Abstract 322: HDL Dynamics in Circulation: Complexity of Protein Distribution and Metabolism Across HDL Size

Introduction: The composition of specific apolipoproteins may determine HDL functions. We present novel mass spectrometry (MS)-based methods that capture the absolute quantities and kinetics of 7 apolipoproteins in 5 HDL size fractions. Methods and Results: Three participants were recruited, infused with a bolus of D3-Leu tracer, and blood was collected for 70 hrs. ApoA-I-containing HDL was prepared by immunoaffinity purification, separated into 5 size fractions, preβ, α3, α2, α1, and α0 by ND-PAGE, and in-gel trypsinized for MS. We monitored 7 proteins that likely affect HDL metabolism - apoA-I, apoA-II, apoA-IV, apoC-III, apoD, apoE and apoM. Each protein pool size had a distinct distribution across the HDL sizes. ApoE and apoM were enriched in larger HDL, whereas apoC-III and apoA-IV were enriched in smaller HDL sizes. We evaluated the tracer enrichment curves of these 7 proteins in the 5 fractions using high resolution parallel reaction monitoring performed on a quadrupole Orbitrap (Thermo). The enrichment curves for each protein varied from each other by slope and time of peak enrichment (Fig. 1a). In contrast, the enrichment curves across HDL sizes for a single protein showed smaller, but likely meaningful, differences in either slope or time of peak enrichment. Irrespective of the HDL size on which it resides, apoE had the fastest FCR, followed by apoA-IV, and apoC-III. ApoA-I/A-II, apoM, and apoD had slower but similar FCRs (Fig. 1b). Conclusions: This study showed distinct distribution and kinetic behaviors of 7 HDL proteins across 5 HDL size fractions that were conserved in the three participants. These findings may help elucidate the functional role of these proteins and the HDL particles that contain them.