Department of Chemistry, Yonsei University, Seoul, 120-749, South Korea.
Analyst. 2011 Jan 21;136(2):388-92. doi: 10.1039/c0an00172d. Epub 2010 Oct 21.
Effects of protein denaturation and formation of protein-sodium dodecyl sulfate (SDS) complexes on protein separation and identification were investigated using hollow fiber flow field-flow fractionation (HF5) and nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS-MS). Denaturation and formation of protein-SDS complexes prior to HF5 separation resulted an increase in the retention of few protein standards due to unfolding of the protein structures and complexation, yielding ~30% increase in hydrodynamic diameter. In addition, low molecular weight proteins which could be lost from the HF membrane due to the pore size limitation showed an increase of peak recovery about 2-6 folds for cytochrome C and carbonic anhydrase. In the case of proteins composed of a number of subunits, denaturation resulted in a decrease in retention due to dissociation of protein subunits. A serum proteome sample, denatured with dithiothreitol and SDS, was fractionated by HF5, and the eluting protein fractions after tryptic digestion were analyzed for protein identification using nLC-ESI-MS-MS. The resulting pools of identified proteins were found to depend on whether the serum sample was treated with or without denaturation prior to the HF5 run due to differences in the aqueous solubility of the proteins. The enhancement of protein solubility by SDS also increased the number of identified membrane proteins (54 vs. 31).
采用中空纤维流动场流分离（HF5）和纳流液相色谱-电喷雾电离-串联质谱（nLC-ESI-MS-MS）研究了蛋白质变性和形成蛋白质-十二烷基硫酸钠（SDS）复合物对蛋白质分离和鉴定的影响。HF5 分离前蛋白质的变性和 SDS 复合物的形成导致少数蛋白质标准品的保留增加，这是由于蛋白质结构展开和复合物形成所致，从而使水动力直径增加约 30%。此外，由于孔径限制，可能会从小孔中丢失 HF 膜的低分子量蛋白质，对于细胞色素 C 和碳酸酐酶，其峰回收增加约 2-6 倍。对于由多个亚基组成的蛋白质，由于蛋白质亚基的解离，变性导致保留时间减少。用二硫苏糖醇和 SDS 变性的血清蛋白质组样品通过 HF5 进行分级，然后对经胰蛋白酶消化后的洗脱蛋白质级分进行纳流液相色谱-电喷雾电离-串联质谱分析，以进行蛋白质鉴定。由于蛋白质在水中的溶解度不同，因此发现所鉴定的蛋白质池取决于在 HF5 运行之前是否对血清样品进行了变性处理。SDS 对蛋白质溶解度的增强也增加了鉴定的膜蛋白数量（54 个比 31 个）。
