Proteomic characterization of six Taiwanese snake venoms: Identification of species-specific proteins and development of a SISCAPA-MRM assay for cobra venom factors

摘要

Deinagkistrodon acutus,Trimeresurus stejnegeri,Protobothrops mucrosquamatus,Daboia russelii siamensis,Bungarus multicinctusandNaja atraare the six medically important venomous snake species in Taiwan. In this study, we characterized and compared their venom protein profiles using proteomic approaches. The major snake venom proteins were identified by GeLC-MS/MS and the total venom proteome was characterized by in-solution digestion coupled with LC-MS/MS. A total of 27–52 proteins, categorized into 23 protein families, were identified in each snake's venom. The major venom components found inViperidaespecies (D. acutus,T. stejnegeri,P. mucrosquamatusandD. russelii) were C-type lectin, snake venom serine proteinase, venom metalloproteinase and phospholipase A2, whereas three-finger toxin and phospholipase A2were the major components detected in the venom ofElapidaesnakes (B. multicinctusandN. atra). This study also provided the first demonstration of some low-abundance proteins in these six snake venoms, including 5′-nucleotidase, glutaminyl-peptide cyclotransferase and phosphodiesterase, among others. Furthermore, we found that cobra venom factor (CVF) is a cobra-specific protein. We produced anti-peptide antibodies against CVF and used it to develop a highly sensitive SISCAPA-MRM assay for quantifying CVF. The limit of detection and lower limit of quantification were 3.2 and 9.6 attomoles, respectively. This assay was used to precisely quantify CVF in 1?μg crude venom proteins from threeNajaspecies and king cobra. The amount of CVF varied from 0.9 to 54.36 femtomoles (equivalent to 0.16–10.03?mg/g of venom protein). Biological significanceThere are six medically significant venomous snakes in Taiwan. The venoms of the fourViperidaespecies (Deinagkistrodon acutus,Trimeresurus stejnegeri,Protobothrops mucrosquamatusandDaboia russelii siamensis) cause local tissue swelling; this symptom is also seen inN. atraenvenomation in humans, potentially complicating the differential diagnosis of envenomation byN. atraandViperidaespecies. Thus, characterization of the venom proteomes of the six Taiwanese snakes, including the relative abundance of the major components and species-specific protein(s) in each venom type, could be useful for future venom research, including the development of new assay(s) for detecting snake species-specific venom protein(s) and new type(s) of antivenom.