全球,据估计,有180万至270万例由蛇咬伤引起的envenoming病例。蛇毒是蛋白质毒素的复杂混合物,脂质,小分子,和盐,这些蛋白质通常会导致蛇咬伤受害者的病理。对于它们的化学表征和鉴定,需要使用分析方法。反相液相色谱-电喷雾质谱联用技术(RP-LC-ESI-MS)因其使用方便,灵敏度,以及LC分离后直接偶联的能力。该方法允许复杂混合物的有效分离和分析物的灵敏检测。另一方面,有时也使用基质辅助激光解吸/电离质谱(MALDI-MS),尽管它通常需要额外的样品制备步骤,它为分析较大的生物分子提供了理想的适用性。在这项研究中,将七个医学上重要的蛇毒蛇毒分离为各自的毒液毒素,并通过ESI-MS进行测量。并行,使用纳米分馏分析,柱后高分辨率分级分离用于收集洗脱毒素用于进一步处理以用于MALDI-MS分析。我们的比较结果表明,从ESI-MS和MALDI-MS方法中观察到的去卷积的蛇毒毒素质量具有良好的敏感性,并且在回收的毒素质量中呈现重叠(在25%和57%之间,取决于分析的毒液)。检测到的高丰度毒素的质量范围在4至28kDa之间。总的来说,在ESI-MS和/或MALDI-MS分析中发现39个质量,大多数在5到9kDa之间(46%),13和15kDa(38%),大小为24和28kDa(13%)。在柱后MS分析旁边,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平行的柱后评估.大多数纳米分馏毒液表现出抗凝血活性,三个毒液还表现出具有明显促凝血活性的毒素(Bothropsasper,Crotalusatrox,和Daboiarusselii)观察到后柱。这项研究的结果强调了ESI-MS和MALDI-MS方法在表征蛇毒毒素方面的互补性,并提供了在多种蛇毒蛇毒中发现的定义毒素质量的补充概述。
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 (Bothrops asper, Crotalus atrox, and Daboia russelii) 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.