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

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&#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

Research

JoVE Journal

Medicine

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

SDS-PAGE/Immunoblot Detection of A&beta; Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

The anomalous folding and polymerization of the &beta;-amyloid (A&beta;) peptide is thought to initiate the neurodegenerative cascade in Alzheimer's disease pathogenesis1. A&beta; is predominantly a 40- or 42-amino acid peptide that is prone to self-aggregation into &beta;-sheet-rich amyloid fibrils that are found in the cores of cerebral senile plaques in Alzheimer's disease. Increasing evidence suggests that low molecular weight, soluble A&beta; multimers are more toxic than fibrillar A&beta; amyloid2. The identification and quantification of low- and high-molecular weight multimeric A&beta; species in brain tissue is an essential objective in Alzheimer's disease research, and the methods employed also can be applied to the identification and characterization of toxic multimers in other proteopathies3. Naturally occurring A&beta; multimers can be detected by SDS-polyacrylamide gel electrophoresis followed by immunoblotting with A&beta;-specific antibodies. However, the separation and detection of multimeric A&beta; requires the use of highly concentrated cortical homogenates and antigen retrieval in small pore-size nitrocellulose membranes. Here we describe a technique for the preparation of clarified human cortical homogenates, separation of proteins by SDS-PAGE, and antigen-epitope retrieval/Western blotting with antibody 6E10 to the N-terminal region of the A&beta; peptide. Using this protocol, we consistently detect A&beta; monomers, dimers, trimers, tetramers, and higher molecular weight multimers in cortical tissue from humans with Alzheimer's pathology.

SDS-PAGE/Immunoblot Detection of A&#946; Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval

We describe a technique for the preparation of clarified human cortical homogenates, protein separation by SDS-PAGE, antigen retrieval and immunoblotting with an antibody to the A&beta; peptide. Using this protocol, we consistently detect monomeric and multimeric A&beta; in cortical tissue from humans with Alzheimer's pathology.

The anomalous folding and polymerization of the &beta;-amyloid (A&beta;) peptide is thought to initiate the neurodegenerative cascade in Alzheimer's ...

Neuroscience

A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high purity material. Here we present a tailored HPLC purification protocol to produce high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides. We have found that the combination of commercially available hydrophobic poly(styrene/divinylbenzene) stationary phase, polymer laboratory reverse phase - styrenedivinylbenzene (PLRP-S) under high pH conditions, enables the attainment of high purity (&gt;95%) A&#946;42 in a single chromatographic run. The purification is highly reproducible and can be amended to both semi-preparative and analytical conditions depending upon the amount of material wished to be purified. The protocol can also be applied to the A&#946;40 peptide with identical success and without the need to alter the method.

Herein we report a tailored HPLC purification protocol that yields high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides, capable of oligomer formation. Amyloid beta is a highly aggregation prone, hydrophobic peptide implicated in Alzheimer&#39;s disease. The amyloidogenic nature of the peptide makes its purification a challenge.

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high ...

Biochemistry

Analysis of &#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and structure by spectrometry and scanning electron microscopy (SEM). A&#946;, which forms neurotoxic amyloid aggregates in Alzheimer&#39;s disease (AD), has been shown to interact with fibrinogen. This A&#946;-fibrinogen interaction makes the fibrin clot structurally abnormal and resistant to fibrinolysis. A&#946;-induced abnormalities in fibrin clotting may also contribute to cerebrovascular aspects of the AD pathology such as microinfarcts, inflammation, as well as, cerebral amyloid angiopathy (CAA). Given the potentially critical role of neurovascular deficits in AD pathology, developing compounds which can inhibit or lessen the A&#946;-fibrinogen interaction has promising therapeutic value. In vitro methods by which fibrin clot formation can be easily and systematically assessed are potentially useful tools for developing therapeutic compounds. Presented here is an optimized protocol for in vitro generation of the fibrin clot, as well as analysis of the effect of A&#946; and A&#946;-fibrinogen interaction inhibitors. The clot turbidity assay is rapid, highly reproducible and can be used to test multiple conditions simultaneously, allowing for the screening of large numbers of A&#946;-fibrinogen inhibitors. Hit compounds from this screening can be further evaluated for their ability to ameliorate A&#946;-induced structural abnormalities of the fibrin clot architecture using SEM. The effectiveness of these optimized protocols is demonstrated here using TDI-2760, a recently identified A&#946;-fibrinogen interaction inhibitor.

