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>

<ol>
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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. 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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. 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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 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 AÃŽÂ²1-42 in CSF Using a Mass Spectrometric Reference Measurement Procedure at JoVE.com

Absolute Quantification of A&#946;1-42 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.

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 ...

absolute-quantification-a1-42-csf-using-mass-spectrometric-reference

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JoVE Journal

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7.5K Views.  The Sahlgrenska Academy at University of Gothenburg, Sahlgrenska University Hospital. A reference measurement procedure for the absolute quantification of Aβ1-42 in human CSF based on solid-phase extraction and liquid chromatography tandem mass spectrometry is described.

Video: Absolute Quantification of Aβ1-42 in CSF Using a Mass Spectrometric Reference Measurement Procedure

Magnetic Resonance Imaging Quantification of Pulmonary Perfusion using Calibrated Arterial Spin Labeling

This demonstrates a MR imaging method to measure the spatial distribution of pulmonary blood flow in healthy subjects 
during normoxia (inspired O2, fraction (FIO2) = 0.21) hypoxia (FIO2 = 0.125), and hyperoxia 
(FIO2 = 1.00). In addition, the physiological responses of the subject are monitored in the MR scan environment. MR images 
were obtained on a 1.5 T GE MRI scanner during a breath hold from a sagittal slice in the right lung at functional residual capacity. An arterial 
spin labeling sequence (ASL-FAIRER) was used to measure the spatial distribution of pulmonary blood flow 1,2 and a multi-echo fast 
gradient echo (mGRE) sequence 3 was used to quantify the regional proton (i.e. H2O) density, allowing the quantification 
of density-normalized perfusion for each voxel (milliliters blood per minute per gram lung tissue). 
With a pneumatic switching valve and facemask equipped with a 2-way non-rebreathing valve, different oxygen concentrations 
were introduced to the subject in the MR scanner through the inspired gas tubing. A metabolic cart collected expiratory gas via expiratory tubing. Mixed expiratory O2 and CO2 concentrations, oxygen consumption, carbon dioxide production, respiratory exchange ratio, 
respiratory frequency and tidal volume were measured. Heart rate and oxygen saturation were monitored using pulse-oximetry. 
Data obtained from a normal subject showed that, as expected, heart rate was higher in hypoxia (60 bpm) than during normoxia (51) or hyperoxia (50) and the arterial oxygen saturation (SpO2) was reduced during hypoxia to 86%. Mean ventilation was 8.31 L/min BTPS during hypoxia, 7.04 L/min during normoxia, and 6.64 L/min during hyperoxia. Tidal volume was 0.76 L during hypoxia, 0.69 L during normoxia, and 0.67 L during hyperoxia. 
Representative quantified ASL data showed that the mean density normalized perfusion was 8.86 ml/min/g during hypoxia, 8.26 ml/min/g during normoxia and 8.46 ml/min/g during hyperoxia, respectively. In this subject, the relative dispersion4, an index of global heterogeneity, was increased in hypoxia (1.07 during hypoxia, 0.85 during normoxia, and 0.87 during hyperoxia) while the fractal dimension (Ds), another index of heterogeneity reflecting vascular branching structure, was unchanged (1.24 during hypoxia, 1.26 during normoxia, and 1.26 during hyperoxia). 
Overview. This protocol will demonstrate the acquisition of data to measure the distribution of pulmonary perfusion noninvasively under conditions of normoxia, hypoxia, and hyperoxia using a magnetic resonance imaging technique known as arterial spin labeling (ASL). 
Rationale: Measurement of pulmonary blood flow and lung proton density using MR technique offers high spatial resolution images which can be quantified and the ability to perform repeated measurements under several different physiological conditions. In human studies, PET, SPECT, and CT are commonly used as the alternative techniques. However, these techniques involve exposure to ionizing radiation, and thus are not suitable for repeated measurements in human subjects.

A MR imaging method to study the distribution of pulmonary blood flow under a variety of physiological conditions, in this case exposure to three different inspired oxygen concentrations: hypoxia, normoxia, and hyperoxia, is described. This technique utilizes human pulmonary physiology research techniques in an MR scanning environment.

This demonstrates a MR imaging method to measure the spatial distribution of pulmonary blood flow in healthy subjects 
during normoxia (inspired O2, ...

Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are associated with obesity, though the precise underlying mechanisms that contribute to the disease are currently being delineated.2,3 Several small animal models of obesity have been developed and are employed in a variety of studies.4 A critical component to these experiments involves the collection of regional and/or total animal fat content data under varied conditions.
Traditional experimental methods available for measuring fat content in small animal models of obesity include invasive (e.g. ex vivo measurement of fat deposits) and non-invasive (e.g. Dual Energy X-ray Absorptiometry (DEXA), or Magnetic Resonance (MR)) protocols, each of which presents relative trade-offs. Current invasive methods for measuring fat content may provide details for organ and region specific fat distribution, but sacrificing the subjects will preclude longitudinal assessments. Conversely, current non-invasive strategies provide limited details for organ and region specific fat distribution, but enable valuable longitudinal assessment. With the advent of dedicated small animal X-ray computed tomography (CT) systems and customized analytical procedures, both organ and region specific analysis of fat distribution and longitudinal profiling may be possible. Recent reports have validated the use of CT for in vivo longitudinal imaging of adiposity in living mice.5,6 Here we provide a modified method that allows for fat/total volume measurement, analysis and visualization utilizing the Carestream Molecular Imaging Albira CT system in conjunction with PMOD and Volview software packages.

