Department of Chemistry, Department of Molecular Biosciences, Proteomics Center of Excellence, Northwestern University, Evanston, Illinois 60208, United States.
Royal Victoria Hospital-McGill University Health Centre, Montreal, Quebec H3A 1W9, Canada.
J Proteome Res. 2023 May 5;22(5):1455-1465. doi: 10.1021/acs.jproteome.2c00804. Epub 2023 Apr 13.
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 = 0.94, = 4 × 10) 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.
高密度脂蛋白（HDL）上含量最丰富的蛋白质是载脂蛋白 A-I（APOA1）和 A-II（APOA2），它们分别有 15 种和 9 种蛋白形式（化学结构变体），是决定 HDL 功能的因素。这些蛋白形式在人血清中的相对丰度与 HDL 胆固醇流出能力和胆固醇含量有关。然而，蛋白形式浓度与 HDL 大小之间的关系尚不清楚。我们采用了一种新的天然凝胶电泳技术，即清晰天然凝胶洗脱液段捕获电泳（CN-GELFrEE）与完整蛋白质的质谱联用，来研究这种关联。将混合血清用 8cm 和 25cm 长的丙烯酰胺凝胶进行分级。Western blot 法测定分子直径，完整质量分析法测定每个级分的蛋白形式谱。8cm 和 25cm 实验分别生成了 19 种和 36 种不同大小的 HDL 级分。蛋白形式的分布随大小而变化。酰化的 APOA1 蛋白形式与较大的 HDL 大小相关（Pearson's = 0.94， = 4 × 10），并且在大于 9.6nm 的颗粒中比在总血清中大约多 4 倍；与 HDL 结合的 APOA1 无酰基化，并且包含前肽 proAPOA1。APOA2 蛋白形式的丰度在 HDL 大小上相似。我们的结果确立了 CN-GELFrEE 作为一种有效的脂质颗粒分离技术，并表明 APOA1 的酰化蛋白形式与较大的 HDL 颗粒相关。
