Snake venoms contain complex mixtures of proteins vital for the survival of venomous snakes. Aligned with their diverse pharmacological activities, the protein compositions of snake venoms are highly variable, and efforts to characterise the primary structures of such proteins are ongoing. Additionally, a significant knowledge gap exists in terms of the higher-order protein structures which modulate venom potency, posing a challenge for successful therapeutic applications. Here we use a multifaceted mass spectrometry approach to characterise proteins from venoms of Collett's snake Pseudechis colletti and the puff adder Bitis arietans. Following chromatographic fractionation and bottom-up proteomics analysis, native mass spectrometry identified, among other components, a non-covalent l-amino acid oxidase dimer in the P. colletti venom and a C-type lectin tetramer in the B. arietans venom. Furthermore, a covalently-linked phospholipase A2 (PLA2) dimer was identified in P. colletti venom, from which the PLA2 species were shown to adopt compact geometries using ion mobility measurements. Interestingly, we show that the dimeric PLA2 possesses greater bioactivity than the monomeric PLA2s. This work contributes to ongoing efforts cataloguing components of snake venoms, and notably, emphasises the importance of understanding higher-order venom protein interactions and the utility of a combined mass spectrometric approach for this task. SIGNIFICANCE: The protein constituents of snake venoms represent a sophisticated cocktail of biologically active molecules ideally suited for further exploration in drug design and development. Despite ongoing efforts to characterise the diverse protein components of such venoms there is still much work required in this area, particularly in moving from simply describing the protein primary sequence to providing an understanding of quaternary structure. The combined proteomic and native mass spectrometry workflow utilised here gives new insights into higher order protein structures in selected snake venoms, and can underpin further investigation into the protein interactions which govern snake venom specificity and potency.

中文翻译：

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使用集成质谱方法询问蛇毒蛋白的高阶结构。

蛇毒中含有复杂的蛋白质混合物，这些蛋白质对毒蛇的生存至关重要。蛇毒的蛋白质组成与其多样的药理活性相适应，是高度可变的，并且正在努力表征这种蛋白质的一级结构。另外，就调节毒力的高级蛋白质结构而言，存在巨大的知识空白，这对成功的治疗应用提出了挑战。在这里，我们使用多方面的质谱分析方法来表征来自Collett的蛇Pseudechis colletti和粉扑添加物Bitis arietans的毒液中的蛋白质。经过色谱分离和自下而上的蛋白质组学分析后，天然质谱法鉴定了P中的非共价I-氨基酸氧化酶二聚体等。Colletti毒液和Arietans毒液中的C型凝集素四聚体。此外，在胶体假单胞菌毒液中鉴定出共价连接的磷脂酶A2（PLA2）二聚体，使用离子迁移率测量表明，PLA2物种​​采用紧凑的几何形状。有趣的是，我们表明二聚PLA2比单体PLA2具有更大的生物活性。这项工作有助于正在进行的对蛇毒成分进行分类的工作，尤其是强调了解高级蛇毒蛋白相互作用的重要性以及组合质谱方法在该任务中的实用性。意义：蛇毒的蛋白质成分代表着复杂的生物活性分子混合物，非常适合在药物设计和开发中进一步探索。尽管正在努力表征这些毒液的各种蛋白质成分，但在该领域仍需要大量工作，特别是在从简单地描述蛋白质一级序列到提供对四级结构的理解方面。此处使用的蛋白质组学和天然质谱相结合的工作流程为选定蛇毒中的高级蛋白质结构提供了新见解，并可以进一步研究控制蛇毒特异性和效力的蛋白质相互作用。