PROJECT SUMMARY Brain amyloid-β levels and amyloid plaque loads in Alzheimer's disease (AD) have been shown to be apolipoprotein E (apoE)-isoform dependent (E4>E3>E2), but the exact mechanisms by which the isoforms (especially E4) differentially contribute to AD pathogenesis are not completely understood. Very little is known about the role of glycosylation of apoE in AD. Current LC-MS approaches for analyzing glycoproteins are quite complex, and cannot delineate glycosylation differences of the individual E2, E3 and E4 isoforms, or the number of glycans attached per apoE molecule. We have developed a new assay for simultaneous detection of apoE isoforms (E2, E3, E4) and apoE glycoforms (O-linked GalNAc(-Sia)-Gal-Sia), based on immunoaffinity enrichment of apoE and subsequent MALDI-TOF mass spectrometry detection of intact apoE. The assay produces data-rich mass spectra that contain signals from the isoforms (E2, E3 E4) and the multiple glycosylated forms of each isoform. In preliminary data from a small cohort of plasma and CSF samples we detected decreased level of glycosylation/sialylation in the E4 isoform compared to E2 and E3 in CSF, which could potentially explain its reduced binding to and removal of amyloid-β plaques. In this Phase I research we will: 1) Optimize the new apoE isoforms/glycoforms assay, and perform extensive analytical validation, testing the assay robustness, accuracy, specificity and reproducibility; and 2) Use the new assay to confirm the initial glycosylation findings and further delineate patterns of decreased glycosylation (E2>E3>E4) using matched plasma and CSF samples from a cohort of cognitively healthy individuals at risk of AD (n~100). Our value proposition is a new, simple, and cost-effective assay for simultaneous phenotyping and glycotyping of apoE that could be utilized for evaluating the role of apoE isoform-specific glycosylation in AD pathogenesis. The glycosylation differences in the apoE isoforms (especially E4) may explain their distinct role in AD, which may lead to development of new glycosylation-based biomarkers for early detection of AD, and apoE isoform- specific targets for AD therapeutics.