Petras Daniel, Heiss Paul, Süssmuth Roderich D, Calvete Juan J
†Institut für Chemie, Technische Universität Berlin, Müller-Breslau-Straße 10, 10623 Berlin, Germany.
‡Laboratorio de Venómica Estructural y Funcional, Instituto de Biomedicina de Valencia, CSIC, 46010 Valencia, Spain.
J Proteome Res. 2015 Jun 5;14(6):2539-56. doi: 10.1021/acs.jproteome.5b00305. Epub 2015 Apr 27.
We report on the first application of top-down mass spectrometry in snake venomics. De novo sequence tags generated by, and ProSight Lite supported analysis of, combined collisional based dissotiations (CID and HCD) recorded in a hybrid LTQ Orbitrap instrument in data-dependent mode identified a number of proteins from different toxin families, namely, 11 three-finger toxins (7-7.9 kDa), a Kunitz-type inhibitor (6.3 kDa), ohanin (11.9 kDa), a novel phospholipase A2 molecule (13.8 kDa), and the cysteine-rich secretory protein (CRISP) ophanin (25 kDa) from Indonesian king cobra venom. Complementary bottom-up MS/MS analyses contributed to the completion of a locus-resolved venom phenotypic map for Ophiophagus hannah, the world's longest venomous snake and a species of medical concern across its wide distribution range in forests from India to Southeast Asia. Its venom composition, comprising 32-35 proteins/peptides from 10 protein families, is dominated by α-neurotoxins and convincingly explains the main neurotoxic effects of human envenoming caused by king cobra bite. The integration of efficient chromatographic separation of the venom's components and locus-resolved toxin identification through top-down and bottom-up MS/MS-based species-specific database searching and de novo sequencing holds promise that the future will be bright for the field of venom research.
我们报道了自上而下质谱法在蛇毒蛋白质组学中的首次应用。在数据依赖模式下，通过混合LTQ Orbitrap仪器记录的基于碰撞解离（CID和HCD）组合产生的从头序列标签以及ProSight Lite支持的分析，鉴定出了来自不同毒素家族的多种蛋白质，即11种三指毒素（7 - 7.9 kDa）、一种库尼茨型抑制剂（6.3 kDa）、ohanin（11.9 kDa）、一种新型磷脂酶A2分子（13.8 kDa）以及来自印度尼西亚眼镜王蛇毒液的富含半胱氨酸的分泌蛋白（CRISP）orphanin（25 kDa）。互补的自下而上的MS/MS分析有助于完成眼镜王蛇（世界上最长的毒蛇，在从印度到东南亚的森林广泛分布范围内是一种受到医学关注的物种）的位点解析毒液表型图谱。其毒液成分由来自10个蛋白质家族的32 - 35种蛋白质/肽组成，以α - 神经毒素为主，令人信服地解释了眼镜王蛇咬伤导致人类中毒的主要神经毒性作用。通过自上而下和自下而上基于MS/MS的物种特异性数据库搜索和从头测序，将毒液成分的高效色谱分离与位点解析毒素鉴定相结合，有望使毒液研究领域的未来前景光明。
