Proteomic Analysis Of Snake Venom From G.intermedius

With the completion of Human Genome Project (HGP), Life Science has entered a new era—post genome time. One of the most remarkable aspects is the thriving of Proteomics. As the direct carrier of the function of genes, the expression profile of genes in specific cells, tissues or organs has made an appeal to many scientists, proteomics has become a hotspot for life sciences. Up to now, proteomics have been widely applied to the investigation of pathogenesis, the searching for tumor biomarkers and the mechanism of how drug works.Snake venom, which is secreted from the snake venom gland, is a complex mixture of many functional enzymes and peptides with pharmacological activities. Though it may paralyze and kill the preys, it is also a precious resource for developing new therapeutic agents. With the blooming of proteomics, and the relative simplicity of the composition of snake venoms, proteomic techniques cut a figure in snake venom investingation. In recent years, Venomic proteomics of nearly seventy species have been reported both from China and abroad, the species ranges from Colubridae, Elapidae to Viperadae (including Crotalini and Agkistrodontini). Also, many valuable proteins have been identified, such as snake venom metalloproteinases (SVMPs), serine proteases, neurotoxins, cardiotoxins, cysteine-rich secretory proteins (CRISP), nerve growth factors (NGF), L-amino acid oxidases, bradykinin potentiating peptide (BPP) and myotoxins. Panorama of these venoms would be helpful for the understanding of the venom evolution, envenomation treatment, and design of anti-venom serum and exploitation of new drugs.G. intermedius distributes widely in northwest China and part of north China, which is the most toxic pit-viper species among six pit-vipers (Genus Gloydius) in our country. At present, there have been reports concerning the snake-bite accidents in the area from Xinjiang, Qinghai and inner Mongolia. The morphology, herpetofauna, feeding and ecology, biochemistry and the toxicology of the venom have been studied and reported previously. However, we still lack a global research of this venom. These would hinder the effective envenomation administration, which is also become a bottleneck for exploitation and application of this resource. Based on these consideration, we plan to make a pilot study on the composition of G. Intermedius venom by using two-dimensional gel electrophoresis (2-DE) combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).The results are as following:1. The molecular weights of rough snake venom was under 94 kDa, mostly distributed within two areas, ie. 12～14 kDa and 30～67 kDa, indicating that the high abundance snake venom proteins located in these two areas. In addition, there were also several strips with mediate dense concentration between 45 kDa and 66 kDa.2. Analysis by MALDI-TOF MS of rough venom reveals sixteen groups of low molecular weight proteins, the Mr ranging from 1 kDa to 15 kDa.3. Separating by 2-DE and characterizing by MALDI-TOF MS, 22 proteins were identified. The pI range of these proteins ranged from 5.2 to 8.3, while the Mr ranged from 14 kDa to 60 kDa. These proteins were identified by searching through protein databases using MASCOT software. These proteins could be cataloged into six protein families, such asâ…¢metalloproteases, serine proteases, phospholipase A₂, L-amino acid oxidase, Cystein-rich secretory protain (CRISP) and nerve growth factor (NGF). Among them, acidic proteins taken the major place, in turn the neutral ones, basic ones rank as the minor.4. A few proteins could not be identified due to either the failure of receiving the peptide mass fingerprinting signal or the poor quality of the PMF obtained. This made the searching in protein database and identifying very difficult. A reliable matching result could not be achieved.The results from the present study show that the molecular weights of main proteins in G. Intermedius venom range from 14 kDa to 67 kDa. Of them, twenty two proteins have been successfully identifyied by using 2-DE combined with MALDI-TOF MS. These proteins were subordinated to six protein families, including metalloproteases, serine proteases, phospholipaseA₂, L-amino acid oxidases, cystein-rich secretory proteins (CRISP) and nerve growth factors. Their pIs ranged from 5.2 to 8.3, and Mr ranged from 14 kDa to 60 kDa. Among them, acidic proteins taken the major place, in turn the neutral ones, basic ones rank as the minor. Six kinds of proteins could not be identified due to either the failure of receiving the peptide mass fingerprinting signal or the poor quality of the PMF obtained. Therefore, a plenary proteomics still depends on other complementary mass spectrometric methods, and information from systematic researches, such as cDNA library. Meanwhile, further studies to characterize the function of proteins identified in the present study are also needed.