Name: a high throughput, multiplexed and targeted proteomic csf assay to quantify neurodegenerative biomarkers and apolipoprotein e isoforms status
Uploaded: 2016-10-20T14:00:00
Description: Watch this Scientific Journal Video about A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status at JoVE.com

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.

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.
<img alt="Figure 1" src="/files/ftp_upload/54541/54541fig1.jpg" /> 
Figure 1. Schematic Illustrating the Overall Process of Creating a Targeted CSF MRM LC-MS/MS Assay....

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Policy Brief: The Global Impact of Dementia 2013-2050. , (2013).</li><li>Hirtz, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+new+quantitative+mass+spectrometry+and+semi-automatic+isofocusing+methods+for+the+determination+of+Apolipoprotein+E+typing.">Development of new quantitative mass spectrometry and semi-automatic isofocusing methods for the determination of Apolipoprotein E typing.</a> Clin Chim Acta. 454, 33-38 (2015).</li><li>Marx, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+proteomics.">Targeted proteomics.</a> Nat Methods. 10 (1), 19-22 (2013).</li><li>Heywood, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+a+peptide+SRM-based+tandem+mass+spectrometry+assay+for+prenatal+screening+of+Down+syndrome.">The development of a peptide SRM-based tandem mass spectrometry assay for prenatal screening of Down syndrome.</a> J Proteomics. 75 (11), 3248-3257 (2012).</li><li>Heywood, W. 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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 -&#160;it is an entirely one-pot reaction method. Due to the small volume of CSF and its limite...

The growing impact of neurodegenerative diseases such as Alzheimer&#39;s Disease, Lewy Body Dementia and Parkinson&#39;s Disease, is becoming a socioeconomic issue in many countries1. There is a need in additional&#160;biomarkers that can be used to identify and classify patients in the early stages of the disease,&#160;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&#160;discovery experiments. These can be further multiplexed over a rapid chromatographic separation (&#60;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&#160;custom peptides synthesis&#160;makes them much more affordable. Heavy peptides, however, are expensive and therefore the markers should&#160;be assessed on a small cohort&#160;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&#160;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.

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			<td height="21" style="height:21px;width:352px;">Acetonitrile (ACN), LC-MS grade&#160;</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">A955-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formic acid, LC-MS grade,</td>
			<td>Fisher</td>
			<td>A117-50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dithiothreitol (DTT)</td>
			<td>Sigma&#160;</td>
			<td style="width:119px;">D5545-5G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrochloric acid, 37% w/w</td>
			<td>VWR&#160;</td>
			<td style="width:119px;">BDH3028-2.5LG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iodoacetamide&#160;&#160;</td>
			<td style="width:71px;">Sigma&#160;</td>
			<td style="width:119px;">I1149-5G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium hydroxide (NaOH)</td>
			<td>Fisher</td>
			<td>S318-500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin, sequencing grade, modified</td>
			<td>Promega</td>
			<td>V5113</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trifluoroacetic acid (TFA), LC-MS grade</td>
			<td>Fisher</td>
			<td>A116-50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Urea</td>
			<td>Sigma</td>
			<td>U0631-500g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Water, LC-MS ULTRA Chromasolv</td>
			<td>Fluka</td>
			<td>14263</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Custom synthesised peptides desalted 1-4mg</td>
			<td style="width:71px;">Genscript</td>
			<td style="width:119px;">custom</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:352px;">heavy labelled amino acid [C13 N15] custom peptides</td>
			<td style="width:71px;">Genscript</td>
			<td style="width:119px;">custom</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:352px;">ASB 14</td>
			<td style="width:71px;">Merck Millipore</td>
			<td style="width:119px;">182750-25gm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Thiourea</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">T7875-500G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Tris base</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">T6066</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">VanGuard precolumn</td>
			<td style="width:71px;">Waters</td>
			<td style="width:119px;">186007125</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Cortecs UPLC C18+ 1.6um&#160; 2.1 x50mm column</td>
			<td style="width:71px;">Waters</td>
			<td style="width:119px;">186007114</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Yeast Enolase&#160;</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">E6126</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:352px;">300ul clear screw top glass vials&#160;&#160;</td>
			<td style="width:71px;">Fisher scientific</td>
			<td style="width:119px;">03-FISV</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:352px;">Y slit screw caps&#160;</td>
			<td style="width:71px;">Fisher scientific</td>
			<td style="width:119px;">9SCK-(B)-ST1X</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:352px;">Freeze dryer</td>
			<td style="width:71px;">Edwards Mudulyo&#160;</td>
			<td style="width:119px;">Mudulyo system</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:352px;">Concentrator/Speed vaccum</td>
			<td style="width:71px;">Eppendof&#160;</td>
			<td style="width:119px;">concentrator&#160; plus 5301</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Xevo -TQ-S mass spectrometer</td>
			<td style="width:71px;">Waters</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Acquity UPLC system</td>
			<td style="width:71px;">Waters</td>
			<td style="width:119px;"></td>
		</tr>
	</tbody>
</table>

a high throughput, multiplexed and targeted proteomic csf assay to quantify neurodegenerative biomarkers and apolipoprotein e isoforms status

Many neurodegenerative diseases are&#160;still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be important in the development of future novel therapies. Often&#160;potential new biomarkers do not make it&#160;into the clinic&#160;due to limitations&#160;in their development and&#160;high costs.&#160;However,&#160;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&#160;for clinical translation into diagnostic laboratories. Traditionally, this platform has been used extensively for measurement of small molecules&#160;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&#160;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.

