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* 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 protein biomarkers from discovery experiments. These can be further multiplexed over a rapid chromatographic separation (<10 min) and assessed. Within this multiplex screen for neurodegenerative biomarkers, we have included the known dementia risk factor apolipoprotein isoform E4 (ApoE4) so we can determine simultaneously its status and level of expression, by that eliminating the need for separate genotyping tests2. LC MS/MS is routinely used as the method of choice for accurately quantitating small molecules over other methods such as ELISA or radioimmunoassay (RIA). This shift in the use of MS technology to analyzing proteins, has been driven mainly by issues with the immuno-based technologies. These include cross-specificity, batch to batch variation, limited shelf life, and high cost. Therefore, targeted proteomics is rapidly becoming a growing alternative to antibody based methods such as Western blotting, RIA and ELISA. However, the ability to multiplex many markers into one assay is the major advantage of this technique over immuno-based methods3. The technique is applicable to many tissues and has been used as a validation strategy for many proteomics studies including plasma4 and urine5,6.
The technique can be applied to any laboratory that has access and expertise in using triple quadrupole mass spectrometers. Peptide design is relatively simple with the growing use of open source databases. The competitive market of custom peptides synthesis makes them much more affordable. Heavy peptides, however, are expensive and therefore the markers should be assessed on a small cohort before moving to a larger scale. There is growing potential for the technique to be used in the clinical diagnostic settings, with most large hospitals having triple quadrupole based platforms which can easily be adapted to run targeted proteomic assays. One such application of the method, making it into the routine diagnostic setting, is its recent application to newborn blood spot screening for sickle cell anemia7.
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. 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 synthesized peptides, a targeted LC-MS/MS multiplex method is created. After evaluation, the assay can be used to assess the efficacy of potential markers of neurodegeneration.
1. Peptide Selection and Design
NOTE: Criteria for a marker peptide is that it must be unique (proteotypic) and representing the quantitative abundance of the protein (quantotypic). To determine if a peptide is unique or not, the 'blast' search tool on the Uniprot website (http://www.uniprot.org/blast/) can be used.
2. Preparation of Standard Peptides
NOTE: In order to select the best quantitative transitions, the detection of the matrix (CSF) needs to be optimized. The most efficient way of optimizing multiplexed peptides is to create pools of the peptides at known concentrations. These pools can then be used for method development and standard curves.
3. Optimization of MS Peptide Detection
4. LC-MRM Method Development
NOTE: Analyze the mixture of synthetic peptides by an Ultra Performance Liquid Chromatography (UPLC) system coupled to triple quadrupole mass spectrometer. Ensure the source is clean. Solvent A is ddH20 with 0.1% FA; Solvent B is ACN with 0.1% FA.
Figure 2. Example of a Dynamic MRM MS Method. Timed channels of peptide transitions can be grouped according to established retention times. Enabling MRMs to be incorporated into a timed fashion as the selected marker peptides elute from the chromatography column, minimizes the number of transitions over a time period and increases the sensitivity of the assay. Please click here to view a larger version of this figure.
5. Addition of Internal Standards
NOTE: As described previously10, stable isotope-labeled internal standards can be included in the assay. Due to the expense of these standards, it is advised to first assess the peptides in the matrix.
6. LC-MRM Assay of CSF Patient Samples
7. Data Analysis
NOTE: Quantitative data is based on the intensity ratio of baseline peak internal standards (heavy labeled peptides). Detailed information regarding SRM/MRM data analysis was previously described10. Ratio data can then be used in a standard curve to determine absolute levels or calculate them from the added concentration of heavy-labeled peptide.
8. Apolipoprotein E Isoform Status
NOTE: To determine ApoE isoform status, the presence of the corresponding peptides can be performed by determining the presence of each isoform.
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...
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...
The authors have nothing to disclose.
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.
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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