English
 
Help Privacy Policy Disclaimer
  Advanced SearchBrowse

Item

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

Karayel, Ö., Tonelli, F., Virreira Winter, S., Geyer, P. E., Fan, Y., Sammler, E. M., et al. (2020). Accurate MS-based Rab10 Phosphorylation Stoichiometry Determination as Readout for LRRK2 Activity in Parkinson's Disease. Molecular and Cellular Proteomics, 19(9), 1546-1560. doi:10.1074/mcp.RA120.002055.

Item is

Basic

show hide
Genre: Journal Article

Files

show Files
hide Files
:
1546.full.pdf (Publisher version), 3MB
Name:
1546.full.pdf
Description:
-
OA-Status:
Visibility:
Public
MIME-Type / Checksum:
application/pdf / [MD5]
Technical Metadata:
Copyright Date:
-
Copyright Info:
© 2020 Karayel et al. Author's Choice—Final version open access under the terms of the Creative Commons CC-BY license.

Locators

show
hide
Description:
DATA AVAILABILITY Proteomics raw data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD015219 and PXD019814 and at Panorama Web (https://panoramaweb.org/pRab10.url). Annotated spectra for the results can be viewed through MS-Viewer using the search key zbcwt6pszv (44).
OA-Status:

Creators

show
hide
 Creators:
Karayel, Özge1, Author           
Tonelli, Francesca2, Author
Virreira Winter, Sebastian1, Author           
Geyer, Philipp E.1, Author           
Fan, Ying2, Author
Sammler, Esther M.2, Author
Alessi, Dario R.2, Author
Steger, Martin1, Author           
Mann, Matthias1, Author           
Affiliations:
1Mann, Matthias / Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Max Planck Society, ou_1565159              
2external, ou_persistent22              

Content

show
hide
Free keywords: ASSAY DEVELOPMENT; ABSOLUTE SILAC; 14-3-3 BINDING; KINASE; QUANTIFICATION; QUANTITATION; ANTIBODIES; PROTEOMICS; MUTATIONS; PROTEINSBiochemistry & Molecular Biology; Biomarker; diagnostic; clinical proteomics; neurodegenerative diseases; parallel reaction monitoring; Parkinson disease; phosphorylation; protein kinases; targeted mass spectrometry; targeted therapies; selected ion monitoring;
 Abstract: 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. 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.

Details

show
hide
Language(s): eng - English
 Dates: 2020-092020
 Publication Status: Published in print
 Pages: 16
 Publishing info: -
 Table of Contents: Acknowledgments:
We thank Sabine Suppmann, Leopold Urich, Stephan Uebel, Stefan Pettera, Martin Spitaler, Nagarjuna Nagaraj, Victoria Sanchez and Antonio Piras from the MPIB Biochemistry Core Facility.
 Rev. Type: Peer
 Degree: -

Event

show

Legal Case

show

Project information

show hide
Project name : -
Grant ID : 6986
Funding program : -
Funding organization : Michael J. Fox Foundation for Parkinson's Research (MJFF)

Source 1

show
hide
Title: Molecular and Cellular Proteomics
Source Genre: Journal
 Creator(s):
Affiliations:
Publ. Info: Bethesda, MD : American Society for Biochemistry and Molecular Biology
Pages: - Volume / Issue: 19 (9) Sequence Number: - Start / End Page: 1546 - 1560 Identifier: ISSN: 1535-9476
CoNE: https://pure.mpg.de/cone/journals/resource/111035577487002