Analytical workflow for rapid screening and purification of bioactives from venom proteomes

被引:15
作者
Otvos, Reka A. [1 ,2 ]
Heus, Ferry [1 ]
Vonk, Freek J. [3 ,5 ,6 ]
Halff, Jenny [1 ]
Bruyneel, Ben [1 ]
Paliukhovich, Iryna [2 ]
Smit, August B. [2 ]
Niessen, Wilfried M. A. [1 ,4 ]
Kool, Jeroen [1 ]
机构
[1] Amsterdam VU Univ, Fac Sci, AIMMS Div BioMol Anal, NL-1083 HV Amsterdam, Netherlands
[2] Vrije Univ Amsterdam, Ctr Neurogen & Cognit Res, Dept Mol & Cellular Neurobiol, NL-1081 HV Amsterdam, Netherlands
[3] Leiden Univ, Inst Biol, Sylvius Lab, NL-2333 BE Leiden, Netherlands
[4] Hyphen MassSpec, NL-2332 XT Leiden, Netherlands
[5] Univ Wales, Sch Biol Sci, Bangor L57 2UW, Gwynedd, Wales
[6] Naturalis Biodivers Ctr, NL-2333 CR Leiden, Netherlands
关键词
On-line microfluidics; Nano-liquid chromatography-mass; spectrometry (nano-LC-MS); Snake venom proteome screening; Venom purification; Lymnaea stagnalis acetylcholine; binding protein (Ls-AChBP); Nicotinic acetylcholine receptor; ACETYLCHOLINE-BINDING-PROTEIN; CRYSTAL-STRUCTURE; ALPHA-NEUROTOXIN; BIOPHARMACEUTICALS; COMPLEX; IMMUNOGENICITY; RECEPTORS; DELIVERY; ASSAY; ACHBP;
D O I
10.1016/j.toxicon.2013.10.013
中图分类号
R9 [药学];
学科分类号
1007 ;
摘要
Animal venoms are important sources for finding new pharmaceutical lead molecules. We used an analytical platform for initial rapid screening and identification of bioactive compounds from these venoms followed by fast and straightforward LC-MS only guided purification to obtain bioactives for further chemical and biological studies. The analytical platform consists of a nano-LC separation coupled post-column to high-resolution mass spectrometry and parallel on-line bioaffinity profiling for the acetylcholine binding protein (AChBP) in a chip based fluorescent enhancement based bioassay. AChBP is a stable structural homologue of the extracellular ligand binding domain of the alpha 7-nicotinic acetylcholine receptor (alpha 7-nAChR). This receptor is an extensively studied medicinal target, previously associated with epilepsy, Alzheimer's, schizophrenia and anxiety. The workflow is demonstrated with the venom of the Naja mossambica mossambica. Two medium affinity AChBP ligands were found. After subsequent LC-MS guided purification of the respective venom peptides, the purified peptides were sequenced and confirmed as Cytotoxin 1 and 2. These peptides were not reported before to have affinity for the AChBP. The purified peptides can be used for further biological studies. (C) 2013 Elsevier Ltd. All rights reserved.
引用
收藏
页码:270 / 281
页数:12
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