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.
Support or Funding Information
American Heart Association: #SDG 27250022, National Institute of Health: #K23 HL133601-01 and #R01 HL081141, and National Institute of General Medical Sciences: #P41 GM108569
