Development of a multiplexed targeted mass spectrometry assay for LRRK2-phosphorylated Rabs and Ser910/Ser935 biomarker sites

Abstract

Mutations that increase the protein kinase activity of LRRK2 are one of the most common causes of familial Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases within their Switch-II motif, impacting interaction with effectors. We describe and validate a new, multiplexed targeted mass spectrometry assay to quantify endogenous levels of LRRK2-phosphorylated Rab substrates (Rab1, Rab3, Rab8, Rab10, Rab35 and Rab43) as well as total levels of Rabs, LRRK2 and LRRK2-phosphorylated at the Ser910 and Ser935 biomarker sites. Exploiting this assay, we quantify for the first time the relative levels of each of the pRab proteins in different cells (mouse embryonic fibroblasts, human neutrophils) and mouse tissues (brain, kidney, lung and spleen). We define how these components are impacted by Parkinson's pathogenic mutations (LRRK2[R1441C] and VPS35[D620N]) and LRRK2 inhibitors. We find that the VPS35[D620N], but not LRRK2[R1441C] mutation, enhances Rab1 phosphorylation in a manner blocked by administration of an LRRK2 inhibitor, providing the first evidence that endogenous Rab1 is a physiological substrate for LRRK2. We exploit this assay to demonstrate that in Parkinson's patients with VPS35[D620N] mutations, phosphorylation of multiple Rab proteins (Rab1, Rab3, Rab8, Rab10 and Rab43) is elevated. We highlight the benefits of this assay over immunoblotting approaches currently deployed to assess LRRK2 Rab signalling pathway.

R1441G but not G2019S mutation enhances LRRK2 mediated Rab10 phosphorylation in human peripheral blood neutrophils

“…In conclusion our findings add compelling evidence that interrogating in vivo LRRK2 dependent pRab10 Thr73 phosphorylation in human peripheral blood neutrophils is a specific, robust and promising biomarker for significant LRRK2 kinase hyperactivation, as with the LRRK2 R1441G mutation or as previously demonstrated with the VPS35 D620N mutation [ 27 ]. Further work is required to explore additional readouts including phosphorylation levels of other RabGTPase substrates of the LRRK2 kinase [ 34 ], assays and biomatrices that would allow detection of more modest LRRK2 kinase activation as with the LRRK2 G2019S mutation and possibly in individuals with iPD. We envision that our assays will be used alongside other markers of the LRRK2 signalling and PD associated pathways across different biomatrices and importantly longitudinally to possibly delineate integrated markers for disease conversion and PD progression.…”

Proteome Landscape of Epithelial-to-Mesenchymal Transition (EMT) of Retinal Pigment Epithelium Shares Commonalities With Malignancy-Associated EMT

“…S-Trap assisted trypsin digestion was performed as described previously (41), with slight modification. Briefly, hiPSC (EP1) derived RPE cells were lysed in 5% SDS lysis buffer in 100 mM Triethylammonium bicarbonate buffer (TEABC pH 7.5) and placed at 95°C for 5 min.…”

“…In order to validate the differentially regulated proteins that were quantified by the TMT labeling method, we performed a label free single shot data-independent acquisition (DIA) mass spectrometry analysis on hRPE monolayers and RPE cells undergoing EMT (12 hr and 48 hr), and analysed the data using a direct DIA strategy with Spectronatu 14.0 (41). Using this approach, we identified 57,728 precursor and 5,276 protein groups at 1% protein level FDR J o u r n a l P r e -p r o o f (supplemental Table S5 ).…”

LRRK2 Targeting Strategies as Potential Treatment of Parkinson’s Disease

“…The next big challenge in the field is the development of reliable biomarkers for accurate detection of LRRK2 activity and monitoring the progression of PD from the early stages. In this respect, antibody or mass spectrometry-based assays that can detect Rab10 phosphorylation in patients' samples are being studied, as well as urinary proteome profiling, as non-invasive analytical methods [132][133][134].…”