besir H. Oesterhelt D. Mann M. Absolute SILAC for accurate quantitation of proteins in complex mixtures down to the attomole level.J. Proteome Res. 2008; 7: 1118-1130Crossref PubMed Scopus (184) Google Scholar, 29Zeiler M. et al.A Protein Epitope Signature Tag (PrEST) library allows SILAC-based absolute quantification and multiplexed determination of protein copy numbers in cell lines.Mol. Cell. Proteomics. 2012; 11 (O111–009613)Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). MS strategies determining stoichiometry that are combined with tailored targeted methods are especially suited for accurate quantification of low levels of a given phosphorylated analyte (30Domon B. Gallien S. Recent advances in targeted proteomics for clinical applications.Proteomics. Clin. Appl. 2015; 9: 423-431Crossref PubMed Scopus (46) Google Scholar). Instruments with an Orbitrap mass analyzer can be operated in targeted scan modes such as high-resolution selected ion monitoring (SIM) or parallel reaction monitoring (PRM). In both methods, precursor ions are isolated with a narrow m/z range by a quadrupole mass filter before introduction into the Orbitrap analyzer, thereby providing increased signal to noise ratio for the ion of interest and allowing attomole-level limits of detection (31Michalski A. et al.Mass spectrometry-based proteomics using Q Exactive, a high-performance benchtop quadrupole Orbitrap mass spectrometer.Mol. Cell. 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Here we describe an accurate and highly-sensitive MS-assay for determining the Rab10-Thr73 phosphorylation stoichiometry and how it changes in Parkinson's disease. We evaluate it by comparing Rab10-Thr73 phosphorylation levels in healthy controls, idiopathic PD patients, and PD patients with defined genetic cause. Our assay enables the detection of subtle changes in Rab10 phosphorylation, which are beyond what is detectable by typical immunoassays. We show that pRab10 stoichiometry when measured precisely using our assay can serve as a robust target engagement and patient stratification marker in clinical studies. The experimental design and statistical rationale for each of the experiments conducted in this study are described in detail in each subsection and figure legend. Briefly, all neutrophil samples isolated from individuals were measured with technical replicates (n = 3). The LOQ experiment and IP experiments were also measured in technical replicates (n = 3). For CV experiments, we repeated MS measurements (analytical), and the workflow in the same gel (intra-assay) or in different gels (inter-assay) using the same phosphoprotein standard (n = 6). Our assay was applied in the neutrophils collected from 14 healthy controls, three idiopathic PD patients, four LRRK2 G2019S and three VPS35 D620N mutation carriers with PD. For setting up and validating our assay, we recruited seven volunteers from the Department of Proteomics and Signal Transduction at the Max Planck Institute of Biochemistry who kindly donated blood for our study. The data shown in Fig. 1, Fig. 2, Fig. 3 and supplemental Figs. S1–S3 are derived blood samples from healthy donors, which provided a written informed consent, with prior approval of the ethics committee of the Max Planck Society.Fig. 2mxSIM accurately determines Rab10-Thr73 phosphorylation stoichiometry. A, Heavy-to-light ratios and the formulas used for protein- and peptide-centric approaches. B, HA-Rab10-pThr73 occupancy in LRRK2-Y1699C-expressing HEK293 cells was determined using the protein- versus peptide-centric approaches after HA-IP. C, HA-Rab10-pThr73 occupancy determined using the peptide-centric approach with either SIL phosphoprotein or SIL peptide standard spike-in after enrichment by anti-Rab10-IP or SDS-PAGE followed by in-gel digestion in HEK293 cells expressing LRRK2-Y1699C (−/+ 200 nm MLi-2, 60 min). D, Endogenous Rab10-pThr73 occupancy determined using SIL peptide standards in mock and LRRK2-Y1699C expressing HEK293 cells (−/+ 200 nm MLi-2, 60 min). Samples from (B), (C), and (D) were analyzed in triplicates using the mxSIM method and the phosphorylation occupancies are presented as means ± SEM. E, Benchmarking our method using Thr73-phosphorylated unlabeled recombinant Rab10 proteins (1-175 aa) as standards. Correlation of the median occupancies determined either by intact mass analysis or by mxSIM in triplicates for two independent experiments. F, SIL phosphorylated and nonphosphorylated Rab10 peptides mixed to mimic 10, 30, 50, 70, and 90% occupancies and spiked into the digested recombinant pRab10 protein were measured using our mxSIM method in triplicates. The solid line represents the median occupancy (75.4 ± 1.5%) whereas the dashed line shows the estimated phosphorylation occupancy of the standard protein (70%) by intact mass analysis. G, CVs were calculated by repeating MS measurements (analytical), the workflow in the same gel (intra-assay) or in different gels (inter-assay) using the same phosphoprotein standard (n = 6).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3Reliable determination of Rab10-pThr73 occupancy in cell lysates. A, Workflow for the Rab10-pThr73 occupancy assay. B, Immunoblotting of WT and R1441G knock-in MEFs treated with the indicated concentrations of MLi-2 for different intervals using monoclonal MJFF-pRAB10 (pThr73) and GAPDH antibodies. C, Rab10-pThr73 occupancies were determined by mxSIM in the same lysates (n = 3). D, Dose–response curve of Rab10-pThr73 occupancy in R1441G knock-in MEFs to generate occupancy based-IC50 values for MLi-2. Each data point represents the median of triplicate measurements in the samples treated with MLi-2 for 90 min. E, Immunoblotting of neutrophils isolated from healthy individuals (DMSO or 30 and 100 nm MLi-2 treated) using monoclonal MJFF-pRAB10 (pThr73) and GAPDH antibodies. F, Rab10-pThr73 occupancies were determined by mxSIM in the same lysates. Normalized Rab10- pThr73 occupancies by subtracting the MLi-2 treated values are shown in red. G, Immunoblotting of the neutrophils isolated from healthy individuals and treated with the indicated concentrations of MLi-2 using monoclonal MJFF-pRAB10 (pThr73) and GAPDH antibodies. H, Individual-specific dose–response curves to generate occupancy based-IC50 for MLi-2. Error bars represent SEM.View Large Image Figure ViewerDownload Hi-res image Download (PPT) For the experiments shown in Fig. 4, neutrophil lysates derived from either idiopathic PD patients, PD patients carrying a heterozygous LRRK2 G2019S or VPS35 D620N mutation or nonPD controls were used that had been previously used for publication ((11Fan Y. Howden A.J.M. Sarhan A.R. Lis P. Ito G. Martinez T.N. Brockmann K. Gasser T. Alessi D.R. Sammler E.M. Interrogating Parkinson's disease LRRK2 kinase pathway activity by assessing Rab10 phosphorylation in human neutrophils.Biochem. J. 2018; 475: 23-44Crossref PubMed Scopus (81) Google Scholar) and (15Mir R. Tonelli F. Lis P. Macartney T. Polinski N.K. Martinez T.N. Chou M.-Y. Howden A.J.M. König T. Hotzy C. Milenkovic I. Brücke T. Zimprich A. Sammler E. Alessi D.R. The Parkinson's disease VPS35 [D620N] mutation enhances LRRK2-mediated Rab protein phosphorylation in mouse and human.Biochem. J. 2018; 475: 1861-1883Crossref PubMed Scopus (100) Google Scholar), respectively, supplemental Table S4). All procedures were performed in compliance with the local ethics review boards and all participants provided informed consent. These lysates were subjected to MS analysis in a blinded experimental set-up, with the identity of the lysates only being revealed after completion of the MS analysis. MLi-2 was purchased from Tocris Bioscience (cat# 5756). DIFP, HA-agarose and trypsin were from Sigma and LysC was from Wako. Microcystin-LR was from Enzo Life Sciences. Complete protease and phosphatase inhibitor tablets were from Roche. SIL peptides in absolute quantities (AQUA) were purchased from Sigma-Aldrich. Anti-GAPDH (#5174), anti-HA (#3724), and anti-Rab10 (#8127) were from Cell Signaling Technologies. Rabbit" @default.
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• W3039492798 title "Accurate MS-based Rab10 Phosphorylation Stoichiometry Determination as Readout for LRRK2 Activity in Parkinson's Disease" @default.
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