Simple and Fast Assay for Apolipoprotein E Phenotyping and Glycotyping: Discovering Isoform-Specific Glycosylation in Plasma and Cerebrospinal Fluid

Background: The mechanisms of how APOE ɛ4 allele ( APOE4) increases the risk of Alzheimer’s disease (AD) pathology have not been fully elucidated. In cerebrospinal fluid (CSF), apoE is heavily glycosylated. Objective: To determine the impact of APOE genotype on the relative abundance of apoE protein isoforms and their specific glycosylation patterns in CSF and plasma via a newly developed mass spectrometric immunoassay (MSIA) assay. Methods: Total glycosylation and isoform-specific glycosylation were analyzed in plasma and CSF from a group of non-demented older individuals ( n = 22), consisting of homozygous ɛ3 and ɛ4 or heterozygous ɛ3/ ɛ4, ɛ2/ ɛ3, or ɛ2/ ɛ4 carriers. The glycan structures were further confirmed after treatment with sialidase. Results: In heterozygous individuals, the apoE3/E2, E4/E2, and E4/E3 isoform ratios were all significantly lower in plasma compared to CSF. For all individuals, a single O-linked glycan was observed in plasma, while two glycans (of the same type) per apoE were observed in CSF. The ratio of glycosylated to total apoE was greater in CSF compared to plasma for all apoE isoforms. In plasma and CSF, a trend of decreasing glycosylation was observed from apoE2 > apoE3 > apoE4. The difference in the percentage of secondary glycosylation in CSF was significantly greater in apoE4 compared to the other isoforms. Conclusion: The new MSIA apoE assay robustly distinguishes among apoE isoforms and glycoforms in plasma and CSF. ApoE4 is the predominant isoform and least glycosylated in CSF. Assessing apoE isoform-specific glycosylation by MSIA may help clarify brain apoE metabolism and AD risk.