Presented here are two methods that can be used individually or in combination to analyze the effect of beta-amyloid on fibrin clot structure. Included is a protocol for creating an in vitro fibrin clot, followed by clot turbidity and scanning electron microscopy methods.

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and ...

A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status

Many neurodegenerative diseases are&#160;still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be important in the development of future novel therapies. Often&#160;potential new biomarkers do not make it&#160;into the clinic&#160;due to limitations&#160;in their development and&#160;high costs.&#160;However,&#160;targeted proteomics using Multiple Reaction Monitoring Liquid Chromatography-tandem/Mass Spectrometry (MRM LC-MS/MS), specifically using triple quadrupole mass spectrometers, is one method that can be used to rapidly evaluate and validate biomarkers&#160;for clinical translation into diagnostic laboratories. Traditionally, this platform has been used extensively for measurement of small molecules&#160;in clinical laboratories, but it is the potential to analyze proteins, that makes it an attractive alternative to ELISA (Enzyme-Linked Immunosorbent Assay)-based methods. We describe here how targeted proteomics can be used to measure multiplexed markers of dementia, including the detection and quantitation of the known risk factor apolipoprotein E isoform 4 (ApoE4).
In order to make the assay suitable for translation, it is designed to be rapid, simple, highly specific and cost effective. To achieve this, every step in the development of the assay must be optimized for the individual proteins and tissues they are analyzed in. This method describes a typical workflow including various tips and tricks to developing a targeted proteomics MRM LC-MS/MS for translation.
The method development is optimized using custom synthesized versions of tryptic quantotypic peptides, which calibrate&#160;the MS for detection and then spiked into CSF to determine correct identification of the endogenous peptide in the chromatographic separation prior to analysis in the MS. To achieve absolute quantitation, stable isotope-labeled internal standard versions of the peptides with short amino acid sequence tags and containing a trypsin cleavage site, are included in the assay.

We describe a high-throughput, multiplex, and targeted proteomic cerebrospinal fluid (CSF) assay developed with potential for clinical translation. The test can quantitate potential markers and risk factors for neurodegeneration, such as the apolipoprotein E variants (E2, E3 and E4), and measure their allelic expression.

Many neurodegenerative diseases are&#160;still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be ...

Sample Preparation for Endopeptidomic Analysis in Human Cerebrospinal Fluid

This protocol describes a method developed to identify endogenous peptides in human cerebrospinal fluid (CSF). For this purpose, a previously developed method based on molecular weight cut-off (MWCO) filtration and mass spectrometric analysis was combined with an offline high-pH reverse phase HPLC pre-fractionation step.
Secretion into CSF is the main pathway for removal of molecules shed by cells of the central nervous system. Thus, many processes in the central nervous system are reflected in the CSF, rendering it a valuable diagnostic fluid. CSF has a complex composition, containing proteins that span a concentration range of 8 - 9 orders of magnitude. Besides proteins, previous studies have also demonstrated the presence of a large number of endogenous peptides. While less extensively studied than proteins, these may also hold potential interest as biomarkers.
Endogenous peptides were separated from the CSF protein content through MWCO filtration. By removing a majority of the protein content from the sample, it is possible to increase the sample volume studied and thereby also the total amount of the endogenous peptides. The complexity of the filtrated peptide mixture was addressed by including a reverse phase (RP) HPLC pre-fractionation step at alkaline pH prior to LC-MS analysis. The fractionation was combined with a simple concatenation scheme where 60 fractions were pooled into 12, analysis time consumption could thereby be reduced while still largely avoiding co-elution.
Automated peptide identification was performed by using three different peptide/protein identification software programs and subsequently combining the results. The different programs were complementary rather than comparable with less than 15% of the identifications overlapped between the three.

