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

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-beta (Abeta) to matrix proteins as well as self-aggregation. Here, we report on a matrix effectresistant method for the measurement of the AD-associated 42 amino acid species of Abeta (Abeta42), together with Abeta40 and Abeta38 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 Abeta peptides as internal standards. The diagnostic performance of the method was evaluated on two independent clinical materials with research volunteers who were cognitively normal andADpatients with mild to moderate dementia. Analytical characteristics of the method include a lower limit of quantification of 62.5 pg/mL for Abeta42 and coefficients of variations below 10%. In a pilot study on AD patients and controls, we verified disease-association with decreased levels of Abeta42 similar to that obtained by ELISA and even better separation was obtained using the Abeta42/Abeta40 ratio. The developed assay is sensitive and is not influenced by matrix effects, enabling absolute quantification of Abeta42, Abeta40, and Abeta38 in CSF, while it retains the ability to distinguish AD patients from controls. We suggest this SRM-based method for Abeta peptide quantification in human CSF valuable for clinical research and trials.JRC.D.2-Standards for Innovation and sustainable Developmen

Structural modelling of the DNAJB6 oligomeric chaperone shows a peptide-binding cleft lined with conserved S/T-residues at the dimer interface

The remarkably efficient suppression of amyloid fibril formation by the DNAJB6 chaperone is dependent on a set of conserved S/T-residues and an oligomeric structure, features unusual among DNAJ chaperones. We explored the structure of DNAJB6 using a combination of structural methods. Lysine-specific crosslinking mass spectrometry provided distance constraints to select a homology model of the DNAJB6 monomer, which was subsequently used in crosslink-assisted docking to generate a dimer model. A peptide-binding cleft lined with S/T-residues is formed at the monomer-monomer interface. Mixed isotope crosslinking showed that the oligomers are dynamic entities that exchange subunits. The purified protein is well folded, soluble and composed of oligomers with a varying number of subunits according to small-angle X-ray scattering (SAXS). Elongated particles (160 × 120 Å) were detected by electron microscopy and single particle reconstruction resulted in a density map of 20 Å resolution into which the DNAJB6 dimers fit. The structure of the oligomer and the S/T-rich region is of great importance for the understanding of the function of DNAJB6 and how it can bind aggregation-prone peptides and prevent amyloid diseases