Name: Absolute Quantification of Aβ1-42 in CSF Using a Mass Spectrometric Reference Measurement Procedure (Video) | JoVE Journal
Uploaded: 2017-03-21T14:00:00.000+00:00
Duration: 8 min 40 s
Description: Learn absolute quantification of Aβ1-42 in human CSF using solid-phase extraction and LC-MS/MS. This validated reference method standardizes Alzheimer's biomarker assays without antibodies for clinical research.

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5 minutes

This work was performed on behalf of the International Federation of Clinical Chemistry Working Group on CSF Proteins, which has the following composition: Kaj Blennow (Chair) and Henrik Zetterberg, Gothenburg University, Sweden; Les Shaw and Magdalena Korecka, University of Philadelphia, PA, USA; Ingrid Zegers, Institute for Reference Materials and Measurements, Geel, Belgium; Piotr Lewczuk, Universit&#228;tsklinikum Erlangen, Germany; and Rand Jenkins, PPD Bioanalytical Laboratory, Richmond, VA, USA; Randall Bateman, Washington University, MO, USA; and H Vanderstichele, Innogenetics NV, Ghent, Belgium. This work was also part of the Global Consortium for Biomarker Standardization CSF Reference Methods Working Group, which is led by Maria C. Carrillo, Ph.D., Senior Director, Medical &amp; Scientific Relations, Alzheimer's Association. The study was supported by The Swedish Research Council (grants #14002, #521-2011-4709, and #2013-2546); the Knut and Alice Wallenberg Foundation; Demensf&#246;rbundet; and Emil and Wera Cornell's, Aina Wallstr&#246;m and Mary-Ann Sj&#246;blom's, Gun and Bertil Stohne's, Magnus Bergvall's, and Gamla Tj&#228;narinnor's Foundations.