A method for mass spectrometric analysis of endogenous peptides in human cerebrospinal fluid (CSF) is presented. By employing molecular weight cut-off filtration, chromatographic pre-fractionation, mass spectrometric analysis and a subsequent combination of peptide identification strategies, it was possible to expand the known CSF peptidome nearly ten-fold compared to previous studies.

This protocol describes a method developed to identify endogenous peptides in human cerebrospinal fluid (CSF). For this purpose, a previously developed ...

An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central nervous system and thus, can be used to analyze the diseased state of the brain or spinal cord without directly accessing these tissues. However, in mice it is difficult to obtain from the cisterna magna due to its closeness to blood vessels, which often contaminate samples. The area for CSF collection in mice is also difficult to dissect to and often only small samples are obtained (maximum of 5-7 &#181;L or less). This protocol describes in detail a technique that improves on current methods of collection to minimize contamination from blood and allow for the abundant collection of CSF (on average 10-15 &#181;L can be collected). This technique can be used with other dissection methods for tissue collection from mice, as it does not impact any tissues during CSF extraction. Thus, the brain and spinal cord are not affected with this technique and remain intact. With greater CSF sample collection and purity, more analyses can be used with this fluid to further aid neuroscience research and better understand diseases affecting the brain and spinal cord.

This protocol describes an improved technique for the abundant collection of cerebrospinal fluid (CSF) with no contamination from blood. With greater sample collection and purity, more analyses can be performed using CSF to further our understanding of diseases that affect the brain and spinal cord.

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central ...

Visualization of Amyloid &#946; Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into extracellular plaques of the brain. The A&#946; peptides, composed of 40 amino acids, are generated from amyloid precursor proteins (APP) by &#946;- and &#947;-secretases. A&#946; is deposited not only in cerebral parenchyma but also in leptomeningeal and cerebral vessel walls, known as cerebral amyloid angiopathy (CAA). While a variety of A&#946; peptides were identified, the detailed production and distribution of individual A&#946; peptides in pathological tissues of AD and CAA have not been fully addressed. Here, we develop a protocol of matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) on human autopsy brain tissues to obtain comprehensive protein mapping. For this purpose, human cortical specimens were obtained from the Brain Bank at the Tokyo Metropolitan Institute of Gerontology. Frozen cryosections are cut and transferred to indium-tin-oxide (ITO)-coated glass slides. Spectra are acquired using the MALDI system with a spatial resolution up to 20 &#181;m. Sinapinic acid (SA) is uniformly deposited on the slide using either an automatic or a manual sprayer. With the current technical advantages of MALDI-IMS, a typical data set of various A&#946; species within the same sections of human autopsied brains can be obtained without specific probes. Furthermore, high-resolution (20 &#181;m) imaging of an AD brain and severe CAA sample clearly shows that A&#946;1-36 to A&#946;1-41 were deposited into leptomeningeal vessels, while A&#946;1-42 and A&#946;1-43 were deposited in cerebral parenchyma as senile plaque (SP). It is feasible to adopt MALDI-IMS as a standard approach in combination with clinical, genetic, and pathological observations in understanding the pathology of AD, CAA, and other neurological diseases based on the current strategy.

Molecular imaging with matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) allows the simultaneous mapping of multiple analytes in biological samples. Here, we present a protocol for the detection and visualization of amyloid &#946; protein on brain tissues of Alzheimer&#39;s disease and cerebral amyloid angiopathy samples using MALDI-IMS.

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into ...

Quantitative Measurement of Intrathecally Synthesized Proteins in Mice

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the CSF protein composition delivers crucial information in basic neuroscience research as well as neurological diseases. One caveat is that proteins measured in CSF may derive from both intrathecal synthesis and transudation from serum, and protein analysis of CSF can only determine the sum of these two components. To discriminate between protein transudation from the blood and intrathecally produced proteins in animal models as well as in humans, CSF protein profiling measurements using conventional protein analysis tools must include the calculation of the albumin CSF/serum quotient (Qalbumin), a marker of the integrity of the blood-brain interface (BBI), and the protein index (Qprotein/Qalbumin), an estimate of intrathecal protein synthesis. This protocol illustrates the entire procedure, from CSF and blood collection to quotients and indices calculations, for the quantitative measurement of intrathecal protein synthesis and BBI impairment in mouse models of neurological disorders.

