Department of Chemistry, Faculty of Science, Ehime University, Matsuyama, Japan.
Electrophoresis. 2009 Oct;30(20):3613-21. doi: 10.1002/elps.200900191.
The process of 0-4% PEG precipitation of high-molecular-mass proteins (around and above 1x10(3) kDa) in human plasma was analyzed by non-denaturing micro 2-DE employing agarose IEF gels in the first dimension, and by MALDI-MS PMF. The PEG 0-2% precipitate could be mostly re-dissolved (fraction PS2%), but some part remained as precipitate (fraction PP2%). Non-denaturing 2-DE followed by MALDI-MS PMF showed that PS2% contained fibrinogen (Fb), fibronectin (FN), and their oligomers as the major components, and von Willebrand factor and coagulation factor XIII as the minor components. PP2% was directly subjected to MALDI-MS PMF and FN and Fb were assigned, suggesting it was formed by co-precipitation of the two proteins. The PEG 2-4% precipitate could be totally dissolved, but after freezing and thawing the solution, a small amount of precipitate appeared. MALDI-MS PMF analysis of the precipitate (PP4%) suggested that its major constituent is complement C1. The soluble fraction of the PEG 2-4% precipitate (PS4%), analyzed by non-denaturing 2-DE and MALDI-MS PMF, contained C4b-binding protein and its complex with complement C4, low-density lipoproteins, IgM, and complement C1 subcomponent q, together with Fb, FN, and their oligomers. When the PS4% fraction was analyzed by a Type-II 2-DE technique (non-denaturing IEF followed by SDS-PAGE, no reducing agent being used), several proteins in molecular mass range from 7x10(1) to 2x10(2) kDa appeared, suggesting that these proteins interacted with the high-molecular-mass proteins, then dissociated in the presence of SDS.
高相对分子质量蛋白质（约 1x10(3) kDa 以上）在人血浆中经 0-4% PEG 沉淀的过程采用琼脂糖 IEF 凝胶第一向非变性微 2-DE 和 MALDI-MS PMF 进行分析。PEG 0-2%沉淀物可大部分重新溶解（PS2%部分），但部分仍为沉淀（PP2%部分）。非变性 2-DE 后再经 MALDI-MS PMF 分析显示 PS2%部分主要包含纤维蛋白原（Fb）、纤维连接蛋白（FN）及其低聚物，以及血管性血友病因子和凝血因子 XIII 作为次要成分。PP2%部分直接进行 MALDI-MS PMF 分析，鉴定出 FN 和 Fb，提示这是两种蛋白质的共沉淀形成的。PEG 2-4%沉淀物可完全溶解，但溶液经冷冻和解冻后，出现少量沉淀。沉淀（PP4%）的 MALDI-MS PMF 分析提示其主要成分是补体 C1。PEG 2-4%沉淀物的可溶性部分（PS4%）经非变性 2-DE 和 MALDI-MS PMF 分析，含有 C4b 结合蛋白及其与补体 C4 的复合物、低密度脂蛋白、IgM 和补体 C1 亚成分 q，以及 Fb、FN 和它们的低聚物。当 PS4%部分经 II 型 2-DE 技术（非变性 IEF 后 SDS-PAGE，不使用还原剂）分析时，在 7x10(1) 至 2x10(2) kDa 分子量范围内出现几种蛋白质，提示这些蛋白质与高分子质量蛋白质相互作用，然后在 SDS 存在下解离。
