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 solutio...

<ol>
	<li>Querfurth, H. W., LaFerla, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease.">Alzheimer's disease.</a> N Engl J Med. 362 (4), 329-344 (2010).</li><li>Braak, H., Braak, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+the+neuropathology+of+Alzheimer's+disease.">Evolution of the neuropathology of Alzheimer's disease.</a> Acta Neurol Scand Suppl. 165, 3-12 (1996).</li><li>Blennow, K., de Leon, M. J., Zetterberg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease.">Alzheimer's disease.</a> Lancet. 368 (9533), 387-403 (2006).</li><li>Dubois, B., 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=Advancing+research+diagnostic+criteria+for+Alzheimer's+disease:+the+IWG-2+criteria.">Advancing research diagnostic criteria for Alzheimer's disease: the IWG-2 criteria.</a> Lancet Neurol. 13 (6), 614-629 (2014).</li><li>Blennow, K., Hampel, H., Weiner, M., Zetterberg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebrospinal+fluid+and+plasma+biomarkers+in+Alzheimer+disease.">Cerebrospinal fluid and plasma biomarkers in Alzheimer disease.</a> Nat Rev Neurol. 6 (3), 131-144 (2010).</li><li>Buchhave, P., 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=Cerebrospinal+fluid+levels+of+beta-amyloid+1-42,+but+not+of+tau,+are+fully+changed+already+5+to+10+years+before+the+onset+of+Alzheimer+dementia.">Cerebrospinal fluid levels of beta-amyloid 1-42, but not of tau, are fully changed already 5 to 10 years before the onset of Alzheimer dementia.</a> Arch Gen Psychiatry. 69 (1), 98-106 (2012).</li><li>Olsson, B., 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=CSF+and+blood+biomarkers+for+the+diagnosis+of+Alzheimer's+disease:+a+systematic+review+and+meta-analysis.">CSF and blood biomarkers for the diagnosis of Alzheimer's disease: a systematic review and meta-analysis.</a> The Lancet Neurology. 15 (7), 673-684 (2016).</li><li>Bjerke, 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=Confounding+factors+influencing+amyloid+Beta+concentration+in+cerebrospinal+fluid.">Confounding factors influencing amyloid Beta concentration in cerebrospinal fluid.</a> Int J Alzheimers Dis. , (2010).</li><li>Dumurgier, J., 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=Intersite+variability+of+CSF+Alzheimer's+disease+biomarkers+in+clinical+setting.">Intersite variability of CSF Alzheimer's disease biomarkers in clinical setting.</a> Alzheimers Dement. 9 (4), 406-413 (2013).</li><li>Mattsson, N., 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=CSF+biomarker+variability+in+the+Alzheimer's+Association+quality+control+program.">CSF biomarker variability in the Alzheimer's Association quality control program.</a> Alzheimers Dement. 9 (3), 251-261 (2013).</li><li>Kang, J. H., Korecka, M., Toledo, J. B., Trojanowski, J. Q., Shaw, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+Utility+and+Analytical+Challenges+in+Measurement+of+Cerebrospinal+Fluid+Amyloid-beta1-42+and+tau+Proteins+as+Alzheimer+Disease+Biomarkers.">Clinical Utility and Analytical Challenges in Measurement of Cerebrospinal Fluid Amyloid-beta1-42 and tau Proteins as Alzheimer Disease Biomarkers.</a> Clin Chem. 59 (6), 903-916 (2013).</li><li>Mattsson, N., 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=Reference+measurement+procedures+for+Alzheimer's+disease+cerebrospinal+fluid+biomarkers:+definitions+and+approaches+with+focus+on+amyloid+beta42.">Reference measurement procedures for Alzheimer's disease cerebrospinal fluid biomarkers: definitions and approaches with focus on amyloid beta42.</a> Biomark Med. 6 (4), 409-417 (2012).</li><li>Ahmadkhaniha, R., Shafiee, A., Rastkari, N., Kobarfard, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+quantification+of+endogenous+androgenic+steroids+in+cattle's+meat+by+gas+chromatography+mass+spectrometry+using+a+surrogate+analyte+approach.">Accurate quantification of endogenous androgenic steroids in cattle's meat by gas chromatography mass spectrometry using a surrogate analyte approach.