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 Table 2. The data generated by this method gives standardized ratios to the relevant stable isotope-labeled internal standard. These values can then be put through a standard curve to determine absolute pmol/100 µl CSF concentrations. These values can then be statistically analyzed for changes in clinical samples.
As described previously8, quantitation of the 74 peptides included in this CSF assay revealed that 25 of these markers were altered significantly in the CSF of dementia patients. To illustrate the effectiveness of this assay, results from previously described dementia markers Pro-Orexin and YKL chitinase 3-like protein (YKL-40)11,12 are given in Figure 4. The integrated ApoE assay identifies the ApoE isoform/allele status of the patient as well. Apo E4 is a known risk factor for Alzheimer's Disease, therefore integrating this into the assay will provide valuable information. The detection of the ApoE isoforms is explained in Figure 5 and is based on the detection of the corresponding peptides for the amino acid changes for isoforms E2 (R158C) and E4 (C112R). Figure 5A shows the peak pattern expected for each isoform combination and Figure 5B shows the result of a CSF tested with the assay on patient samples.
Figure 3. Representative Overlaid MRM Chromatograms. Reprint from Heywood et al.8 Biomarkers significant for the neurodegenerative disorders Lewy Body Dementia and Alzheimer's Disease8 are shown over a 10 min LC gradient. Please click here to view a larger version of this figure.
Figure 4. Example Data. Reprint from Heywood et al.8 Graphs show how the described MRM LC-MS/MS method can reliably quantitate and discriminate from controls the known neurodegenerative markers such as YKL chitinase 3-like protein (YKL-40)11,12 and the AD marker Pro-Orexin 13. AD = Alzheimer's Disease, LBD = Lewy Body Dementia and PD = Parkinson's Disease. Data previously published. Please click here to view a larger version of this figure.
Figure 5. Illustration of How the Apo E Isoform Status of a Patient Can Be Determined. A. Indicates the peptides covering the 112 amino acid sequence LGADMEDVCGR for neutral (E3a) or LGADMEDVR for presence of E4 and for position 158 to detect RLAVYQAGAR the neutral (E3b) or CLAVYQAGAR for the E2 isoform. B. Peptides from the ApoE sequence are shown in the left hand panel. The different combinations of the peptides detected in the CSF can indicate the ApoE isoform status. Please click here to view a larger version of this figure.
Time | Flow (ml/min) | % A | % B | Curve |
Initial | 0.8 | 97 | 3 | initial |
0.2 | 0.8 | 97 | 3 | 6 |
7 | 0.8 | 60 | 40 | 6 |
7.01 | 0.8 | 0.1 | 99.9 | 6 |
8 | 0.8 | 0.1 | 99.9 | 6 |
8.01 | 0.8 | 97 | 3 | 1 |
10 | 0.8 | 97 | 3 | 1 |
Table 1. UPLC Gradient Settings for a 10 min Method. A = ddH20 0.1% FA, B= ACN, 0.1% FA
Table 2. Peptides Included in the CSF MRM LC MS/MS Assay. Shown are all the included peptides used in the method, which are described and published 8. Indication of whether the marker was reliably detected in 100 µl of CSF is indicated. Transitions labeled in bold are the ones used for quantitative data. Please click here to download this table.
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 limited complexity (compared to plasma), it has been found that these steps can be removed from the final protocol, thereby simplifying the assay and making it more suitable for translation into diagnostic setting. The inclusions of a pre-column to the main analytical column and of the solvent delays in the MS method, appear to be sufficient to maintain sensitivity during the MS run for more than 500 samples. As the assay-UPLC running time is short, it is important to unequivocally identify the correct peak in the CSF digest matrix.
This can only be achieved with the use of spiked synthesized peptides and a standard curve. If there is an uncertainty of a peptide matched to the correct chromatographic peak then it may be useful to run the sample through multiple transitions and check the transitions intensity pattern with the synthetic standards. All of our assays contain at least 2 peptides transitions to confirm the identity of the peptide.
The assay has been developed for markers detected in up to 100 µl of CSF. For some other markers of dementia, a larger volume of CSF may be needed. Another limitation of the assay is the issue of dynamic range of protein expression. Some abundant markers need to be injected on the mass spectrometer in a smaller amounts whilst some low abundant markers require a larger injection volumes. Therefore the sample may need to be injected twice.
The significance of this technique is the ability to multiplex and the increased specificity of the 3 levels of identification over antibodies (retention time, precursor and product m/z). From the perspective of clinical translation, the biggest advantage is the potential cost saving on antibodies, though an initial outlay for the mass spectrometry equipment and trained personnel is required. Another asset is the speed in which the method can be translated onto the triple quadrupole based systems. This speeds up significantly the ability to translate and validate an assay compared to all immuno-based techniques. Finally, as this platform and expertise has been routinely used to measure small molecules clinically, many large hospital centers already have this infrastructure in place. In the field of neurodegeneration there is a lot of focus and need for new biomarkers in CSF and serum. This method provides a platform for a high-throughput assay where future markers can be added (and removed) to be assessed for efficacy, etc. This technique has further application to other tissues for ApoE isoform identification, such as plasma which has already been described2 and even bloodspots.
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.
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 |