Mladic Marija, Zietek Barbara M, Iyer Janaki Krishnamoorthy, Hermarij Philip, Niessen Wilfried M A, Somsen Govert W, Kini R Manjunatha, Kool Jeroen
Division of BioAnalytical Chemistry, Amsterdam Institute for Molecules Medicines and Systems, VU University Amsterdam, De Boelelaan 1083, 1081HV, Amsterdam, The Netherlands.
Department of Biological Sciences, National University of Singapore, 14 Science Drive 4 117543, Singapore.
Toxicon. 2016 Feb;110:79-89. doi: 10.1016/j.toxicon.2015.12.008. Epub 2015 Dec 19.
Snake venoms comprise complex mixtures of peptides and proteins causing modulation of diverse physiological functions upon envenomation of the prey organism. The components of snake venoms are studied as research tools and as potential drug candidates. However, the bioactivity determination with subsequent identification and purification of the bioactive compounds is a demanding and often laborious effort involving different analytical and pharmacological techniques. This study describes the development and optimization of an integrated analytical approach for activity profiling and identification of venom constituents targeting the cardiovascular system, thrombin and factor Xa enzymes in particular. The approach developed encompasses reversed-phase liquid chromatography (RPLC) analysis of a crude snake venom with parallel mass spectrometry (MS) and bioactivity analysis. The analytical and pharmacological part in this approach are linked using at-line nanofractionation. This implies that the bioactivity is assessed after high-resolution nanofractionation (6 s/well) onto high-density 384-well microtiter plates and subsequent freeze drying of the plates. The nanofractionation and bioassay conditions were optimized for maintaining LC resolution and achieving good bioassay sensitivity. The developed integrated analytical approach was successfully applied for the fast screening of snake venoms for compounds affecting thrombin and factor Xa activity. Parallel accurate MS measurements provided correlation of observed bioactivity to peptide/protein masses. This resulted in identification of a few interesting peptides with activity towards the drug target factor Xa from a screening campaign involving venoms of 39 snake species. Besides this, many positive protease activity peaks were observed in most venoms analysed. These protease fingerprint chromatograms were found to be similar for evolutionary closely related species and as such might serve as generic snake protease bioactivity fingerprints in biological studies on venoms.
蛇毒是肽和蛋白质的复杂混合物，在猎物被蛇毒咬伤后会调节其多种生理功能。蛇毒的成分作为研究工具和潜在的药物候选物进行研究。然而，对生物活性进行测定，随后鉴定和纯化生物活性化合物是一项要求很高且通常很费力的工作，涉及不同的分析和药理技术。本研究描述了一种综合分析方法的开发和优化，该方法用于针对心血管系统、特别是凝血酶和因子Xa酶的毒液成分进行活性分析和鉴定。所开发的方法包括对粗蛇毒进行反相液相色谱（RPLC）分析，并同时进行质谱（MS）和生物活性分析。该方法中的分析和药理部分通过在线纳米分级分离相联系。这意味着在将其高分辨率纳米分级分离（每孔6秒）到高密度384孔微量滴定板上并随后对板进行冷冻干燥后评估生物活性。对纳米分级分离和生物测定条件进行了优化，以保持液相色谱分辨率并实现良好的生物测定灵敏度。所开发的综合分析方法成功应用于快速筛选蛇毒中影响凝血酶和因子Xa活性的化合物。并行的精确质谱测量提供了观察到的生物活性与肽/蛋白质质量之间的相关性。这导致在一项涉及39种蛇毒液的筛选活动中鉴定出了几种对药物靶标因子Xa具有活性的有趣肽。除此之外，在大多数分析的毒液中观察到许多阳性蛋白酶活性峰。发现这些蛋白酶指纹色谱图对于进化上密切相关的物种是相似的，因此可能作为毒液生物学研究中的通用蛇蛋白酶生物活性指纹。