Elevated spinal fluid protein levels can either be the result of diffusion of plasma protein across an altered blood-brain barrier or intrathecal synthesis. An optimized testing protocol is presented in this article that helps to discriminate both cases and provides quantitative measurements of intrathecally synthesized proteins.

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the ...

Full- versus Sub-Regional Quantification of Amyloid-Beta Load on Mouse Brain Sections

Extracellular accumulation of amyloid-beta (A&#946;) plaques is one of the major pathological hallmarks of Alzheimer's disease (AD), and is the target of the only FDA-approved disease-modifying treatment for AD. Accordingly, the use of transgenic mouse models that overexpress the amyloid precursor protein and thereby accumulate cerebral A&#946; plaques are widely used to model human AD in mice. Therefore, immunoassays, including enzyme-linked immunosorbent assay (ELISA) and immunostaining, commonly measure the A&#946; load in brain tissues derived from AD transgenic mice. Though the methods for A&#946; detection and quantification have been well established and documented, the impact of the size of the region of interest selected in the brain tissue on A&#946; load measurements following immunostaining has not been reported. Therefore, the current protocol aimed to compare the A&#946; load measurements across the full- and sub-regions of interest using an image analysis software. The steps involved in brain tissue preparation, free-floating brain section immunostaining, imaging, and quantification of A&#946; load in full- versus sub-regions of interest are described using brain sections derived from 13-month-old APP/PS1 double transgenic male mice. The current protocol and the results provide valuable information about the impact of the size of the region of interest on A&#946;-positive area quantification, and show a strong correlation between the A&#946;-positive area obtained using the full- and sub-regions of interest analyses for brain sections derived from 13-month-old male APP/PS1 mice that show widespread A&#946; deposition.

The present protocol describes and compares the procedure to perform a full-region or sub-region of interest analysis of sagittal mouse brain sections to quantify amyloid-beta load in the APP/PS1 transgenic mouse model of Alzheimer's disease.

Extracellular accumulation of amyloid-beta (A&#946;) plaques is one of the major pathological hallmarks of Alzheimer's disease (AD), and is the target of ...

Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease (AD), amyloid-beta peptides form protofibrils in the extracellular space, which act as seeds that gradually grow and mature into large amyloid plaques. Despite this basic understanding, current knowledge of the amyloid fibril structure, composition, and deposition patterns in the brain is limited. One major barrier has been the inability to isolate highly purified amyloid fibrils from brain extracts. Affinity purification and laser capture microdissection-based approaches have been previously used to isolate amyloids but are limited by the small quantity of material that can be recovered. This novel, robust protocol describes the biochemical purification of amyloid plaque cores using sodium dodecyl sulfate (SDS) solubilization with sucrose density gradient ultracentrifugation and ultrasonication and yields highly pure fibrils from AD patients and AD model brain tissues. Mass spectrometry (MS)-based bottom-up proteomic analysis of the purified material represents a robust strategy to identify nearly all the primary protein components of amyloid fibrils. Previous proteomic studies of proteins in the amyloid coronae have revealed an unexpectedly large and functionally diverse collection of proteins. Notably, after refining the purification strategy, the number of co-purifying proteins was reduced by more than 10-fold, indicating the high purity of the isolated SDS insoluble material. Negative staining and immuno-gold electron microscopy allowed confirmation of the purity of these preparations. Further studies are required to understand the spatial and biological attributes that contribute to the deposition of these proteins into amyloid inclusions. Taken together, this analytical strategy is well-positioned to increase the understanding of amyloid biology.

This biochemical purification method with mass spectrometry-based proteomic analysis facilitates the robust characterization of amyloid fibril cores, which may accelerate the identification of targets for preventing Alzheimer's disease.

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease ...

Absolute Quantification of Aβ_1-42 in CSF Using a Mass Spectrometric Reference Measurement Procedure

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SDS-PAGE/Immunoblot Detection of Aβ Multimers in Human Cortical Tissue Homogenates using Antigen-Epitope Retrieval