MSACL 2023 Abstract

INTRODUCTION: Apolipoprotein E (ApoE) is a glycoprotein that plays a key role in transporting cholesterol and other lipids in the brain, redistributing them to cells, and facilitating cellular uptake. APOE gene exists in three different alleles in populations: ε2 (Cys112, Cys158), ε3 (Cys112, Arg158), and ε4 (Arg112, Arg158), producing six possible genotypes: three homozygous (ε2/ε2, ε3/ε3, and ε4/ε4) and three heterozygous (ε2/ε3, ε2/ε4, and ε4/ε4). The ε4 allele is the major genetic risk factor of Alzheimer’s disease (AD), while ε2 protects against the disease. The mechanisms by which the ApoE genotype impacts AD progression and development still need to be clarified. An LC-MS-based proteomic method for the determination of the genotypes by analyzing ApoE isoform-specific peptides was proposed recently (PMID:24392642). Here, we present a simplified version of the method without using isotopically labeled internal standard (SIS) peptides.

OBJECTIVES: Development of simplified multiple reaction monitoring (MRM) LC-MS assays to identify ApoE genotypes from plasma samples without using SIS-peptides.

METHODS: We used 172 samples from an AD‐related cohort of plasma samples that had been previously genotyped for ApoE using TaqMan allelic discrimination assay. Plasma proteins were reduced, alkylated, and digested with trypsin, and the digests were analyzed by UPLC-MRM-MS. MRM-MS was used to simultaneously monitor the four isoform-specific tryptic peptides (CLAVYQAGAR -- apoE2; LGADMEDVCGR -- apoE2 and 3; LAVYQAGAR -- apoE3 and 4; LGADMEDVR -- apoE4) plus one tryptic peptide that was common to all isoforms (LGPLVEQGR -- total apoE), which was used as the internal standard. The analysis was performed using the Shimadzu Nexera XR UHPLC system coupled to the Sciex QTrap 6500+ mass spectrometer operating in the positive-ion mode. Chromatographic separation was performed using a Zorbax Eclipse Plus C18 column (2.1 × 150 mm, 1.8 μm) with a 15-minute total run time. A 96-well plate was used for sample preparation and analysis in a high-throughput format. Isoform-specific peptides were detected using the chromatographic peak-area ratios of the corresponding peptides to the total ApoE internal standard peptide. The determined ApoE genotype for each sample was reported using an Excel macro written in-house.
RESULTS: An UPLC-MRM-MS assay for the determination of the ApoE genotype without using SIS peptides was established. Instead of using the standard SIS-based approach, we monitored the signal ratios of isoform-specific peptides to that of a peptide common to all isoforms (the total ApoE peptide), which was used in this case as an analog of the internal standard. The ApoE genotype was assigned based on the presence or absence of the isoform-specific peptides. 172 patient plasma samples with previously determined genotypes by the genetic testing method were analyzed. Genotypes were successfully determined for all samples. In our analysis, six samples demonstrated discrepancies with the genetic test results. These discrepancies will be further investigated on the matter of the potential presence of additional mutations in the samples' DNA sequences. The assay can add valuable ApoE genotype determination while performing conventional targeted proteomics analyses. Adding isoform-specific peptide transitions without the need for synthesizing and optimizing corresponding SIS-peptide internal standards allows ApoE genotype information to be obtained during routine targeted proteomics analyses of plasma samples using standard kits.

CONCLUSION: A simplified UPLC-MRM-MS assay for the determination of the ApoE genotypes in patient plasma samples, without the use of stable isotope-labeled internal standard peptides, was developed. The assay can be used for ApoE genotype determination along with standard targeted proteomics and metabolomics analyses. It can be a valuable tool in clinical research on AD and can be used to add valuable ApoE genotype information to large-cohort proteomics studies.