NOTE: This protocol requires aliquots of at least 50 &#181;L, with a concentration of 50 &#181;g/mL for each A&#946; peptide, as starting material. The A&#946; peptides should be dissolved in 20% acetonitrile (ACN) and 4% concentrated ammonia solution in deionized water (v/v) and stored at 80 &#176;C.
Caution: See Table 1 for safety information.
1. Preparation of the Solutions
<ol>
	<li>Prepare 100 mL of 20% ACN and 4% concentrated ammonia solution in deionized water (v/v) by diluting 20 mL of ACN and 4 mL of concentrated ammonia (~25%) in deionized water. Adjust the final volume to 100 mL with deionized water. Make fresh daily.</li>
	<li>Prepare 50 mL of 5 M guanidine-hydrochloride by dissolving 26.08 g of guanidine-hydrochloride in deionized water to a final volume of 50 mL. Store at 20 &#176;C and make fresh monthly.</li>
	<li>Prepare 200 mL of 4% phosphoric acid in deionized water (v/v) by diluting 9.4 mL of concentrated phosphoric acid (~85%) in deionized water. Adjust the final volume to 200 mL with deionized water. Store in the refrigerator and make fresh weekly.</li>
	<li>Prepare 50 mL of 75% ACN and 10% concentrated ammonia solution (v/v) in deionized water by diluting 37.5 mL of ACN and 5 mL of concentrated ammonia (~25%) in deionized water. Adjust the final volume to 50 mL with deionized water. Make fresh daily.</li>
	<li>Thaw at least 2.5 mL of human CSF for the calibrators, obtained from de-identified leftover samples from routine clinical analysis.</li>
	<li>Prepare artificial CSF containing 150 mM Na, 3.0 mM K, 1.4 mM Ca, 0.8 mM Mg, 1.0 mM P, and 155 mM Cl in deionized water and add bovine serum albumin to a final concentration of 4 mg/mL. Only 1 mL is needed per analysis, but prepare a large volume, aliquot it, and store for future use.</li>
</ol>
2. Preparation of the Calibrators
<ol>
	<li>Prepare 0.5 mL of 4 &#181;g/mL 15N-A&#946;1-42 peptide by adding 40 &#181;L of 50 &#181;g/mL 15NA&#946;142 to 0.46 mL of 20% ACN and 4% concentrated ammonia in a 0.5-mL microcentrifuge tube. Mix on a vortex mixer for 1 min.</li>
	<li>Prepare 2 mL of 100 ng/mL 15N-A&#946;1-42 peptide by adding 50 &#181;L of the 4 &#181;g/mL 15NA&#946;142 to 1.95 mL of 20% ACN and 4% concentrated ammonia in a 2-mL microcentrifuge tube. Mix on a vortex mixer for 1 min.</li>
	<li>Prepare six calibrator solutions (A-F) by mixing the volumes of each solution indicated in Table 2. Use 0.5, 1.5, and 2 mL microcentrifuge tubes. Mix on a vortex mixer for 1 min.</li>
	<li>Prepare the final calibrators (in duplicate) in 0.5 mL microcentrifuge tubes by adding the corresponding calibration solutions and human CSF according to Table 3. Mix on a vortex mixer for 1 min.</li>
</ol>
3. Preparation of the Internal Standard
<ol>
	<li>Prepare 2 mL of 0.8 &#181;g/mL 13C-A&#946;1-42 peptide by adding 32 &#181;L of 50 &#181;g/mL 13CA&#946;142 to 1.968 mL of 20% ACN and 4% concentrated ammonia in a 2-mL microcentrifuge tube. Mix on a vortex mixer for 1 min.</li>
	<li>Prepare 5 mL of 16 ng/mL 13C-A&#946;1-42 peptide by adding 0.1 mL of 0.8 &#181;g/mL to 4.9 mL of 20% ACN and 4% concentrated ammonia in a 5 mL microcentrifuge tube. Mix on a vortex mixer for 1 min.</li>
</ol>
4. Preparation of the Response Factor Sample
NOTE: The response factor (RF) determination is performed to determine the concentration of the labeled peptide used for calibration (15N-A&#946;1-42). This requires that the concentration of the native A&#946;1-42 peptide has been determined using amino acid analysis (AAA). Thus, the volume and concentration of native A&#946;1-42 peptide aliquots need to fulfill the requirements of the AAA.
<ol>
	<li>Prepare 0.5 mL of 4 &#181;g/mL native (unlabeled)A&#946;1-42 by adding 40 &#181;L of 50 &#181;g/mL native A&#946;1-42 to 0.46 mL of 20% ACN and 4% concentrated ammonia in a 0.5-mL microcentrifuge tube. Mix on a vortex mixer for 1 min.</li>
	<li>Prepare a 2-mL 40 ng/mL mix of native and 15N-A&#946;1-42 by adding 20 &#181;L of 4 &#181;g/mL native A&#946;1-42 and 20 &#181;L of 4&#181;g/mL 15N-A&#946;1-42 to 1.96 mL of 20% ACN and 4% concentrated ammonia in a 2 mL microcentrifuge tube. Mix on a vortex mixer for 1 min.</li>
	<li>Add 20 &#181;L of the 40 ng/mL mix to 0.38 mL of artificial CSF in a 0.5 mL microcentrifuge tube. Prepare duplicates and mix on a vortex mixer for 1 min.</li>
</ol>
5. Sample Preparation
NOTE: Thaw the samples to be measured at room temperature on a roller.
<ol>
	<li>Add 0.18 mL of each calibrator, response factor, and unknown sample (including quality control [QC] samples, if used) to a 1 mL protein 96-deep-well plate, according to Figure 1 (assuming a full plate is used). Make sure to add the samples in, or close to, the bottom of the wells.</li>
	<li>Add 20 &#181;L of internal standard to each well (i.e., calibrators, response factors, QCs, and unknowns); it is crucial to release the drop on the side of the well close to the surface of the sample without submerging the pipette tip.</li>
	<li>Add 0.2 mL of 5 M guanidine-hydrochloride to each well.</li>
	<li>Place the sample plate on a microplate shaker and mix the samples for 45 min at 1,100 rpm. The optimal frequency might differ depending on instrumentation. Set the frequency and amplitude of the mixer so that the solutions are thoroughly mixed and no drops of internal standard or CSF are left unmixed on the side of the wells.</li>
	<li>Add 0.2 mL of 4% phosphoric acid to each well. Vortex mix briefly.</li>
</ol>
6. Solid Phase Extraction
