Department of Chemistry and Pharmaceutical Sciences, Division of BioAnalytical Chemistry, Faculty of Science, Amsterdam Institute of Molecular and Life Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands.
Centre for Analytical Sciences Amsterdam (CASA), 1012 WX Amsterdam, The Netherlands.
Toxins (Basel). 2024 Aug 21;16(8):370. doi: 10.3390/toxins16080370.
Worldwide, it is estimated that there are 1.8 to 2.7 million cases of envenoming caused by snakebites. Snake venom is a complex mixture of protein toxins, lipids, small molecules, and salts, with the proteins typically responsible for causing pathology in snakebite victims. For their chemical characterization and identification, analytical methods are required. Reversed-phase liquid chromatography coupled with electrospray ionization mass spectrometry (RP-LC-ESI-MS) is a widely used technique due to its ease of use, sensitivity, and ability to be directly coupled after LC separation. This method allows for the efficient separation of complex mixtures and sensitive detection of analytes. On the other hand, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is also sometimes used, and though it typically requires additional sample preparation steps, it offers desirable suitability for the analysis of larger biomolecules. In this study, seven medically important viperid snake venoms were separated into their respective venom toxins and measured by ESI-MS. In parallel, using nanofractionation analytics, post-column high-resolution fractionation was used to collect the eluting toxins for further processing for MALDI-MS analysis. Our comparative results showed that the deconvoluted snake venom toxin masses were observed with good sensitivity from both ESI-MS and MALDI-MS approaches and presented overlap in the toxin masses recovered (between 25% and 57%, depending on the venom analyzed). The mass range of the toxins detected in high abundance was between 4 and 28 kDa. In total, 39 masses were found in both the ESI-MS and/or MALDI-MS analyses, with most being between 5 and 9 kDa (46%), 13 and 15 kDa (38%), and 24 and 28 kDa (13%) in size. Next to the post-column MS analyses, additional coagulation bioassaying was performed to demonstrate the parallel post-column assessment of venom activity in the workflow. Most nanofractionated venoms exhibited anticoagulant activity, with three venoms additionally exhibiting toxins with clear procoagulant activity (, , and ) observed post-column. The results of this study highlight the complementarity of ESI-MS and MALDI-MS approaches for characterizing snake venom toxins and provide a complementary overview of defined toxin masses found in a diversity of viper snake venoms.
据估计，全球每年有 180 万至 270 万例蛇咬伤中毒病例。蛇毒是一种复杂的蛋白质毒素、脂质、小分子和盐的混合物，其中蛋白质通常是导致蛇咬伤患者出现病理的原因。为了对其进行化学表征和鉴定，需要使用分析方法。反相液相色谱-电喷雾电离质谱联用（RP-LC-ESI-MS）是一种广泛应用的技术，因为它易于使用、灵敏度高，并且可以在 LC 分离后直接进行。该方法能够高效分离复杂混合物并对分析物进行灵敏检测。另一方面，基质辅助激光解吸/电离质谱（MALDI-MS）有时也会用到，尽管它通常需要额外的样品制备步骤，但它非常适合分析较大的生物分子。在这项研究中，七种具有重要医学意义的蝰蛇毒液被分离成各自的毒液毒素，并通过 ESI-MS 进行测量。同时，使用纳流分析技术，在柱后进行高分辨率分级，收集洗脱毒素，以便进一步进行 MALDI-MS 分析。我们的比较结果表明，ESI-MS 和 MALDI-MS 两种方法都能很好地检测到解卷积的蛇毒毒素质量，并在回收的毒素质量上存在重叠（取决于分析的毒液，在 25%至 57%之间）。高丰度检测到的毒素的质量范围在 4 至 28 kDa 之间。总共在 ESI-MS 和/或 MALDI-MS 分析中发现了 39 种质量，其中大多数在 5 至 9 kDa（46%）、13 至 15 kDa（38%）和 24 至 28 kDa（13%）之间。除了柱后 MS 分析外，还进行了额外的凝血生物测定，以证明在工作流程中对毒液活性进行柱后平行评估。大多数纳流分离的毒液具有抗凝活性，其中三种毒液在柱后还显示出具有明显促凝活性的毒素（，和）。这项研究的结果突出了 ESI-MS 和 MALDI-MS 方法在表征蛇毒毒素方面的互补性，并提供了对多种蝰蛇毒液中发现的定义明确的毒素质量的互补概述。
