An automated clinical mass spectrometric method for identification and quantification of variant and wild-type amyloid-β 1-40 and 1-42 peptides in CSF

Abstract

Introduction: We developed an automated liquid chromatography-tandem mass spectrometry high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for multiplex quantification of wild-type (wt) amyloid β (Aβ) peptides 1-40 (Aβ40) and 1-42 (Aβ42) and detection of variant Aβ peptides in cerebrospinal fluid.

Methods: The multiplex Aβ HPLC-MS/MS assay was validated in a clinically accredited laboratory following regulatory guidelines, with Aβ42 calibration assigned to the ERM/IFCC certified reference material; sequence variants were additionally multiplexed into the method.

Results: Sample preparation was fully automated on a liquid handler. The assay quantified wt-Aβ42 and wt-Aβ40 and detected sequence variants, when present, within the Aβ42 sequence.

Discussion: Extension of the HPLC-MS/MS approach for quantification of wt-Aβ42 and wt-Aβ40 to include known sequence variants increases analytical accuracy of the mass spectrometric approach and enables identification of cases of autosomal dominant Alzheimer's disease. Development of an automated workflow and selection of appropriate instrumentation enabled deployment of this method in routine clinical testing.

Keywords: Alzheimer's disease; amyloid‐β peptides; automation; autosomal dominant Alzheimer's disease; cerebrospinal fluid; certified reference material; mass spectrometry; variant.

© 2020 The Authors. Alzheimer's & Dementia: Diagnosis, Assessment & Disease Monitoring published by Wiley Periodicals, Inc. on behalf of Alzheimer's Association.

Figures

FIGURE 1

The transmembrane amyloid precursor protein (APP) is cleaved by β‐ and γ‐secretase to form the 1‐40 and 1‐42 residue isoforms of amyloid beta (Aβ). In addition to proteolytic isoforms, there are known amino acid variants, both pathogenic and benign, within the Aβ42 sequence

FIGURE 2

Manual and automated sample preparation prior to high performance liquid chromatography tandem mass spectrometry analysis demonstrated comparable performance for both (A and B) amyloid β 1‐42 (Aβ42) and (C and D) amyloid β 1‐40 (Aβ40). A and C, Shaded region represents the 95% confidence interval (CI) of the slope of the linear regression. B and D, Black dashed lines represent the 95% CI of the mean difference (solid line)

FIGURE 3

Amyloid β peptide 1‐42 (Aβ42) certified reference material (CRM)‐assigned calibrators versus calibrators pre‐CRM assignment

FIGURE 4

The multiplex amyloid beta (Aβ) high performance liquid chromatography tandem mass spectrometry assay enabled identification of cases (A) homozygous for wild‐type amyloid‐precursor protein (wt‐APP) alleles and (B‐J) heterozygous for an Alzheimer's disease autosomal dominant APP variant, as demonstrated by the multiple reaction monitoring chromatograms. A, In the homozygous sample, a single wt‐Aβ40 peak (blue) is observed. B‐J, In the heterozygous samples, both the wt‐Aβ40 (blue) and var‐Aβ40 (magenta) peaks are observed, the latter corresponding to the following pathogenic variants: (B) Arctic (E22G), (C) Dutch (E22Q), (D) English (H6R), (E) Flemish (A21G), (F) Iowa (D23N), (G) Italian (E22K), (H) Osaka (E22∆), (I) Piedmont (L34V), and (J) Tottori (D7N)

FIGURE 5

ROC curves using the multiplex amyloid β (Aβ) high performance liquid chromatography tandem mass spectrometry assay for: tau/Aβ42 (dark blue), Aβ42 (green), Aβ42/Aβ40 (light blue), and Aβ40 (orange) with total tau measured by enzyme‐linked immunosorbent assay