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.