Mladic Marija, de Waal Tessa, Burggraaff Lindsey, Slagboom Julien, Somsen Govert W, Niessen Wilfried M A, Manjunatha Kini R, Kool Jeroen
Division of BioAnalytical Chemistry, Amsterdam Institute for Molecules Medicines and Systems, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV, Amsterdam, The Netherlands.
hyphen MassSpec, Herenweg 95, 2361 EK, Warmond, The Netherlands.
Anal Bioanal Chem. 2017 Oct;409(25):5987-5997. doi: 10.1007/s00216-017-0531-3. Epub 2017 Aug 11.
This study presents an analytical method for the screening of snake venoms for inhibitors of the angiotensin-converting enzyme (ACE) and a strategy for their rapid identification. The method is based on an at-line nanofractionation approach, which combines liquid chromatography (LC), mass spectrometry (MS), and pharmacology in one platform. After initial LC separation of a crude venom, a post-column flow split is introduced enabling parallel MS identification and high-resolution fractionation onto 384-well plates. The plates are subsequently freeze-dried and used in a fluorescence-based ACE activity assay to determine the ability of the nanofractions to inhibit ACE activity. Once the bioactive wells are identified, the parallel MS data reveals the masses corresponding to the activities found. Narrowing down of possible bioactive candidates is provided by comparison of bioactivity profiles after reversed-phase liquid chromatography (RPLC) and after hydrophilic interaction chromatography (HILIC) of a crude venom. Additional nanoLC-MS/MS analysis is performed on the content of the bioactive nanofractions to determine peptide sequences. The method described was optimized, evaluated, and successfully applied for screening of 30 snake venoms for the presence of ACE inhibitors. As a result, two new bioactive peptides were identified: pELWPRPHVPP in Crotalus viridis viridis venom with IC = 1.1 μM and pEWPPWPPRPPIPP in Cerastes cerastes cerastes venom with IC = 3.5 μM. The identified peptides possess a high sequence similarity to other bradykinin-potentiating peptides (BPPs), which are known ACE inhibitors found in snake venoms.
本研究提出了一种用于筛选蛇毒中血管紧张素转换酶（ACE）抑制剂的分析方法及其快速鉴定策略。该方法基于一种在线纳米分馏方法，该方法将液相色谱（LC）、质谱（MS）和药理学整合在一个平台上。在对粗毒进行初步LC分离后，引入柱后分流，实现并行MS鉴定和在384孔板上的高分辨率分馏。随后将这些板冷冻干燥，并用于基于荧光的ACE活性测定，以确定纳米级分馏物抑制ACE活性的能力。一旦确定了具有生物活性的孔，并行MS数据就会揭示与所发现活性相对应的质量。通过比较粗毒在反相液相色谱（RPLC）和亲水相互作用色谱（HILIC）后的生物活性图谱，可缩小可能的生物活性候选物范围。对具有生物活性的纳米级分馏物的含量进行额外的纳升液相色谱-串联质谱（nanoLC-MS/MS）分析，以确定肽序列。所描述的方法经过优化、评估，并成功应用于筛选30种蛇毒中是否存在ACE抑制剂。结果，鉴定出两种新的生物活性肽：绿曼巴蛇毒中的pELWPRPHVPP，IC₅₀ = 1.1 μM；角蝰蛇毒中的pEWPPWPPRPPIPP，IC₅₀ = 3.5 μM。所鉴定的肽与其他缓激肽增强肽（BPPs）具有高度的序列相似性，BPPs是在蛇毒中发现的已知ACE抑制剂。
