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A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status

Published: October 20th, 2016



1Centre for Translational Omics, Genetics and Genomic Medicine Deptartment, Great Ormond Street Institute of Child Health, University College London, 2Dementia Research Centre, Institute of Neurology, University College London, 3Clinical Neurochemistry Laboratory, Institute of Neuroscience and Physiology, Department of Psychiatry and Neurochemistry, The Sahlgrenska Academy, University of Gothenburg, 4Neurology Unit, Department of Pathophysiology and Transplantation, University of Milan, 5Great Ormond Street Hospital for Children, University College London
* These authors contributed equally

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.

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.

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....

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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 1
Figure 1. Schematic Illustrating the Overall Process of Creating a Targeted CSF MRM LC-MS/MS Assay........

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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.......

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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.......

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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.


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Name Company Catalog Number Comments
Acetonitrile (ACN), LC-MS grade  Fisher A955-1
Formic acid, LC-MS grade, Fisher A117-50
Dithiothreitol (DTT) Sigma  D5545-5G
Hydrochloric acid, 37% w/w VWR  BDH3028-2.5LG
Iodoacetamide   Sigma  I1149-5G
Sodium hydroxide (NaOH) Fisher S318-500
Trypsin, sequencing grade, modified Promega V5113
Trifluoroacetic acid (TFA), LC-MS grade Fisher A116-50
Urea Sigma U0631-500g
Water, LC-MS ULTRA Chromasolv Fluka 14263
Custom synthesised peptides desalted 1-4mg Genscript custom
heavy labelled amino acid [C13 N15] custom peptides Genscript custom
ASB 14 Merck Millipore 182750-25gm
Thiourea Sigma T7875-500G
Tris base Sigma T6066
VanGuard precolumn Waters 186007125
Cortecs UPLC C18+ 1.6um  2.1 x50mm column Waters 186007114
Yeast Enolase  Sigma E6126
300ul clear screw top glass vials   Fisher scientific 03-FISV
Y slit screw caps  Fisher scientific 9SCK-(B)-ST1X
Freeze dryer Edwards Mudulyo  Mudulyo system
Concentrator/Speed vaccum Eppendof  concentrator  plus 5301
Xevo -TQ-S mass spectrometer Waters
Acquity UPLC system Waters

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