NOTE: In all washing, loading, and elution steps, apply the lowest possible vacuum after adding the solution and gradually increase as needed to load or elute the solution. Disable the vacuum between each loading and elution step.
<ol>
	<li>Put a reservoir tray for waste under a mixed-mode, cation-exchange, 96-well solid phase extraction (SPE) plate in the extraction plate manifold chamber.</li>
	<li>Condition the SPE sorbent by adding 0.2 mL of methanol to each well.</li>
	<li>Equilibrate the sorbent by adding 0.2 mL of 4% phosphoric acid to each well.</li>
	<li>Transfer all samples (about 0.62 mL in each well) from the deep-well plate to the SPE plate. Use an eight-channel pipette when transferring the samples from the deep-well plate to the SPE plate; it is not crucial to transfer the entire or equal volumes of all samples, since the samples contain an internal standard that will compensate for variations.</li>
	<li>Wash the sorbent after the samples have passed through by adding 0.2 mL of 4% phosphoric acid to each well.</li>
	<li>After the washing solvent has eluted from the sorbent, replace the reservoir tray with a collection plate or tubes.</li>
	<li>Elute the sample from the sorbent by twice adding 50 &#181;L of 75% ACN/10% concentrated ammonia, and note that this solution requires a very low vacuum to pass through the sorbent. Remember to disable the vacuum between each addition.
	<ol>
		<li>OPTIONAL: Seal the collection plate or tubes and freeze them at -80 &#176;C. Remove the seal from the collection plate or tubes before proceeding to step 6.8.2.</li>
		<li>Dry the eluates by using vacuum centrifugation (without applying heat); this can take from one to several hours, depending on the vacuum centrifuge.</li>
		<li>Seal the containers and freeze them at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
7. Liquid Chromatography
<ol>
	<li>Prepare mobile phase A (5% ACN and 0.3% concentrated ammonia in deionized water [v/v]), B (4% deionized water and 0.1% concentrated ammonia in ACN [v/v]), and needle wash (50% ACN and 4% concentrated ammonia in deionized water [v/v]).
	<ol>
		<li>For 500 mL of mobile phase A, add 25 mL of ACN and 1.5 mL of concentrated ammonia to deionized water. Adjust the final volume to 500 mL with deionized water.</li>
		<li>For 500 mL of mobile phase B, add 500 &#181;L of concentrated ammonia and 25 mL of deionized water to ACN. Adjust the final volume to 500 mL with ACN.</li>
		<li>Prepare 250 mL of needle wash by adding 120 mL of ACN and 10 mL of concentrated ammonia to deionized water. Adjust the final volume to 250 mL with deionized water.</li>
		<li>Put the mobile phases A and B and needle wash bottles open in a sonication bath for 20 min before use with the LC system</li>
	</ol>
	</li>
	<li>Dissolve each sample with 25 &#181;L of 20% ACN and 4% concentrated ammonia solution and place them on a shaker for 20 min. Centrifuge down the samples and place them in the autosampler (keep at 7 &#176;C).</li>
	<li>Inject 20 &#181;L of sample on a 1 x 250 mm2 polystyrene-divinylbenzene (reversed-phase) monolithic column maintained at 50 &#176;C.
	<ol>
		<li>Use the LC gradient shown in Table 4 with a flow rate of 0.3 mL/min. Divert the first two and the last five min to waste (post-column) using a divert valve to reduce the contamination of the mass spectrometer.</li>
	</ol>
	</li>
</ol>
8. Mass Spectrometric Analysis
NOTE: These parameters were used for a quadrupole-orbitrap hybrid mass spectrometer equipped with aheated electrospray ionization source.
<ol>
	<li>Set the parameters for the ion source according to Table 5.</li>
	<li>Set the MS instrument to isolate the 4+ charge states of native A&#946;1-42 (1,129.48 mass-to-charge ratio [m/z]), 15N-A&#946;1-42 (1,143.00 m/z), and 13C-A&#946;1-42 (1,179.50 m/z) in the quadrupole mass analyzer with an isolation width of 2.5 m/z.</li>
	<li>Fragment the isolated peptides in the collision cell with a normalized collision energy (NCE) of 17.0. This might need to be tuned for each instrument, even of the same type (and especially if using other types of instruments, such as a triple quadrupole MS).</li>
	<li>Record the fragment spectra with a resolution of 17.500, with an automatic gain control target of 2 x 105 charges and a maximum injection time of 250 ms.</li>
</ol>
9. Data Processing
<ol>
	<li>Use the sum of the product ions (with a mass tolerance of &#177; 250 milli mass units [mmu]) in Table 6 to calculate the chromatographic areas for each peptide. Note that the ion types and numbers are only shown for native A&#946;1-42 product ions, since they are the same for both 15N-A&#946;1-42 and 13C-A&#946;1-42.</li>
	<li>Determine the average response factor of the two response factor samples by dividing the area under the curve (chromatographic peak) of 15N-A&#946;1-42 with the area under the curve of native A&#946;1-42.</li>
	<li>Adjust the concentration of the 15N-A&#946;1-42 used for calibration by multiplying it with the response factor calculated in step 9.2.</li>
	<li>Construct a calibration curve by plotting the area ratios of 15N-A&#946;1-42 to 13C-A&#946;1-42 from the two sets of calibrators against the concentration.</li>
	<li>Calculate the slope and intercept of the calibration curve using linear regression.</li>
	<li>Calculate the area ratio of native A&#946;1-42 to the internal standard (13C-A&#946;1-42) for unknown samples.</li>
	<li>Extrapolate the concentration of unknown samples from the calibration curve using the slope and intercept obtained in step 9.5.</li>
</ol>

