Discipline of Chemistry, School of Physics, Chemistry and Earth Sciences, The University of Adelaide, Adelaide, SA 5005, Australia.
Australian Proteome Analysis Facility, Macquarie University, Macquarie Park, NSW 2109, Australia.
J Proteome Res. 2024 Oct 4;23(10):4601-4613. doi: 10.1021/acs.jproteome.4c00486. Epub 2024 Sep 4.
Snake venoms are comprised of bioactive proteins and peptides that facilitate severe snakebite envenomation symptoms. A comprehensive understanding of venom compositions and the subtle heterogeneity therein is important. While bottom-up proteomics has been the well-established approach to catalogue venom compositions, top-down proteomics has emerged as a complementary strategy to characterize venom heterogeneity at the intact protein level. However, top-down proteomics has not been as widely implemented in the snake venom field as bottom-up proteomics, with various emerging top-down methods yet to be developed for venom systems. Here, we have explored three main top-down mass spectrometry methodologies in a proof-of-concept study to characterize selected three-finger toxin and phospholipase A proteoforms from the forest cobra () venom. We demonstrated the utility of a data-independent acquisition mode "MS" for untargeted fragmentation on a chromatographic time scale and its improvement in protein sequence coverage compared to conventional targeted tandem mass spectrometry analysis. We also showed that protein identification can be further improved using a hybrid fragmentation approach, combining electron-capture dissociation and collision-induced dissociation. Lastly, we reported the promising application of multifunctional cyclic ion mobility separation and post-ion mobility fragmentation on snake venom proteins for the first time.
蛇毒由生物活性蛋白和肽组成，可导致严重的蛇咬伤中毒症状。全面了解毒液成分及其细微异质性非常重要。虽然自下而上的蛋白质组学已成为编目毒液成分的既定方法，但自上而下的蛋白质组学已成为在完整蛋白质水平上表征毒液异质性的补充策略。然而，与自下而上的蛋白质组学相比，自上而下的蛋白质组学在蛇毒领域的应用并不广泛，各种新兴的自上而下的方法尚未针对毒液系统开发出来。在这里，我们在概念验证研究中探索了三种主要的自上而下的质谱方法，以表征来自森林眼镜蛇（）毒液的选定三指毒素和磷脂酶 A 同工型。我们证明了“MS”数据非依赖性采集模式在色谱时间尺度上进行非靶向碎片化的实用性，以及与传统靶向串联质谱分析相比，其在蛋白质序列覆盖度方面的改善。我们还表明，使用电子俘获解离和碰撞诱导解离相结合的混合碎片化方法可以进一步提高蛋白质鉴定的效果。最后，我们首次报道了多功能循环离子迁移分离和离子迁移后碎片化在蛇毒蛋白中的应用前景。
