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.
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.
Alzheimers sygdom (AD) er den mest almindelige form for demens og påvirker omkring 35 millioner mennesker verden 1. De neuropatologiske kendetegnende for sygdommen udbredt opfattelse at ligge i centrum for AD patogenese er intracellulære neurofibrillære tråde af hyperphosphoryleret tau protein 2 og ekstracellulære plaques består af aggregeret amyloid-beta (Ap) peptider 3. I tråd med dette, har vurderingen af plak patologi in vivo ved biomarkører for nylig blevet medtaget i de diagnostiske kriterier for AD 4 forskning. For CSF målinger af Ap 1-42, adskillige immunoassays er tilgængelige og anvendes i mange kliniske laboratorier 5. Koncentrationen af Ap 1-42 i CSF er ca. 50% lavere i AD-patienter end i kognitivt normale ældre, hvilket afspejler aflejringen af peptidet i plaques i BRain 6, 7.
Disse biomarkører er hovedsageligt analyseret ved anvendelse immunoassays (dvs. antistofbaserede teknikker), men disse assays kan være påvirket af matrixeffekter 8. Brugen af immunoassays på forskellige teknologiske platforme og manglen på analysen standardisering 9, 10 gør indførelsen af globale cut-off koncentrationer vanskelige 11, 12. En analytisk valideret RMP ville tillade en ensartet kalibrering af forskellige assay platforme, ideelt resulterer i bedre sammenlignelighed på tværs af analytiske platforme og i bedre kontrol over faktorer, der bidrager til den samlede måling variabilitet.
Den absolutte kvantificering af Ap 1-42 ved hjælp af den udviklede LC-MS / MS-metode overvinder mange af de problemer der er forbundet med antistof-baserede techniques. Metoden, der er opført som en RMP af det blandede udvalg for Sporbarhed i Laboratory Medicine (JCTLM database identifikationsnummer C11RMP9), vil blive anvendt til at bestemme den absolutte koncentration af Ap 1-42 i et certificeret referencemateriale (CRM) at harmonisere CSF Ap 1 -42 målinger på tværs af teknikker og analytiske platforme. Den beskrevne arbejdsgang skal være relevant for udviklingen af kandidat referencemetoder for peptider og proteiner inden for andre områder af medicin.
For den beskrevne fremgangsmåde, i stedet for at bruge en surrogat matrix anvendte vi surrogat analyt tilgang 13, 14, 15, 16, som muliggør kalibrering i human CSF. Surrogat analyt fremgangsmåde involverer to forskellige isotopmærkede standarder. On (15 NAβ 142) anvendes til at generere kalibreringskurven i human CSF, mens en anden (13 CAβ 142) …
The authors have nothing to disclose.
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.
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 |