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JP and EP reports no disclosures. HZ has served on the advisory boards of Roche Diagnostics, Eli Lilly, and Pharmasum Therapeutics. KB has served as a consultant or on the advisory boards for Fujirebio Europe, IBL International, and Roche Diagnostics. HZ and KB are co-founders of Brain Biomarker Solutions in Gothenburg AB, a GU Venture-based platform company at the University of Gothenburg.

For the described method, instead of using a surrogate matrix, we used the surrogate analyte approach13,14,15,16, which enables calibration in human CSF. The surrogate analyte approach involves two different isotopically labeled standards. One (15NA&#946;142) is used to generate the calibration curve in human CSF, while another (13CA&#946;142) is used...

Alzheimer&#39;s disease (AD) is the most common form of dementia and affects about 35 million people worldwide1. The neuropathological hallmarks of the disease widely believed to lie at the core of AD pathogenesis are intracellular neurofibrillary tangles of hyperphosphorylated tau protein2 and extracellular plaques consisting of aggregated amyloid-beta (A&#946;) peptides3. In line with this, the assessment of plaque pathology in vivo by biomarkers has recently been included in the research diagnostic criteria for AD4. For CSF measurements of A&#946;1-42, several immunoassays are available and are used in many clinical laboratories5. The concentration of A&#946;1-42 in CSF is approximately 50% lower in AD patients than in the cognitively normal elderly, reflecting the deposition of the peptide in plaques in the brain6,7.
These biomarkers are mainly analyzed using immunoassays (i.e., antibody-based techniques), but these assays may be influenced by matrix effects8. The use of immunoassays on different technology platforms and the lack of assay standardization9,10 makes the introduction of global cut-off concentrations difficult11,12. An analytically validated RMP would permit the uniform calibration of different assay platforms, ideally resulting in better comparability across analytical platforms and in better control of factors contributing to the overall measurement variability.
The absolute quantification of A&#946;1-42 using the developed LC-MS/MS method overcomes many of the issues associated with antibody-based techniques. The method, listed as an RMP by the Joint Committee for Traceability in Laboratory Medicine (JCTLM database identification number C11RMP9), will be used to determine the absolute concentration of A&#946;1-42 in a Certified Reference Material (CRM) to harmonize CSF A&#946;1-42 measurements across techniques and analytical platforms. The described workflow should be of relevance for the development of candidate reference methods for peptides and proteins within other areas of medicine.

