1 research outputs found
Preparative Electrophoresis for HDL Particle Size Separation and Intact-Mass Apolipoprotein Proteoform Analysis
The
most abundant proteins on high-density lipoproteins
(HDLs),
apolipoproteins A-I (APOA1) and A-II (APOA2), are determinants of
HDL function with 15 and 9 proteoforms (chemical-structure variants),
respectively. The relative abundance of these proteoforms in human
serum is associated with HDL cholesterol efflux capacity, and cholesterol
content. However, the association between proteoform concentrations
and HDL size is unknown. We employed a novel native-gel electrophoresis
technique, clear native gel-eluted liquid fraction entrapment electrophoresis
(CN-GELFrEE) paired with mass spectrometry of intact proteins to investigate
this association. Pooled serum was fractionated using acrylamide gels
of lengths 8 and 25 cm. Western blotting determined molecular diameter
and intact-mass spectrometry determined proteoform profiles of each
fraction. The 8- and 25 cm experiments generated 19 and 36 differently
sized HDL fractions, respectively. The proteoform distribution varied
across size. Fatty-acylated APOA1 proteoforms were associated with
larger HDL sizes (Pearson’s R = 0.94, p = 4 × 10–7) and were approximately
four times more abundant in particles larger than 9.6 nm than in total
serum; HDL-unbound APOA1 was acylation-free and contained the pro-peptide
proAPOA1. APOA2 proteoform abundance was similar across HDL sizes.
Our results establish CN-GELFrEE as an effective lipid–particle
separation technique and suggest that acylated proteoforms of APOA1
are associated with larger HDL particles