The overall goal of this reference measurement procedure is to harmonize measurements of a beta in human CSF. This method can help standardize assays for the measurement of CSF a beta 42 which is a biomarker for central amyloid pathology and neuropathologic hallmark of Alzheimer's disease. The main advantage of this technique is that it is thoroughly validated and doesn't use antibodies for quantification of AVR 142.
The implications of this technique extend toward therapy of Alzheimer's disease because it would be used to harmonize assays and facilitate the introduction of general cut off levels in clinical trials. While this method can provide insight into amyloid pathology in humans, it can also be applied to other mortal organisms such as transgenic mice and nonhuman primates to invenerate new drugs. Generally, individuals new to this method will struggle because AVR 142 is very hydrophobic and can easily stick to surfaces such as pipet tips and tubes.
First, add 40 microliters of N15 a beta peptide to 0.46 milliliters of 20%acetonitrile and 4%concentrated ammonia in a 0.5 milliliter microcentrifuge tube. Mix the peptide solution on a vortex mixer for one minute. Add 50 microliters of the peptide solution to 1.95 milliliters of 20%acetonitrile and 4%concentrated ammonia in a two milliliter microcentrifuge tube.
Mix the peptide solution on a vortex mixer for one minute. Next, prepare six calibrator solutions by mixing the appropriate volumes of each solution in microcentrifuge tubes. After mixing the calibrator solutions, prepare the final calibrators in duplicate by mixing the appropriate volumes of the corresponding calibrator solutions and human CSF in 0.5 milliliter microcentrifuge tubes.
Mix each solution on a vortex mixer for one minute. To prepare the internal standard, first add 32 microliters of 13C a beta peptide to 1.968 milliliters of 20%acetonitrile and 4%concentrated ammonia in a two milliliter microcentrifuge tube. Mix the peptide solution on a vortex mixer for one minute.
Next, add 0.1 milliliters of the peptide solution to 4.9 milliliters of 20%acetonitrile and 4%concentrated ammonia in a five milliliter microcentrifuge tube. Mix the peptide solution on a vortex mixer for one minute. To prepare the response factor sample, add 40 microliters of native a beta peptide to 0.46 milliliters of 20%acetonitrile and 4%concentrated ammonia in a 0.5 milliliter microcentrifuge tube.
After mixing, add 20 microliters of the peptide solution and 20 microliters of four micrograms per milliliter, 15N a beta peptide to 1.96 milliliters of 20%acetonitrile and 4%concentrated ammonia in a two milliliter microcentrifuge tube. Mix the peptide solution on a vortex mixer for one minute. Following this, add 20 microliters of the peptide solution to 0.38 milliliters of artificial CSF in a 0.5 milliliter microcentrifuge tube.
After preparing duplicates, mix each solution on a vortex mixer for one minute. After thawing the samples on a roller, add 0.18 milliliters of each calibrator, response factor and unknown sample to a one milliliter protein 96 deep well plate, making sure to add the samples in or close to the bottom of the wells. Now, add 20 microliters of internal standard to each well by releasing a drop of the standard on the side of the well close to the surface of the sample without submerging the pipet tip.
Then, add 0.2 milliliters of five molar guanadine hydrochloride to each well. Place the sample plate on a microplate shaker and mix the samples for 45 minutes at 1100 rpm. After mixing, add 0.2 milliliters of 4%phosphoric acid to each well.
Then, place the sample plate on the microplate shaker and mix briefly at 1000 rpm. Place a reservoir tray for waste under a mixed mode cation exchange 96 well solid phase extraction, or SPE plate in the extraction plate manifold chamber. Next, condition the SPE sorbent by adding 0.2 milliliters of methanol to each well.
Then, equilibrate the sorbent by adding 0.2 milliliters of 4%phosphoric acid to each well. Using an eight channel pipet, transfer all samples from the deep well plate to the SPE plate. Following this, wash the sorbent twice after the samples have passed through by adding 0.2 milliliters of 4%phosphoric acid to each well.
After the washing solvent has eluted from the sorbent, replace the reservoir tray with tubes. Now, elute the sample from the sorbent by adding two 50 microliter aloquats of 75%acetonitrile and 10%concentrated ammonia, noting that this solution requires a very low vacuum to pass through the sorbent. Dry the eluates by using vacuum centrifugation without applying heat.
Finally, seal the tubes and freeze them at 80 degrees celsius until analysis. The calibrators are close to the regression line with low standard deviations. If calibration is nonlinear, the run should be discarded and the deviation is most likely due to incorrect pipetting techniques and/or errors in the dilution of calibrators.
The coefficient of variation of replicates should preferably be below 10%The native 15N and 13C a beta elute from the LC column simultaneously with close to symmetrical peaks and without significant tailing. At least 10 measurements should be performed for each peak and can be adjusted with a maximum injection time in the method. The peptides can be measured simultaneously during the MS analysis unless the method sensitivity is suboptimal, in which only peptides of interest should be measured for each injection.
Once mastered, this technique can be done in four hours if it's performed properly. While attempting this procedure, it's important to remember to always saturate the pipet tips before transferring the peptide solution. It's also important to use tubes with as little void volume as possible.
After its development, this technique paved the way for researchers in the field of Alzheimer's disease to explore concordance with other methods, such as amyloid PET imaging. It has also been used to assigned a beta 142 concentrations to CSF based reference materials. After watching this video, you should have a good understanding of how to prepare calibrators in human CSF and using solid phase extraction prior to LCMS analysis to measure the absolute concentration of a beta 142 in samples.
Don't forget that working with human CSF can be extremely hazardous and precautions such as gloves, protective glasses and other safety measures should always be taken while performing this procedure.
