La quantification absolue de Aß<sub> 1-42</sub> Dans le LCR aide d'une référence Procédure de mesure de spectrométrie de masse
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 maladie d'Alzheimer (MA) est la forme la plus courante de démence et affecte environ 35 millions de personnes à travers le monde 1. Les caractéristiques neuropathologiques de la maladie largement soupçonné d'être au cœur de la pathogenèse de la MA sont des enchevêtrements neurofibrillaires intracellulaires de protéine tau hyperphosphorylée 2 et plaques extracellulaires composées de agrégées bêta-amyloïde (Aß) peptides 3. Conformément à cela, l'évaluation de la plaque pathologie in vivo par des biomarqueurs a récemment été inclus dans les critères diagnostiques de recherche pour AD 4. Pour les mesures de LCR de Aß 1-42, plusieurs analyses immunologiques sont disponibles et sont utilisés dans de nombreux laboratoires cliniques 5. La concentration de Aß 1-42 dans le LCR est d' environ 50% plus faible chez les patients AD que chez les sujets âgés normaux sur le plan cognitif, ce qui reflète le dépôt du peptide dans les plaques dans le brain 6, 7.
Ces biomarqueurs sont principalement analysés en utilisant des immunoessais (ie, les techniques à base d' anticorps), mais ces essais peuvent être influencés par les effets de matrice 8. L'utilisation d'immunoessais sur différentes plates – formes technologiques et le manque de dosage normalisation 9, 10 permet l'introduction de concentrations de coupure mondiales difficiles 11, 12. Un RMP analytiquement validé permettrait l'étalonnage uniforme des différentes plates-formes de dosage, idéalement résultant en une meilleure comparabilité entre les plates-formes d'analyse et un meilleur contrôle des facteurs qui contribuent à la variabilité globale de mesure.
La quantification absolue de Aß 1-42 en utilisant la méthode LC-MS / MS développé surmonte un grand nombre des problèmes associés à la techn à base d' anticorpsiques. La méthode, répertorié comme un PGR par le Comité commun pour la traçabilité en médecine de laboratoire (JCTLM base de données le numéro d' identification de C11RMP9), sera utilisé pour déterminer la concentration absolue de Aß 1-42 dans un matériau de référence certifié (CRM) pour harmoniser CSF Aß 1 mesures -42 à travers des techniques et des plates – formes analytiques. Le workflow décrit devrait être de l'intérêt pour le développement de méthodes de référence des candidats pour des peptides et des protéines dans d'autres domaines de la médecine.
Protocol
Representative Results
Discussion
Pour le procédé décrit, au lieu d'utiliser une matrice de substitution, on a utilisé la méthode de remplacement de l' analyte 13, 14, 15, 16, ce qui permet un calibrage dans le LCR humain. L'approche de l'analyte de substitution implique deux normes marqués par des isotopes différents. L' une (15 NAβ 142) est utilisé pour générer la courbe d…
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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