<table><tbody><tr><td>Eppendorf Protein LoBind microcentrifuge tubes, 0.5 mL</td><td>Eppendorf</td><td>022431081</td><td></td></tr><tr><td>Eppendorf Protein LoBind microcentrifuge tubes, 1.5 mL</td><td>Eppendorf</td><td>022431081</td><td></td></tr><tr><td>Eppendorf Protein LoBind microcentrifuge tubes, 2 mL</td><td>Eppendorf</td><td>022431102</td><td></td></tr><tr><td>Eppendorf Protein LoBind microcentrifuge tubes, 5 mL</td><td>Eppendorf</td><td>0030 108.310</td><td></td></tr><tr><td>Eppendorf Protein LoBind Deepwell Plates 96</td><td>Eppendorf</td><td>951032905</td><td></td></tr><tr><td>Micronic 0.75 mL polypropylene tubes, V-bottom</td><td>Micronic</td><td>MPW32071BC3</td><td>Tubes used for collecting SPE eluate, but other collection devices can be used such as 96-well plates.</td></tr><tr><td>Micronic Split TPE Capcluster for 96 ind. tubes</td><td>Micronic</td><td>MP53026</td><td>Caps for Micronic tubes.</td></tr><tr><td>Micronic Loborack-96 white w high cover bar coded</td><td>Micronic</td><td>MPW51015BC3</td><td>Holder for Micronic tubes.</td></tr><tr><td>Biohit Optifit tips 1.2 mL</td><td>Sartorius</td><td>791210</td><td>Used to reach all the way to the bottom of the LoBind deepwell plates (96-well). Other narrow tips might work as well.</td></tr><tr><td>Waters Oasis MCX 96-well &amp;micro;Elution Plate</td><td>Waters</td><td>186001830BA&amp;nbsp;</td><td></td></tr><tr><td>Waters Plate manifold reservoir tray</td><td>Waters</td><td>WAT058942</td><td></td></tr><tr><td>Waters Extraction Plate Manifold for Oasis 96-Well Plates</td><td>Waters</td><td>186001831</td><td></td></tr><tr><td>Ammonium hydroxide solution, puriss. p.a., reag. ISO, reag. Ph. Eur., ~25% NH3 basis</td><td>Sigma-Aldrich</td><td>30501-1L-D</td><td></td></tr><tr><td>Acetonitrile, Far UV HPLC Gradient grade, 2.5 L</td><td>Fisher Scientific</td><td>A/0627/17X</td><td></td></tr><tr><td>ortho-Phosphoric acid 85%, ACS,ISO,Reag. Ph Eur</td><td>Merck Millipore</td><td>1005731000</td><td></td></tr><tr><td>Guanidine Hydrochloride, 500 g</td><td>Thermo Scientific</td><td>24110</td><td></td></tr><tr><td>Thermo Scientific Q-exactive Hybrid Quadrupole Orbitrap Mass Spectrometer</td><td>Thermo Scientific</td><td>IQLAAEGAAPFALGMAZR</td><td>With bundled Dionex Ultimate 3000 LC &amp;amp; autosampler.</td></tr><tr><td>Dionex ProSwift RP-4H Monolith Column (1.0 x 250mm)</td><td>Dionex</td><td>066640</td><td></td></tr><tr><td>Bovine Serum Albumin, lyophilized powder, suitable for (for molecular biology), Non-acetylated</td><td>Sigma-Aldrich</td><td>B6917</td><td></td></tr><tr><td>Beta-Amyloid (1-42), Ultra Pure, TFA</td><td>rPeptide</td><td>A-1002-2</td><td></td></tr><tr><td>15N Beta-Amyloid (1-42), Uniformly labeled</td><td>rPeptide</td><td>A-1102-2</td><td></td></tr><tr><td>13C Beta-Amyloid (1-42), Uniformly labeled</td><td>rPeptide</td><td>A-1106-2</td><td></td></tr><tr><td>Microplate shakers, TiMix 2</td><td>Edmund B&amp;uuml;hler</td><td>6110 000</td><td></td></tr></tbody></table>

absolute quantification of a&#946;1-42 in csf using a mass spectrometric reference measurement procedure

Absolute Quantification of A&#946;1-42 in CSF Using a Mass Spectrometric Reference Measurement Procedure

Alzheimer's disease (AD) is the most common neurodegenerative disease among the elderly and accounts for 60-80% of all cases of dementia. Currently, the diagnosis of AD is based on cognitive tests and mental state exams, but the peptide amyloid-beta (A&#946;) in cerebrospinal fluid (CSF) is increasingly used in clinical trials and settings. As for most protein and peptide biomarkers, quantification is performed using antibody-based techniques, such as enzyme-linked immunosorbent assay (ELISA). However, intra- and inter-laboratory variability in these assays hamper its use as a diagnostic marker in clinical routine.
An antibody-independent Reference Measurement Procedure (RMP) was developed based on solid-phase extraction (SPE) and liquid chromatography (LC)-tandem mass spectrometry (MS/MS), where stable, isotope-labeled A&#946; peptides were used as internal standards, enabling absolute quantification. A high-resolution quadrupole-orbitrap hybrid instrument was used for the measurements. The method allows for the quantification of CSF A&#946;1-42 between 150-4,000 pg/mL.

The plate setup in Figure 1 is used for a full plate of samples. If fewer unknown samples are to be analyzed, the second calibrator, RF, and QC sets should be placed after the first half of the unknown samples.
As seen in Figure 2, the calibrators are close to the regression line, with low standard deviations. This method has a lower level of quantification of 150 pg/mL and an upper level of qua...

A reference measurement procedure for the absolute quantification of A&#946;1-42 in human CSF based on solid-phase extraction and liquid chromatography tandem mass spectrometry is described.

Alzheimer's disease (AD) is the most common neurodegenerative disease among the elderly and accounts for 60-80% of all cases of dementia. Currently, the ...

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.

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