Manabe Takashi, Jin Ya
Department of Chemistry, Graduate School of Science and Engineering, Ehime University, Matsuyama, Japan.
Electrophoresis. 2007 Jun;28(12):2065-79. doi: 10.1002/elps.200600691.
Previously, we have reported on the analysis of human plasma proteins on a nondenaturing micro-2-DE (mu2-DE) gel, using in-gel digestion followed by MALDI-MS and PMF [1]. Many of the spots on the mu2-DE gel showed apparent masses much larger than the calculated masses of their assigned polypeptides, suggesting noncovalent or covalent interactions between the polypeptides. In the present study, we aimed to further analyze the plasma protein spots on a nondenaturing mu2-DE gel, on which protein/polypeptide interactions have been suggested. The proteins in the spots were extracted under alkaline conditions and subjected to 3-D separation using SDS-PAGE in microslab gel format (muSDS gel) with or without the sample treatment of reduction-alkylation. The clear bands in each lane of the muSDS gels demonstrated the successful extraction of proteins from the relevant gel spot and visualized the relative contents of the polypeptides in the spot. Most of the bands were assigned by in-gel digestion followed by MALDI-MS and PMF (MASCOT/Swiss-Prot). The large discrepancy between the apparent mass value of a protein spot and the estimated mass values of the polypeptide bands on a nonreducing muSDS gel strongly suggested noncovalent polypeptide interactions. The differences in the polypeptide separation patterns on the muSDS gels, between with and without the treatment of reduction-alkylation, confirmed polypeptide disulfide bonding. The method employed here, aiming to integrate information on the proteins separated on nondenaturing 2-DE gels with that on the interactions between polypeptides, would help the comprehensive understanding of complex protein systems.
此前，我们报道了在非变性微二维电泳（mu2-DE）凝胶上对人血浆蛋白进行分析，采用胶内消化，随后进行基质辅助激光解吸电离质谱（MALDI-MS）和肽质量指纹图谱（PMF）分析[1]。mu2-DE凝胶上的许多斑点显示的表观质量远大于其指定多肽的计算质量，这表明多肽之间存在非共价或共价相互作用。在本研究中，我们旨在进一步分析非变性mu2-DE凝胶上的血浆蛋白斑点，该凝胶上已表明存在蛋白质/多肽相互作用。在碱性条件下提取斑点中的蛋白质，并使用微板凝胶形式的十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE，muSDS凝胶）进行三维分离，样品进行或不进行还原烷基化处理。muSDS凝胶各泳道中的清晰条带表明从相关凝胶斑点中成功提取了蛋白质，并可视化了斑点中多肽的相对含量。大多数条带通过胶内消化，随后进行MALDI-MS和PMF（MASCOT/瑞士蛋白质数据库）鉴定。蛋白质斑点的表观质量值与非还原muSDS凝胶上多肽条带的估计质量值之间的巨大差异强烈表明存在非共价多肽相互作用。在muSDS凝胶上，还原烷基化处理与未处理之间多肽分离模式的差异证实了多肽二硫键的存在。这里采用的方法旨在整合在非变性二维电泳凝胶上分离的蛋白质信息与多肽之间相互作用的信息，这将有助于全面理解复杂的蛋白质系统。
