High-Throughput Venomics

In this study, we present high-throughput (HT) venomics, a novel analytical strategy capable of performing a full proteomic analysis of a snake venom within 3 days. This methodology comprises a combination of RP-HPLC-nanofractionation analytics, mass spectrometry analysis, automated in-solution tryptic digestion, and high-throughput proteomics. In-house written scripts were developed to process all the obtained proteomics data by first compiling all Mascot search results for a single venom into a single Excel sheet. Then, a second script plots each of the identified toxins in so-called Protein Score Chromatograms (PSCs). For this, for each toxin, identified protein scores are plotted on the y-axis versus retention times of adjacent series of wells in which a toxin was fractionated on the x-axis. These PSCs allow correlation with parallel acquired intact toxin MS data. This same script integrates the PSC peaks from these chromatograms for semiquantitation purposes. This new HT venomics strategy was performed on venoms from diverse medically important biting species; Calloselasma rhodostoma, Echis ocellatus, Naja pallida, Bothrops asper, Bungarus multicinctus, Crotalus atrox, Daboia russelii, Naja naja, Naja nigricollis, Naja mossambica, and Ophiophagus hannah. Our data suggest that high-throughput venomics represents a valuable new analytical tool for increasing the throughput by which we can define venom variation and should greatly aid in the future development of new snakebite treatments by defining toxin composition.本研究提出了高通量（HT）毒组学（venomics）——一种可在3天内完成蛇毒全蛋白质组分析的新型分析策略。该方法整合了反相高效液相色谱-纳米分级分离分析、质谱分析、自动化溶液内胰蛋白酶消化以及高通量蛋白质组学技术。我们开发了自研脚本以处理所有获取的蛋白质组学数据：首先将单一蛇毒的全部Mascot搜索结果整合至单个Excel工作表中；随后，通过第二段脚本将每一种已鉴定毒素绘制于所谓的蛋白质得分色谱图（"Protein Score Chromatograms", PSCs）中。具体而言，针对每种毒素，将其鉴定得到的蛋白质得分作为纵轴，与毒素被分级分离的相邻孔板系列的保留时间作为横轴进行绘图。此类蛋白质得分色谱图可与并行获取的完整毒素质谱数据进行关联分析。该脚本还可整合这些色谱图中的PSC峰，以实现半定量分析。我们将这一新型HT毒组学策略应用于多种具有医学重要性的毒蛇毒液：马来亚红口蝮（Calloselasma rhodostoma）、眼斑锯鳞蝰（Echis ocellatus）、浅红眼镜蛇（Naja pallida）、巨蝮（Bothrops asper）、银环蛇（Bungarus multicinctus）、西部菱斑响尾蛇（Crotalus atrox）、罗素蝰（Daboia russelii）、印度眼镜蛇（Naja naja）、黑颈眼镜蛇（Naja nigricollis）、莫桑比克眼镜蛇（Naja mossambica）以及眼镜王蛇（Ophiophagus hannah）。本研究数据表明，高通量毒组学是一种极具价值的新型分析工具，可提升我们解析毒液变异的通量，并通过明确毒素组成，极大助力未来新型蛇伤治疗手段的开发。