Fat content analysis is routinely conducted in studies utilizing murine obesity models. Emerging methods in small animal CT imaging and analysis are providing for longitudinal detail rich fat content analysis. Here we detail step by step procedures for performing small animal CT imaging, analysis, and visualization.

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are ...

Epidural Intracranial Pressure Measurement in Rats Using a Fiber-optic Pressure Transducer

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and cardiac arrest. This elevation may result in further neurological injury, in the form of transtentorial herniation1,2,3,4, midbrain compression, neurological deficit or increased cerebral infarct2,4. Current therapies are often inadequate to control elevated ICP in the clinical setting5,6,7 . Thus there is a need for accurate methods of ICP measurement in animal models to further our understanding of the basic mechanisms and to develop new treatments for elevated ICP.
In both the clinical and experimental setting ICP cannot be estimated without direct measurement. Several methods of ICP catheter insertion currently exist. Of these the intraventricular catheter has become the clinical 'gold standard' of ICP measurement in humans8. This method involves the partial removal of skull and the instrumentation of the catheter through brain tissue. Consequently, intraventricular catheters have an infection rate of 6-11%9. For this reason, subdural and epidural cannulations have become the preferred methods in animal models of ischemic injury. 
Various ICP measurement techniques have been adapted for animal models, and of these, fluid-filled telemetry catheters10 and solid state catheters are the most frequently used11,12,13,14,15. The fluid-filled systems are prone to developing air bubbles in the line, resulting in false ICP readings. Solid state probes avoid this problem (Figure 1). An additional problem is fitting catheters under the skull or into the ventricles without causing any brain injury that might alter the experimental outcomes. Therefore, we have developed a method that places an ICP catheter contiguous with the epidural space, but avoids the need to insert it between skull and brain. 
An optic fibre pressure catheter (420LP, SAMBA Sensors, Sweden) was used to measure ICP at the epidural location because the location of the pressure sensor (at the very tip of the catheter) was found to produce a high fidelity ICP signal in this model. There are other manufacturers of similar optic fibre technologies13 that may be used with our methodology. Alternative solid state catheters, which have the pressure sensor located at the side of the catheter tip, would not be appropriate for this model as the signal would be dampened by the presence of the monitoring screw. 
Here, we present a relatively simple and accurate method to measure ICP. This method can be used across a wide range of ICP related animal models.

A novel technique to record the pressures within the skull is described. The minimally invasive method uses a fibre-optic pressure sensing system to accurately measure intracranial pressure (ICP) in anaesthetized rats without causing significant brain trauma. The technique may be used in a wide range of experimental models.

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United States. Tacrolimus is a narrow therapeutic index drug and as such requires therapeutic drug monitoring and dose adjustment based on its whole blood trough concentrations. To facilitate home therapeutic drug and adherence monitoring, the collection of dried blood spots is an attractive concept. After a finger stick, the patient collects a blood drop on filter paper at home. After the blood is dried, it is mailed to the analytical laboratory where tacrolimus is quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in combination with a simple manual protein precipitation step and online column extraction.
For tacrolimus analysis, a 6-mm disc is punched from the saturated center of the blood spot. The blood spot is homogenized using a bullet blender and then proteins are precipitated with methanol/0.2 M ZnSO4 containing the internal standard D2,13C-tacrolimus. After vortexing and centrifugation, 100 &#181;l of supernatant is injected into an online extraction column and washed with 5 ml/min of 0.1 formic acid/acetonitrile (7:3, v:v) for 1 min. Hereafter, the switching valve is activated and the analytes are back-flushed onto the analytical column (and separated using a 0.1% formic acid/acetonitrile gradient). Tacrolimus is quantified in the positive multi reaction mode (MRM) using a tandem mass spectrometer.
The assay is linear from 1 to 50 ng/ml. Inter-assay variability (3.6%-6.1%) and accuracy (91.7%-101.6%) as assessed over 20 days meet acceptance criteria. Average extraction recovery is 95.5%. There are no relevant carry-over, matrix interferences and matrix effects. Tacrolimus is stable in dried blood spots at RT and at +4 &#176;C for 1 week. Extracted samples in the autosampler are stable at +4 &#176;C for at least 72 hr.

Here we describe a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay to quantify the immunosuppressant tacrolimus in dried blood spots using a simple manual protein precipitation step and online column extraction.

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United ...

