Institute of Biomedical and Pharmaceutical Sciences, Guangdong University of Technology, Guangzhou, Guangdong 510006, China.
Faculty of Science, Ehime University, Matsuyama 790-0825, Japan.
J Chromatogr B Analyt Technol Biomed Life Sci. 2020 May 1;1144:122104. doi: 10.1016/j.jchromb.2020.122104. Epub 2020 Apr 3.
Subunit structures of proteins are essential for their properties and functions, but there is a lack of method for global detection of the status of proteins being monomers or homo-oligomers. In this work, we report on a new method to simultaneously speculate hundreds of monomeric or homo-oligomeric subunit structures of cellular proteins, based on in-depth analysis of native 2D protein maps. Previously we have reported on the analysis of soluble proteins of human bronchial muscle cells (HBSMC) by combining nondenaturing 2DE, grid gel-cutting and quantitative LC-MS/MS. Totally 4323 proteins were detected and for each protein the quantity distribution on the gel was reconstructed as a native 2D map. In this work, this large dataset of maps were further mined with bioinformatic analysis. The native 2D maps of 1901 HBSMC proteins that were detected in at least five out of the grid-cut 972 gel squares were examined and 658 proteins that showed one major quantity-peak distribution were subjected to further analysis. After excluding those that mainly formed hetero-oligomeric structures, the monomeric or homo-oligomeric subunit structures of 505 proteins were speculated. The quotient of the apparent molecular mass of the quantity-peak position on the native 2D map divided by the theoretical molecular mass was calculated for each protein, to speculate the number of monomers which constituted its subunit structure. The suggested composition was then compared with the "Subunit structure" record of the protein in UniProtKB. When the database record included possible interactions with other proteins, their native 2D maps were extracted from the native map dataset, presented together and compared to confirm the prominent subunit structure. With this new approach, the monomeric or homo-oligomeric subunit structures of 401 proteins were speculated. Among them, 162 proteins had the speculated subunit structures coinciding with their database records, and 91 proteins with matched database records as being monomers or homo-oligomers but mismatched at the numbers of the composing monomers. For 148 proteins that did not have database record, their subunit structures were newly speculated. We expect this method, combining nondenaturing 2DE separation with in-depth proteomic and bioinformatic analysis, would suggest a means to achieve large-scale information on monomeric and homo-oligomeric subunit structures of cellular proteins.
蛋白质的亚基结构对于其性质和功能至关重要，但目前缺乏一种方法来全面检测蛋白质是单体还是同聚体。在这项工作中，我们报告了一种新的方法，可以基于对天然 2D 蛋白质图谱的深入分析，同时推测数百种细胞蛋白质的单体或同聚体亚基结构。此前，我们已经报告了通过结合非变性 2DE、网格胶切割和定量 LC-MS/MS 分析人支气管平滑肌细胞（HBSMC）可溶性蛋白质的情况。总共检测到 4323 种蛋白质，并且为每种蛋白质在凝胶上的数量分布重建了天然 2D 图谱。在这项工作中，这个大型图谱数据集进一步通过生物信息学分析进行了挖掘。在网格切割的 972 个凝胶方块中至少有 5 个检测到的 1901 种 HBSMC 蛋白质的天然 2D 图谱进行了检查，并且对显示一个主要数量峰分布的 658 种蛋白质进行了进一步分析。排除那些主要形成异聚体结构的蛋白质后，推测了 505 种蛋白质的单体或同聚体亚基结构。对于每个蛋白质，计算天然 2D 图谱上数量峰位置的表观分子量与理论分子量的商，以推测构成其亚基结构的单体数量。然后将建议的组成与 UniProtKB 中蛋白质的“亚基结构”记录进行比较。当数据库记录包含与其他蛋白质可能的相互作用时，从天然图谱数据集中提取它们的天然 2D 图谱，一起呈现并进行比较以确认突出的亚基结构。使用这种新方法，推测了 401 种蛋白质的单体或同聚体亚基结构。其中，162 种蛋白质的推测亚基结构与其数据库记录相符，91 种蛋白质的数据库记录显示为单体或同聚体，但组成单体的数量不匹配。对于 148 种没有数据库记录的蛋白质，新推测了它们的亚基结构。我们希望这种结合非变性 2DE 分离与深入蛋白质组学和生物信息学分析的方法，为获得细胞蛋白质单体和同聚体亚基结构的大规模信息提供一种手段。
