A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

We describe a high-throughput, multiplex, and targeted proteomic cerebrospinal fluid (CSF) assay developed with potential for clinical translation. The test can quantitate potential markers and risk factors for neurodegeneration, such as the apolipoprotein E variants (E2, E3 and E4), and measure their allelic expression.

Abstract

Many neurodegenerative diseases are still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be important in the development of future novel therapies. Often potential new biomarkers do not make it into the clinic due to limitations in their development and high costs. However, targeted proteomics using Multiple Reaction Monitoring Liquid Chromatography-tandem/Mass Spectrometry (MRM LC-MS/MS), specifically using triple quadrupole mass spectrometers, is one method that can be used to rapidly evaluate and validate biomarkers for clinical translation into diagnostic laboratories. Traditionally, this platform has been used extensively for measurement of small molecules in clinical laboratories, but it is the potential to analyze proteins, that makes it an attractive alternative to ELISA (Enzyme-Linked Immunosorbent Assay)-based methods. We describe here how targeted proteomics can be used to measure multiplexed markers of dementia, including the detection and quantitation of the known risk factor apolipoprotein E isoform 4 (ApoE4).

In order to make the assay suitable for translation, it is designed to be rapid, simple, highly specific and cost effective. To achieve this, every step in the development of the assay must be optimized for the individual proteins and tissues they are analyzed in. This method describes a typical workflow including various tips and tricks to developing a targeted proteomics MRM LC-MS/MS for translation.

The method development is optimized using custom synthesized versions of tryptic quantotypic peptides, which calibrate the MS for detection and then spiked into CSF to determine correct identification of the endogenous peptide in the chromatographic separation prior to analysis in the MS. To achieve absolute quantitation, stable isotope-labeled internal standard versions of the peptides with short amino acid sequence tags and containing a trypsin cleavage site, are included in the assay.

Introduction

The growing impact of neurodegenerative diseases such as Alzheimer's Disease, Lewy Body Dementia and Parkinson's Disease, is becoming a socioeconomic issue in many countries1. There is a need in additional biomarkers that can be used to identify and classify patients in the early stages of the disease, and to monitor any potential new treatments. The overall goal of this method is to create a generic pipeline for a streamlined, economic and faster way of validating potential CSF markers of neurodegeneration. The rationale is to use targeted proteomics or peptide MRM LC-MS/MS as an easily amendable method to assess multiple potential protei....

Protocol

NOTE: A schematic of the overall protocol described here is given in Figure 1. All samples used for the development of this method are surplus clinical diagnostic samples and have ethical approval from the London Bloomsbury Ethics committee.

figure-protocol-362
Figure 1. Schematic Illustrating the Overall Process of Creating a Targeted CSF MRM LC-MS/MS Assay. Candidate marker peptides for evaluation are selected from protein targets. Through the use of custom synthesi....

Results

Using the method described above, a high throughput 10 min multiplex assay consisting of 74 peptides from 54 proteins was developed, as an assay for markers of the neurodegenerative disorders Alzheimer's Disease and Lewy Body Dementia (LBD)8. Figure 3 shows a multiplex chromatogram published previously8 of the significant peptide markers from the assay. The peptides included in the assay and their quantitative transitions are given in Tab.......

Discussion

As with all MS based assays, the critical steps in the method are the determination of the appropriate and accurate amounts of internal standards. If absolute quantitation is being used, then the correct amounts of spiked peptides in the standard curve are also critical.

Our assay does not require the precipitation of the CSF or the use of any type of clean up or desalting steps prior to MS analysis - it is an entirely one-pot reaction method. Due to the small volume of CSF and its limite.......

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was funded and facilitated through the GOSomics research initiative by the National Institute for Health Research, Biomedical Research Centre at Great Ormond Street Hospital and the UCL Biological Mass Spectrometry Centre at the UCL Institute of Child Health with kind donations from the Szeban Peto Foundation. The Dementia Research Centre is an Alzheimer's Research UK coordinating centre. The authors acknowledge the support of Alzheimer's Research UK, the NIHR Queen Square Dementia Biomedical Research Unit, UCL/H Biomedical Research Centre, and Leonard Wolfson Experimental Neurology Centre.

....

Materials

NameCompanyCatalog NumberComments
Acetonitrile (ACN), LC-MS grade FisherA955-1
Formic acid, LC-MS grade,FisherA117-50
Dithiothreitol (DTT)Sigma D5545-5G
Hydrochloric acid, 37% w/wVWR BDH3028-2.5LG
Iodoacetamide  Sigma I1149-5G
Sodium hydroxide (NaOH)FisherS318-500
Trypsin, sequencing grade, modifiedPromegaV5113
Trifluoroacetic acid (TFA), LC-MS gradeFisherA116-50
UreaSigmaU0631-500g
Water, LC-MS ULTRA ChromasolvFluka14263
Custom synthesised peptides desalted 1-4mgGenscriptcustom
heavy labelled amino acid [C13 N15] custom peptidesGenscriptcustom
ASB 14Merck Millipore182750-25gm
ThioureaSigmaT7875-500G
Tris baseSigmaT6066
VanGuard precolumnWaters186007125
Cortecs UPLC C18+ 1.6um  2.1 x50mm columnWaters186007114
Yeast Enolase SigmaE6126
300ul clear screw top glass vials  Fisher scientific03-FISV
Y slit screw caps Fisher scientific9SCK-(B)-ST1X
Freeze dryerEdwards Mudulyo Mudulyo system
Concentrator/Speed vaccumEppendof concentrator  plus 5301
Xevo -TQ-S mass spectrometerWaters
Acquity UPLC systemWaters

References

  1. Prince, P. M., Guerchet, D. M., Prina, D. M., et al. . Policy Brief: The Global Impact of Dementia 2013-2050. , (2013).
  2. Hirtz, C., et al. Development of new quantitative mass spectrometry and semi-automatic isofocusing me....

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Explore More Articles

Targeted ProteomicsMultiplexed AssayCerebrospinal FluidNeurodegenerative BiomarkersApolipoprotein E IsoformsRapid And Cost effectiveClinical TranslationDiagnosisTreatment MonitoringPeptide StandardsTrypsin DigestionInternal Standard

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2025 MyJoVE Corporation. All rights reserved