Aβ의 절대 정량<sub> 1-42</sub> CSF의 질량 분석 참조 측정 절차를 사용하여
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
알츠하이머 병 (AD)은 치매의 가장 흔한 형태이며, 전 세계적으로 한 약 35 만명에 영향을 미친다. 널리 AD의 발병 기전의 핵심 거짓말을 믿고 병의 신경 병리학 적 특징은 집계 아밀로이드 – 베타 (Aβ) 펩타이드 3 구성된 hyperphosphorylated 타우 단백질이 세포 내 신경 섬유 엉킴 및 세포 외 플라크 있습니다. 이에 맞춰, 바이오 마커에 의해 생체 내에서 플라크 병리의 평가는 최근에 AD 4에 대한 연구 진단 기준에 포함되었습니다. Aβ 1-42의 CSF 측정을 위해 여러 가지 면역 사용할 수 있으며 많은 임상 실험실 (5)에 사용됩니다. CSF에서 Aβ 1-42의 농도는 BR에 플라크의 펩티드의 침착을 반영인지 정상 노인보다 AD 환자의 약 50 % 낮다아인 (6, 7).
이러한 생체는 주로 면역 (예를 들어, 항체 – 기반 기술)을 사용하여 분석하지만, 이러한 분석은 매트릭스의 효과 (8)에 의해 영향을받을 수있다. 다른 기술 플랫폼의 면역 분석 및 표준화 9의 부재의 사용은 10 11 12 어려운 글로벌 컷오프 농도로 도입한다. 해석 적 검증 RMP는 이상적으로 분석 플랫폼에서 더 나은 비교와 전체 측정 변동에 기여하는 요인의 더 나은 제어의 결과로, 다른 분석 플랫폼의 균일 보정을 허용합니다.
현상 된 LC-MS / MS 방법을 사용하여 Aβ 1-42의 절대 정량 항체 기반 TECHN와 관련된 많은 문제들을 극복iques. 실험 의약 추적을위한 합동위원회 (JCTLM 데이터베이스 식별 번호 C11RMP9)에 의해 RMP로 나열있어서, CSF Aβ 한 조화하는 인증 표준 물질 (CRM)에서 Aβ 1-42의 절대 농도를 결정하는 데 사용될 기술 및 분석 플랫폼에서 -42 측정. 설명 된 워크 플로는 의학의 다른 영역에서 펩타이드 및 단백질 후보 참조 방법의 개발을위한 관련이 있어야한다.
Protocol
Representative Results
Discussion
상술 한 방법 대신 대용 행렬을 이용하여, 우리는 인간 CSF의 교정을 가능하게 대리 분석 방법 13, 14, 15, 16을 사용 하였다. 대리 분석 방법은 두 가지 동위 원소 표지 된 표준을 포함한다. 또 (13 CAβ 142) 내부 표준으로 사용되는 동안 온 (15 NAβ 142), 인간 CSF의 보정 곡선을 …
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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