Absolutt Kvantifisering av Ap<sub> 1-42</sub> I CSF Ved hjelp av en massespektrometrisk Reference målingsprosedyre
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
Alzheimers sykdom (AD) er den vanligste formen for demens, og rammer om lag 35 millioner mennesker over hele verden en. De nevropatologiske kjennetegn ved sykdommen antatt å ligge i kjernen av AD patogenesen er intracellulære neurofibrillary floker av hyperfosforylisert tau protein 2 og ekstracellulære plakk som består av aggregert amyloid-beta (Ap) peptider 3. I tråd med dette har vurderingen av plakk patologi in vivo av biomarkører nylig blitt inkludert i forsknings diagnostiske kriteriene for AD 4. For CSF målinger av Ap 1-42 rekke immunologiske analyser tilgjengelig og brukes i mange kliniske laboratorier 5. Konsentrasjonen av Ap 1-42 i CSF er omtrent 50% lavere i AD pasienter enn i de kognitivt normale eldre, reflekterer avsetning av peptidet i plakk i brain 6, 7.
Disse biomarkører er i hovedsak analysert ved hjelp av immunologiske analyser (dvs. antistoffbaserte teknikker), men disse analysene kan være påvirket av matrix effekter 8. Bruken av immunoassays på forskjellige teknologiplattformer og mangelen på analysen standardisering 9, 10 som gjør innføringen av globale cut-off-konsentrasjoner vanskelige 11, 12. En analytisk validert RMP ville tillate ensartet kalibrering av forskjellige analyse plattformer, helst noe som resulterer i bedre sammenlignbarhet på tvers analytiske plattformer og i bedre kontroll av faktorer som bidrar til den totale måle variabilitet.
Den absolutte kvantifisering av Ap 1-42 bruker den utviklede LC-MS / MS metode vinner mange av problemene forbundet med antistoff-baserte techniques. Metoden, som er oppført som en RMP av Fellesutvalget for sporbarhet i laboratoriemedisin (JCTLM database identifikasjonsnummer C11RMP9), vil bli brukt til å bestemme den absolutte konsentrasjonen av Ap 1-42 i en Certified Reference Material (CRM) for å harmonisere CSF Ap 1 -42 målinger på tvers av teknikker og analytiske plattformer. Den beskrevne arbeidsflyten bør være av relevans for utvikling av kandidaten referansemetoder for peptider og proteiner i andre områder av medisin.
Protocol
Representative Results
Discussion
For den beskrevne fremgangsmåte, i stedet for å bruke et surrogat matrise, vi brukte surrogat analytten tilnærmingen 13, 14, 15, 16, som muliggjør kalibrering i humant CSF. Surrogat analytt tilnærming innebærer to forskjellige isotopisk merkede standarder. En (15 NAβ 142) blir brukt til å generere kalibreringskurven i human CSF, mens en annen (13 CAβ <…
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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