Detection and identification of huwentoxin-IV interacting proteins by biotin-avidin chemistry combined with mass spectrometry

Journal of venomous animals and toxins

Hai Yu¹  Yizhong Yan¹  Xianchun Wang¹  Hui Liu²  Zhigui Duan² 

[1] Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education, College of Life Sciences, Hunan Normal University, Changsha, People's Republic of China

关键词: Biotin labeling;    Huwentoxin-IV;    Far-western blotting;    Interacting protein;    Mass spectrometry;   

DOI  :  10.1186/1678-9199-20-18

学科分类：药理学

来源: BioMed Central

【 摘 要 】

Numerous spider toxins are of interest as tools for neurophysiological research or as lead molecules for the development of pharmaceuticals and insecticides. Direct detection and identification of the interacting proteins of a spider toxin are helpful for its action-mechanism analysis and practical application. The present study employed a combinative strategy for the analysis of interacting proteins of huwentoxin-IV (HWTX-IV), a peptidic neurotoxin from the venom of the spider Selenocosmia huwena. HWTX-IV was first lightly labeled with biotin under the optimized mild experimental conditions and the toxin labeled with a single biotin group (monobiotinylated HWTX-IV) was demonstrated by electrophysiological experiments to retain its original bioactivity and was used in combination with far-western blotting to detect its interacting proteins. Comparative experiments indicated that some membrane proteins from rat neuromuscular junction preparations bind to monobiotinylated HWTX-IV after being transferred onto a PVDF membrane from the SDS-gel. With capillary high performance liquid chromatography-tandem mass spectrometry, several membrane proteins with which HWTX-IV potentially interacted were identified from the preparations and then bioinformatically analyzed. This work has provided not only a new insight into the action mechanism of HWTX-IV but also a reference technology for the relevant researches.

【 授权许可】

CC BY