Abstract Body: High density lipoproteins (HDL) are heterogeneous and proteomically diverse, making them difficult to study structurally. Most previous work has been limited to reconstituted HDL which can be better controlled compositionally. However, given the role of the major HDL protein apolipoprotein A-I (APOA1) in facilitating the docking of other proteins, it is important to study its structure in plasma HDL. We have immunopurified two HDL subspecies; LPA1 (contains APOA1 but no apolipoprotein A2 (APOA2)) and LPA1A2 (contains both APOA1 and APOA2). For each subspecies we isolated both large (~11 nm) and small (~9 nm) size fractions by size exclusion chromatography. These were analyzed for lipid and protein composition and subjected to isotope-assisted formaldehyde cross-linking by mass spectrometry (XL-MS). There were 87 distinct cross-links found across all four samples with the large LPA1 fraction (LPA1-L) having the most at 62 crosslinks found in every replicate. LPA1 small (LPA1-S), LPA1A2 large, and LPA1A2 small showed ~30 crosslinks each. Interestingly, only 14 crosslinks (8 of those being intermolecular) were common to all four samples, indicating significant conformational differences exist between the particles. To provide a context for the XL-MS data, we also performed cryoEM studies on the LPA1 particles. An inherent hurdle for using cryoEM on HDL is the lack of fiducial markers for image alignment. We developed a novel anti-APOA1 antibody fragment, Fab16, which binds helix 5 of APOA1 as demonstrated by hydrogen deuterium exchange mass spectrometry. Up to 4 Fab16 molecules bound the LPA1-L particles while only 2 bind LPA1-S particles. Interestingly, the pattern of Fab 16 association on the LPA1-L particles was not consistent with trefoil or any other double belt-related models for APOA1 structure proposed previously. Thus far, the resolution of the cryoEM maps (~10 Å) is too low to derive a good idea of how APOA1 is organized on plasma HDL. Current work is focusing on using additional Fab fragments as well as Fab-orienting nanobodies to combine with the XL-MS data to derive a comprehensive understanding of APOA1 structure on plasma HDL.