AIMMS Division of BioMolecular Analysis, Faculty of Sciences, VU University Amsterdam, De Boelelaan 1081, 1083 HV Amsterdam, The Netherlands.
Department of Biophysical Chemistry, Institute for Molecules and Materials, Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands.
Biology (Basel). 2014 Feb 13;3(1):139-56. doi: 10.3390/biology3010139.
A nano-flow high-resolution screening platform, featuring a parallel chip-based microfluidic bioassay and mass spectrometry coupled to nano-liquid chromatography, was applied to screen animal venoms for nicotinic acetylcholine receptor like (nAChR) affinity by using the acetylcholine binding protein, a mimic of the nAChR. The potential of this microfluidic platform is demonstrated by profiling the Conus textile venom proteome, consisting of over 1,000 peptides. Within one analysis (<90 min, 500 ng venom injected), ligands are detected and identified. To show applicability for non-peptides, small molecular ligands such as steroidal ligands were identified in skin secretions from two toad species (Bufo alvarius and Bufo marinus). Bioactives from the toad samples were subsequently isolated by MS-guided fractionation. The fractions analyzed by NMR and a radioligand binding assay with α7-nAChR confirmed the identity and bioactivity of several new ligands.
一种纳米流高分辨率筛选平台，采用基于芯片的平行微流控生物测定和质谱与纳流液相色谱相结合的方法，利用乙酰胆碱结合蛋白（nAChR 的模拟物）筛选动物毒液对烟碱型乙酰胆碱受体样（nAChR）的亲和力。该微流控平台的潜力通过对超过 1000 种肽的 Conus 纺织毒液蛋白质组进行分析得到证明。在一次分析中（<90 分钟，500 纳克毒液注入），可以检测和鉴定配体。为了展示对非肽的适用性，从两种蟾蜍物种（Bufo alvarius 和 Bufo marinus）的皮肤分泌物中鉴定出了甾体配体等小分子配体。随后通过 MS 引导的分级分离从蟾蜍样本中分离出生物活性物质。通过 NMR 分析和与α7-nAChR 的放射性配体结合测定对经分析的馏分进行了分析，证实了几种新配体的身份和生物活性。
