New plasma LC-MS/MS assays for the quantitation of beta-amyloid peptides and identification of apolipoprotein E proteoforms for Alzheimer’s disease risk assessment

Objectives Early detection of Alzheimer’s disease (AD) represents an unmet clinical need. Beta-amyloid (Aβ) plays an important role in AD pathology, and the Aβ42/40 peptide ratio is a good indicator for amyloid deposition. In addition, variants of the APOE gene are associated with variable AD risk. Here we describe the development and validation of high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays for plasma Aβ40 and Aβ42 quantitation, as well as apolipoprotein E (ApoE) phenotype determination as a surrogate for APOE genotype. Methods Aβ40 and Aβ42 were simultaneously immunoprecipitated (IP) from plasma, proteolytically digested, and quantitated by LC-MS/MS. ApoE proteoform status was qualitatively assessed by targeting tryptic peptides from the ApoE2, ApoE3, and ApoE4 proteoforms. Both assays were validated according to CLIA guidelines. Results Within-run precision was 1.8 to 4.2% (Aβ40), 1.9 to 7.2% (Aβ42), and 2.6 to 8.3% (Aβ42/40 ratio). Between-run precision was 3.5 to 5.9% (Aβ40), 3.8 to 8.0% (Aβ42), and 3.3 to 8.7% (Aβ42/40 ratio). Both Aβ40 and Aβ42 were linear from 10 to 2,500 pg/mL. Identified ApoE proteoforms had 100% concordance with APOE genotypes. Conclusion We have developed a precise, accurate, and sensitive high-throughput LC-MS/MS assay for plasma Aβ40, Aβ42, and proteoforms of ApoE.