2 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
A Targeted, Differential Top-Down Proteomic Methodology for Comparison of ApoA‑I Proteoforms in Individuals with High and Low HDL Efflux Capacity
Top-down proteomics (TDP) allows
precise determination/characterization of the different proteoforms
derived from the expression of a single gene. In this study, we targeted
apolipoprotein A-I (ApoA-I), a mediator of high-density-lipoprotein
cholesterol efflux (HDL-E), which is inversely associated with coronary
heart disease risk. Absolute ApoA-I concentration and allelic variation
only partially explain interindividual HDL-E variation. Therefore,
we hypothesize that differences in HDL-E are associated with the abundances
of different ApoA-I proteoforms. Here, we present a targeted TDP methodology
to characterize ApoA-I proteoforms in serum samples and compare their
abundances between individuals. We characterized 18 ApoA-I proteoforms
using selected-ion monitoring coupled to electron-transfer dissociation
mass spectrometry. We then compared the abundances of these proteoforms
between two groups of four participants, representing the individuals
with highest and lowest HDL-E values within the Chicago Healthy Aging
Study (<i>n</i> = 420). Six proteoforms showed significantly
(<i>p</i> < 0.0005) higher intensity in high HDL-E individuals:
canonical ApoA-I [fold difference (fd) = 1.17], carboxymethylated
ApoA-I (fd = 1.24) and, with highest difference, four fatty acylated
forms: palmitoylated (fd = 2.16), oleoylated (fd = 2.08), arachidonoylated
(fd = 2.31) and one bearing two modifications: palmitoylation and
truncation (fd = 2.13). These results demonstrate translational potential
for targeted TDP in revealing, with high sensitivity, associations
between interindividual proteoform variation and physiological differences
underlying disease risk