</a> Anal Chim Acta. 631 (1), 80-86 (2009).</li><li>Jemal, M., Schuster, A., Whigan, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+chromatography/tandem+mass+spectrometry+methods+for+quantitation+of+mevalonic+acid+in+human+plasma+and+urine:+method+validation,+demonstration+of+using+a+surrogate+analyte,+and+demonstration+of+unacceptable+matrix+effect+in+spite+of+use+of+a+stable+isotope+analog+internal+standard.">Liquid chromatography/tandem mass spectrometry methods for quantitation of mevalonic acid in human plasma and urine: method validation, demonstration of using a surrogate analyte, and demonstration of unacceptable matrix effect in spite of use of a stable isotope analog internal standard.</a> Rapid Commun Mass Spectrom. 17 (15), 1723-1734 (2003).</li><li>Leinenbach, A., 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=Mass+spectrometry-based+candidate+reference+measurement+procedure+for+quantification+of+amyloid-beta+in+cerebrospinal+fluid.">Mass spectrometry-based candidate reference measurement procedure for quantification of amyloid-beta in cerebrospinal fluid.</a> Clin Chem. 60 (7), 987-994 (2014).</li><li>Li, W., Cohen, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+endogenous+analytes+in+biofluid+without+a+true+blank+matrix.">Quantitation of endogenous analytes in biofluid without a true blank matrix.</a> Anal Chem. 75 (21), 5854-5859 (2003).</li><li>Perret-Liaudet, A., 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=Cerebrospinal+fluid+collection+tubes:+a+critical+issue+for+Alzheimer+disease+diagnosis.">Cerebrospinal fluid collection tubes: a critical issue for Alzheimer disease diagnosis.</a> Clin Chem. 58 (4), 787-789 (2012).</li><li>Lewczuk, P., 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=Effect+of+sample+collection+tubes+on+cerebrospinal+fluid+concentrations+of+tau+proteins+and+amyloid+beta+peptides.">Effect of sample collection tubes on cerebrospinal fluid concentrations of tau proteins and amyloid beta peptides.</a> Clin Chem. 52 (2), 332-334 (2006).</li><li>Pannee, J., 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=Reference+measurement+procedure+for+CSF+amyloid+beta+(A&#946;)1-42+and+the+CSF+A&#946;1-42+/A&#946;1-40+ratio+-+a+cross-validation+study+against+amyloid+PET.">Reference measurement procedure for CSF amyloid beta (A&#946;)1-42 and the CSF A&#946;1-42 /A&#946;1-40 ratio - a cross-validation study against amyloid PET.</a> J Neurochem. 139 (4), 651-658 (2016).</li><li>Thienpont, L. M., Van Houcke, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traceability+to+a+common+standard+for+protein+measurements+by+immunoassay+for+in-vitro+diagnostic+purposes.">Traceability to a common standard for protein measurements by immunoassay for in-vitro diagnostic purposes.</a> Clin Chim Acta. 411 (23-24), 2058-2061 (2010).</li><li>Vesper, H. W., Thienpont, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traceability+in+laboratory+medicine.">Traceability in laboratory medicine.</a> Clin Chem. 55 (6), 1067-1075 (2009).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#181;Elution Plate</td>
			<td>Waters</td>
			<td>186001830BA&#160;</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 &#38; 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&#252;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

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...

Watch this Scientific Journal Video about Absolute Quantification of AÎ²1-42 in CSF Using a Mass Spectrometric Reference Measurement Procedure at JoVE.com

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

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.

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 ...

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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7.5K vistas.  The Sahlgrenska Academy at University of Gothenburg, Sahlgrenska University Hospital. 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'...