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&#39;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...

Watch this Scientific Journal Video about A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status at JoVE.com

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

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&#160;still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be ...

The overall goal of this method is to quickly and affordably quantitate multiple biomarkers in cerebrospinal fluid, or CSF, to assess their use towards diagnosis and treatment monitoring. This is a new assay that allows us to measure biomarkers that were not possible to measure before. And we think this can be used for diagnostic purposes and also to differentiate between different neurodegenerative diseases and potentially also give prognostic information.The main advantage of this technique is that it's designed to be rapid, simple in sample preparation, highly specific, and cost effective for translation into the clinical lab. The implications of this technique extend toward therapy or diagnosis, as this high-throughput test can be used in future clinical trials for novel therapies in neurodegeneration. This methodology can also be applied to other diseases and tissues, such as urine for kidney disorders, plasma for cardiomyopathy, and cells for pluripotency.To begin this procedure, resuspend the desired synthetic peptides to a one-milligram-per-milliliter stock concentration, according to the manufacturer's instructions. Prepare one-in-10 dilutions of the peptide from the stock concentration, and pool 1, 000 picomoles of each peptide into a low-binding microcentrifuge tube. Dry the pooled peptide sample in a SpeedVac concentrator.When finished, store the sample at minus 20 degrees Celsius. Next, aliquot 100 microliters of CSF into low-binding microcentrifuge tubes. Freeze-dry the CSF samples.Resuspend an aliquot of the pooled peptide sample in digestion buffer to obtain concentrations of 10 and one picomole per microliter. Now, spike the pooled peptide sample into the freeze-dried 100-microliter aliquots of CSF, at zero, one, two, five, 10, and 15 picomolar concentrations. Then, add 20 nanograms of intact unrelated protein to act as an internal standard and control for the digestion efficiency of trypsin.Add digest buffer to the CSF aliquots to a final volume of 20 microliters, and vortex the samples. Following this, digest the samples per standard protocol by adding 1.5 microliters of dithiothreitol, and shaking at room temperature for one hour. Following this, add three microliters of iodoacetamide to the samples and shake at room temperature for 45 minutes in the dark.Then, add 165 microliters of double-distilled water. Add 10 microliters of a 0.1-microgram-per-microliter, sequencing-grade modified trypsin solution, resuspended in 50-millimolar ammonium bicarbonate buffer to the samples. Incubate the samples in a water bath at 37 degrees Celsius overnight.Then, stop the digestion by freezing the samples. After defrosting the CSF digests on ice, centrifuge at 16, 000 times g for 10 minutes. When finished, transfer 60 microliters of each digest into a 300-microliter glass insert vile.Using an LC-MS system, equipped with a C18 UPLC column, inject the highest concentration from the standard curve point using a 10-minute, one-to-40%acetonitrile linear gradient. Once the run is complete, open the resulting chromatogram in the software, and note the retention time and the top two most intense transitions per peptide. Based on this information, update a previously created multiple reaction monitoring, or MRM method with timed channels to measure peptides.To maintain sensitivity, keep each channel with points per peak greater than eight and dwell time greater than 0.01 seconds for at least one transition for each peptide. Include solvent delays in the MRM method by selecting solvent delays in method events in the MS method file. It is critical to correctly match the correct peak in a short HPLC method.This can be done using the spiked peptides in the matrix that are used for a standard curve and observing the multiple transitions during the method development. Finally, manually review the data annotation to ensure accuracy. Analyze each peptide and ratio to an appropriate stable isotope-labeled internal standard.A chromatogram of the significant peptide markers from the high-throughput CSF assay is shown here. The data gives standardized ratios to the relevant stable isotope-labeled internal standard, which can be used to determine absolute CSF concentrations and statistically analyzed for changes in clinical samples. Quantitation of the 74 peptides included in this CSF assay revealed that 25 were altered significantly in the CSF of dementia patients, and results from dementia markers pro-orexin and YKL chitinase-3-like protein are displayed here.ApoE4 is a known risk factor for Alzheimer's disease, and the detection of ApoE isoforms is based on the detection of the corresponding peptides for the amino acid changes for isoforms E2 and E4.The peak pattern expected for each isoform combination is shown here. Once the method is set up, the assay can be done in a day if it is performed properly. After the development of this method, it was possible to measure a new set of biomarkers with high analytical sensitivity and specificity, and this will make it possible for researchers to assess neurodegenerative diseases in a new manner.After watching this video, you should have a good understanding of how to design and optimize an MRM assay for sensitivity and specificity. Don't forget that working with DTE, termite, formic acid, and human tissue can be extremely dangerous and precautions, such as wearing gloves, should be applied during this procedure.

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