Aβ的绝对定量<sub> 1-42</sub>在CSF使用质谱参考测量程序
Summary
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.
Abstract
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 (Aβ) 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 Aβ 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 Aβ1-42 between 150-4,000 pg/mL.
Introduction
阿尔茨海默氏病（AD）是老年痴呆症的最常见的形式，影响了全球100约35万人。疾病广泛认为在躺在床上的AD发病的芯的神经病学特征是tau蛋白的蛋白2的细胞内神经原纤维缠结和细胞外斑块由聚集的淀粉样-β（Aβ）肽3。本着这一精神，体内斑块病理的生物标志物评估最近已包含在公元4研究的诊断标准。对于Aβ1-42的CSF测量，几个免疫测定可以使用，在许多临床实验室5被使用。 的Aβ1-42的CSF中的浓度是在AD患者比在认知正常老年人下大约50％，这反映了肽在斑中的BR的沉积AIN 6,7。
这些生物标志物重点分析使用免疫（ 即，基于抗体的技术），但这些试验可以由基体效应8的影响。使用不同技术平台的免疫测定和缺乏测定标准化9，10，使引入全球截止浓度难以11,12。一种分析验证RMP将允许不同的检测平台统一校准，最好导致跨平台的分析比较好和更好的控制有助于整体测量变异的因素。
使用开发的LC-MS / MS方法的Aβ1-42的绝对定量克服了与基于抗体的TECHN有关的问题iques。该方法被列为联合委员会溯源实验室医学（JCTLM数据库标识号C11RMP9），一个RMP将被用来确定一个标准物质（CRM）Aβ1-42的绝对浓度协调CSFAβ1跨越技术和分析平台-42测量。所描述的工作流程应该是相关的用于医学的其他领域内的肽和蛋白质候选参考方法的发展。
Protocol
Representative Results
Discussion
对于所描述的方法，而不是使用一个代理矩阵，我们使用了替代的分析物的方法13，14，15，16，它使校正在人CSF。代孕的方式分析涉及到两个不同的同位素标记的标准。酮（15NAβ142）被用来产生于人类CSF中的校准曲线，而另一个（13CAβ142）用作内标。然后未知的内源性…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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, Universitätsklinikum 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 & 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; Demensförbundet; and Emil and Wera Cornell’s, Aina Wallström and Mary-Ann Sjöblom’s, Gun and Bertil Stohne’s, Magnus Bergvall’s, and Gamla Tjänarinnor’s Foundations.
Materials
| Eppendorf Protein LoBind microcentrifuge tubes, 0.5 mL | Eppendorf | 022431081 | |
| Eppendorf Protein LoBind microcentrifuge tubes, 1.5 mL | Eppendorf | 022431081 | |
| Eppendorf Protein LoBind microcentrifuge tubes, 2 mL | Eppendorf | 022431102 | |
| Eppendorf Protein LoBind microcentrifuge tubes, 5 mL | Eppendorf | 0030 108.310 | |
| Eppendorf Protein LoBind Deepwell Plates 96 | Eppendorf | 951032905 | |
| Micronic 0.75 mL polypropylene tubes, V-bottom | Micronic | MPW32071BC3 | Tubes used for collecting SPE eluate, but other collection devices can be used such as 96-well plates. |
| Micronic Split TPE Capcluster for 96 ind. tubes | Micronic | MP53026 | Caps for Micronic tubes. |
| Micronic Loborack-96 white w high cover bar coded | Micronic | MPW51015BC3 | Holder for Micronic tubes. |
| Biohit Optifit tips 1.2 mL | Sartorius | 791210 | Used to reach all the way to the bottom of the LoBind deepwell plates (96-well). Other narrow tips might work as well. |
| Waters Oasis MCX 96-well µElution Plate | Waters | 186001830BA | |
| Waters Plate manifold reservoir tray | Waters | WAT058942 | |
| Waters Extraction Plate Manifold for Oasis 96-Well Plates | Waters | 186001831 | |
| Ammonium hydroxide solution, puriss. p.a., reag. ISO, reag. Ph. Eur., ~25% NH3 basis | Sigma-Aldrich | 30501-1L-D | |
| Acetonitrile, Far UV HPLC Gradient grade, 2.5 L | Fisher Scientific | A/0627/17X | |
| ortho-Phosphoric acid 85%, ACS,ISO,Reag. Ph Eur | Merck Millipore | 1005731000 | |
| Guanidine Hydrochloride, 500 g | Thermo Scientific | 24110 | |
| Thermo Scientific Q-exactive Hybrid Quadrupole Orbitrap Mass Spectrometer | Thermo Scientific | IQLAAEGAAPFALGMAZR | With bundled Dionex Ultimate 3000 LC & autosampler. |
| Dionex ProSwift RP-4H Monolith Column (1.0 x 250mm) | Dionex | 066640 | |
| Bovine Serum Albumin, lyophilized powder, suitable for (for molecular biology), Non-acetylated | Sigma-Aldrich | B6917 | |
| Beta-Amyloid (1-42), Ultra Pure, TFA | rPeptide | A-1002-2 | |
| 15N Beta-Amyloid (1-42), Uniformly labeled | rPeptide | A-1102-2 | |
| 13C Beta-Amyloid (1-42), Uniformly labeled | rPeptide | A-1106-2 | |
| Microplate shakers, TiMix 2 | Edmund Bühler | 6110 000 |
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