A Selected Reaction Monitoring (SRM)-Based Method for Absolute Quantification of Aβ₃₈, Aβ₄₀, and Aβ₄₂ in Cerebrospinal Fluid of Alzheimer's Disease Patients and Healthy Controls

Abstract

Cerebrospinal fluid (CSF) biomarkers for Alzheimer's disease (AD) are increasingly used in research centers, clinical trials, and clinical settings. However, their broad-scale use is hampered by lack of standardization across analytical platforms and by interference from binding of amyloid-β (Aβ) to matrix proteins as well as self-aggregation. Here, we report on a matrix effect-resistant method for the measurement of the AD-associated 42 amino acid species of Aβ (Aβ₄₂), together with Aβ₄₀ and Aβ₃₈ in human CSF based on mass spectrometric quantification using selected reaction monitoring (SRM). Samples were prepared by solid-phase extraction and quantification was performed using stable-isotope labeled Aβ peptides as internal standards. The diagnostic performance of the method was evaluated on two independent clinical materials with research volunteers who were cognitively normal and AD patients with mild to moderate dementia. Analytical characteristics of the method include a lower limit of quantification of 62.5 pg/mL for Aβ₄₂ and coefficients of variations below 10%. In a pilot study on AD patients and controls, we verified disease-association with decreased levels of Aβ₄₂ similar to that obtained by ELISA and even better separation was obtained using the Aβ₄₂/Aβ₄₀ ratio. The developed assay is sensitive and is not influenced by matrix effects, enabling absolute quantification of Aβ₄₂, Aβ₄₀, and Aβ₃₈ in CSF, while it retains the ability to distinguish AD patients from controls. We suggest this SRM-based method for Aβ peptide quantification in human CSF valuable for clinical research and trials.