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

Network Analysis of Foramen Ovale Electrode Recordings in Drug-resistant Temporal Lobe Epilepsy Patients

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control seizures. However, a significant minority of patients continues to exhibit post-operative seizures, even in those cases in which the suspected source of seizures has been correctly localized and resected. The protocol presented here combines a clinical procedure routinely employed during the pre-operative evaluation of temporal lobe epilepsy (TLE) patients with a novel technique for network analysis. The method allows for the evaluation of the temporal evolution of mesial network parameters. The bilateral insertion of foramen ovale electrodes (FOE) into the ambient cistern simultaneously records electrocortical activity at several mesial areas in the temporal lobe. Furthermore, network methodology applied to the recorded time series tracks the temporal evolution of the mesial networks both interictally and during the seizures. In this way, the presented protocol offers a unique way to visualize and quantify measures that considers the relationships between several mesial areas instead of a single area.

This protocol describes a procedure to track the evolution of mesial network measures in temporal lobe epilepsy (TLE) patients. It is based on the combination of intracranial recordings with a novel numerical technique for data analysis. Specifically, we present a protocol for network analyses of foramen ovale recordings.

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control ...

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry (LCM-LC/MS)

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel therapeutics are urgently required. The ability of dosed TB drugs to reach and sterilize bacteria within poorly-vascularized necrotic regions (caseum) of pulmonary granulomas is crucial for successful therapeutic intervention. Effective therapeutic regimens must therefore contain drugs with favorable caseum penetration properties. Current LC/MS methods for quantifying drug levels in biological tissues have limited spatial resolution capabilities, making it difficult to accurately determine absolute drug concentrations within small tissue compartments such as those found within necrotic granulomas. Here we present a protocol combining laser capture microdissection (LCM) of pathologically-distinct tissue regions with LC/MS quantification. This technique provides absolute quantification of drugs within granuloma caseum, surrounding cellular lesion and uninvolved lung tissue and, therefore, accurately determines whether bactericidal concentrations are being achieved. In addition to tuberculosis research, the technique has many potential applications for spatially-resolved quantification of drugs in diseased tissues.

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry LCM-LC/MS

Here, we describe a protocol using laser capture microdissection coupled with LC/MS analysis to spatially-quantify drug distributions within pulmonary tuberculosis granulomas. The approach has broad applicability to quantifying drug concentrations within tissues at high spatial detail.

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel ...

Measurement of Carotenoids in Perifovea using the Macular Pigment Reflectometer

The macular pigment reflectometer (MPR) objectively measures the overall macular pigment optical density (MPOD) and further provides the lutein optical density (L-OD) and zeaxanthin optical density (Z-OD) in the central 1 degree of the fovea. A modification of the technique was developed to evaluate in vivo carotenoid density eccentric to the fovea. An adjustable track system with red LED lights was placed 6.1 m away from the participant to facilitate ocular fixation. Lights were spaced appropriately to create increments of 1 degree retinal disparity during the reflectometry measurements. All reflectometry measurements were obtained with pupillary dilation. The mean MPR-MPOD value for the central measurement was 0.593 (SD 0.161) with an L-OD to Z-OD ratio of 1:2.61. The MPR-MPOD value at 1 degree was 0.248 and the mean MPR-MPOD value at 2 degrees in the parafoveal region was 0.143. The L-OD to Z-OD ratio at 1 degree and 2 degrees off center was 1.38:1.0 and 2.08:1.0, respectively. The results demonstrate that MPOD measurements obtained using the MPR decrease as a function of retinal eccentricity and that there is a higher concentration of zeaxanthin centrally compared to lutein. The L-OD to Z-OD ratio changes with foveal eccentricity, with twice more lutein than zeaxanthin at 2 degrees off center. Our technique successfully provides a quick in vivo method for the measurement of macular pigment optical density at various foveal eccentricities. The results agree with prior published in vivo and in vitro xanthophyll carotenoid density distribution measurements.

We present a protocol to determine the levels of overall macular pigment, lutein, and zeaxanthin optical density in the central and parafoveal regions of the retina. The protocol includes a novel adjustable track system used to measure macular pigment optical density in the foveal eccentricity.

The macular pigment reflectometer (MPR) objectively measures the overall macular pigment optical density (MPOD) and further provides the lutein optical ...

Using an Automated Hirschberg Test App to Evaluate Ocular Alignment

A smartphone app has been developed to perform the automated photographic Hirschberg test for objective measurement of ocular misalignment. By computing the difference in corneal reflection generated by the phone camera flash relative to the iris center based on high resolution images, the app can measure misalignment with a much higher precision than the naked eye performing the Hirschberg test. It has been validated in a previous clinical evaluation study by comparing to the clinical gold standard-prism and alternate cover test. The goal of this article is to describe the testing techniques regarding how to use the app to measure ocular alignment for different fixation distances, without or with cover to break fusion, as well as angle kappa, so that users can use the app to perform equivalent tests typically done in clinic using prisms.

We present a protocol of using a smartphone app to perform Hirschberg test for measuring manifest and intermittent ocular misalignment (strabismus) under near and far fixation conditions.

A smartphone app has been developed to perform the automated photographic Hirschberg test for objective measurement of ocular misalignment. By computing ...