来自蛇的毒液是对多种酶和受体具有强亲和力的高活性蛋白质的丰富来源。在毒气引起的许多不同的毒性中,神经毒性在蛇以及有毒的海蜗牛和昆虫对猎物的麻痹中起着重要作用。为了提高毒液毒性分析的分析发现成分,本文介绍了微流控高分辨率筛选(HRS)的实施,以从毒液中获得神经毒性指纹,从而有助于鉴定毒液的神经毒性成分。为了演示此工作流程,使用乙酰胆碱结合蛋白(AChBP)模拟神经毒性蛋白的靶标,对47个蛇毒进行了分析。特别是烟碱乙酰胆碱受体(nAChRs)。在微流控HRS系统中,纳米液体色谱(nanoLC)分离与AChBP分析和平行质谱(MS)在线连接。对几乎所有有神经毒性的蛇毒进行了测试,我们获得了生物活性指纹,显示主要和次要的生物活性区域含有与三指毒素(3FTxs)一致的质量,然而,viperid和Colubrid毒液几乎没有或没有可检测的生物活性。我们的发现表明,毒液与AChBP的相互作用与不同蛇种在人类毒液后观察到的神经毒性的严重程度相关。我们进一步,作为原理的证明,通过nanoLC-MS/MS表征了来自蛇毒(Daboiarusselli)和elapid(Aspidelapsscutatusscutatus)蛇的生物活性毒液肽,揭示了不同的毒素类别与AChBP相互作用,并且这种结合与基于钙通量细胞的测定中α7-nAChR的抑制相关。本文描述的在线柱后结合测定和随后的毒素表征方法为快速研究神经毒性蛇毒蛋白提供了新的体外分析平台。
Venoms from snakes are rich sources of highly active proteins with potent affinity towards a variety of enzymes and receptors. Of the many distinct toxicities caused by envenomation, neurotoxicity plays an important role in the paralysis of prey by snakes as well as by venomous sea snails and insects. In order to improve the analytical discovery component of venom toxicity profiling, this paper describes the implementation of microfluidic high-resolution screening (HRS) to obtain neurotoxicity fingerprints from venoms that facilitates identification of the neurotoxic components of envenomation. To demonstrate this workflow, 47 snake venoms were profiled using the acetylcholine binding protein (
AChBP) to mimic the target of neurotoxic proteins, in particular nicotinic acetylcholine receptors (nAChRs). In the microfluidic HRS system, nanoliquid chromatographic (nanoLC) separations were on-line connected to both
AChBP profiling and parallel mass spectrometry (MS). For virtually all neurotoxic elapid snake venoms tested, we obtained bioactivity fingerprints showing major and minor bioactive zones containing masses consistent with three-finger toxins (3FTxs), whereas, viperid and colubrid venoms showed little or no detectable bioactivity. Our findings demonstrate that venom interactions with
AChBP correlate with the severity of neurotoxicity observed following human envenoming by different snake species. We further, as proof of principle, characterized bioactive venom peptides from a viperid (Daboia russelli) and an elapid (Aspidelaps scutatus scutatus) snake by nanoLC-MS/MS, revealing that different toxin classes interact with the
AChBP, and that this binding correlates with the inhibition of α7-nAChR in calcium-flux cell-based assays. The on-line post-column binding assay and subsequent toxin characterization methodologies described here provide a new in vitro analytic platform for rapidly investigating neurotoxic snake venom proteins.