Adaptation of Native GELFrEE for HDL Particle Size Subtype Separation and Differential Apolipoprotein Proteoform Quantification

Abstract

High‐density lipoproteins (HDL) are central to cholesterol transport, and higher concentration of HDL‐bound cholesterol (HDL‐C) is associated with lower coronary artery disease (CAD) risk. Recent reports refute a causal link between these variables, suggesting better understanding of HDL variation is necessary. HDL particles differ in size and size subtypes differ in proteome, lipidome, and functional characteristics. Apolipoprotein A‐I (ApoA‐I), a major determinant of HDL structure and biochemistry, is made up of 15 distinct proteoforms. ApoA‐I proteoforms correlate with HDL efflux, a measure of cholesterol transport efficiency, but it is unknown if ApoA‐I proteoforms vary with HDL particle size. Herein, we adapt a native separation methodology to inquire on the relationship between HDL size subtypes, their function, and apolipoprotein proteoform profiles.Pooled serum (20μl) from 30 individuals with high, medium and low HDL‐C levels, were loaded to CN‐GELFrEE, a native electrophoretic technique, to separate HDL particles by size. Mid‐resolution and high‐resolution modes were employed. Immunoassays were done to characterize ApoA‐I content and average particle size of the electrophoretic fractions. Proteoform quantification was performed on ApoA‐I‐containing fractions by top‐down LC‐MS. SIM scans were devised for higher sensitivity in ApoA‐I proteoform detection. Custom software matched, scored and quantified proteoform‐specific spectra, and calculated association to particle size.In both resolution modes, average fraction sizes were roughly linearly correlated to fraction collection time, and size‐range overlap between fractions was small. In mid‐resolution mode 3‐4 fractions contained ApoA‐I, while high‐resolution mode yielded 36 distinct HDL size fractions. Fractions roughly spanned the range between 5 to 11nm, similar to previously reported ranges for HDL particles.Proteoform quantification revealed significant variation of proteoform profiles in different size‐ranges of HDL. Fatty‐acylated ApoA‐I, a species previously correlated to higher cholesterol efflux, had higher relative abundance (~1.5x to 2x) in the pre‐beta‐1 (5‐7.1nm) and alpha‐3 (9‐11nm) size ranges of HDL, while glycated and oxidized proteoforms were significantly more abundant (~3x to 4x) in the medium size ranges (alpha‐4, alpha‐3 and alpha‐2). Interestingly, the truncated proteoform of ApoA‐I had no significant differences in abundance between different particle sizes.Our experimental data suggest that the profile of ApoA‐I proteoforms in HDL particles is size‐dependent and thus ApoA‐I proteoforms may be important markers or mediators of HDL size regulation. Notably, the differences in PTM prevalence from medium sizes to large and pre‐beta subtypes may be markers of the pathway of HDL maturation and/or different functions of each subtype.