La cuantificación absoluta de Aß<sub> 1-42</sub> En el LCR utilizando un procedimiento de medición de referencia de espectrometría de masas
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
La enfermedad de Alzheimer (EA) es la forma más común de demencia y afecta a unos 35 millones de personas en todo el mundo 1. Los signos neuropatológicos característicos de la enfermedad se cree ampliamente que se encuentran en el núcleo de patogénesis de la EA son los ovillos neurofibrilares intracelulares de proteína Tau hiperfosforilada y 2 placas extracelulares que consisten en agregados de beta-amiloide (Aß) péptidos 3. En línea con esto, la evaluación de la patología de la placa in vivo mediante biomarcadores recientemente se ha incluido en los criterios diagnósticos de investigación para 4 dC. Para las mediciones de LCR de 1-42, varios inmunoensayos están disponibles y se utilizan en muchos laboratorios clínicos 5. La concentración de 1-42 en CSF es de aproximadamente 50% menor en los pacientes de AD que en los ancianos cognitivamente normales, lo que refleja la deposición del péptido en las placas en el brain 6, 7.
Estos biomarcadores se analizan utilizando principalmente inmunoensayos (por ejemplo, técnicas basadas en anticuerpos), pero estos ensayos pueden estar influenciadas por los efectos de matriz 8. El uso de inmunoensayos en diferentes plataformas tecnológicas y la falta de estandarización de ensayo 9, 10 hace que la introducción de las concentraciones globales de corte difíciles 11, 12. Un PGR analítico validado permitiría la calibración uniforme de diferentes plataformas de ensayo, lo ideal sería que resulta en una mejor comparabilidad entre las plataformas de análisis y un mejor control de los factores que contribuyen a la variabilidad general de medición.
La cuantificación absoluta de 1-42 usando el método LC-MS / MS desarrollado supera muchos de los problemas asociados con la tecn basada en anticuerposiques. El método, que aparece como un PGR por el Comité Conjunto para la Trazabilidad en Medicina de Laboratorio (JCTLM base de datos de identificación del número C11RMP9), se utiliza para determinar la concentración absoluta de 1-42 en un material de referencia certificado (CRM) para armonizar CSF Aß 1 mediciones de -42 a través de técnicas y plataformas analíticas. El flujo de trabajo descrito debe ser de relevancia para el desarrollo de métodos de referencia candidato para péptidos y proteínas dentro de otras áreas de la medicina.
Protocol
Representative Results
Discussion
Para el método descrito, en vez de usar una matriz sustituta, se utilizó el enfoque sustituto analito 13, 14, 15, 16, que permite la calibración en CSF humano. El enfoque analito sustituto implica dos estándares marcados isotópicamente diferentes. Un (15 NAβ 142) se utiliza para generar la curva de calibración en CSF humano, mientras que otro (13 CAβ <s…
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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