Snake bites are a serious health problem in many tropical and subtropical regions [1]. For instance, approx. 15000–20000 people are estimated to die annually in India due to snake envenomation [2]. The development of novel medical therapies to treat venomous snake bites is of great importance, and biochemical characterization of snake venom is imperative, because the underlying treatable pathogenesis is dependent on the venom's composition. In addition, previous studies have shown that some components of snake venom have beneficial attributes in the treatment of various pathophysiological conditions. Enzymes from cobra venom show promise in the treatment and/or prevention of Parkinson's and Alzheimer's diseases [3,4], and the venom from snakes in the viper family has been shown to promote tumour reduction [5]. Hence the elucidation of specific proteomic profiles of snake venom could have vast implications for medicine.
Snake venom is composed mainly of proteins and peptides, which possess a variety of biological activities. Snake venom is broadly divided into three categories based on toxicity from envenomation. These categories are: (i) haemotoxins, which promote haemorrhaging primary to extensive local swelling and necrosis, (ii) neurotoxins, which disable muscle contraction and paralyse the heart as well as hinder respiration, and (iii) cardiotoxins, which elicit specific toxicity to cardiac and muscle cells, causing irreversible depolarization of cell membranes [6].
The cobra and viper are two of the world's most poisonous snakes and are indigenous to the countries of Asia and Oceania. Cobra venom is mainly categorized as a cytotoxin [7], whereas vipers employ mostly haemotoxic venom [8]. The specific composition of snake venom varies considerably from species to species [9]. This combination of toxins is the determinant of venom toxicity. Obviously, to elucidate the mechanism of venom toxicity, it is necessary to identify and characterize venomous proteins in an individual snake. The global analysis of snake venom presents a challenge for analytical techniques, which demands an approach that effectively separates a complex mixture of proteins and peptides with varying size and charge. Traditionally, venomous proteins and peptides are measured either by antibody recognition or by peptide N-terminal sequencing. Both methods are inadequate for global analysis of snake venom, therefore a new strategy must have the power to universally separate and identify the proteins and the peptides in snake venoms.
During the last several years, the field of proteomics has evolved considerably [10]. With the availability of genomic sequences, the power of 2DE (two-dimensional electrophoresis) and multiple-dimensional chromatographies to separate complex mixtures of proteins, advances in MS, and the development of computational methods, it is now possible to globally identify proteins expressed in a cell under a given set of conditions. Several reports have described the analysis of snake venom using proteomic strategies [11–15]. At the first Swiss Proteomics Society congress (SPS'01), a total of eight laboratory groups participated in an exercise examining protein identification using different mass spectrometric approaches [16]. One of the samples for the exercise was snake venom from the Brazilian snake, Bothrops jararaca, provided by Dr D. C. Pimenta (Institute Butantan, São Paulo, Brazil). A total of 12 different MS approaches and five different protein search engines were employed in these laboratories. Although databases for venom proteins and peptides are incomplete, the venomous toxins were identified successfully by all participants regardless of the methodology used to identify them. The results from this exercise revealed the scope and efficiency of the current proteomic techniques in identifying venom proteins.
We are interested in developing an approach to distinguish snake species based upon proteomic characterization of their venom. In the present study, we have chosen two snakes, a cobra and a viper, the two major species of snake found in southern China [17]. The present study utilized four different proteomic strategies. The first involves direct tryptic digestion, followed by HPLC coupled with ion-trap tandem MS (shotgun-LC-MS/MS). The second uses 1DE [one-dimensional gel electrophoresis (SDS/PAGE)] to separate venomous proteins, then LC-MS/MS for identification of the separated proteins (1DE-LC-MS/MS). The third applies GF (gel filtration) to separate venom proteins followed by 1DE-LC-MS/MS. The last includes two steps for protein separation, GF (gel filtration chromatography) and 2DE, followed by protein identification with MALDI–TOF (matrix-assisted laser-desorption ionization–time-of-flight) MS (2DE-MALDI–TOF-MS). Using these strategies, we report here for the first time the complete proteomic profiles of venom from two different species of snake, in which 124 and 74 proteins and peptides, in cobra and viper venom respectively, have been verified. It is not surprising that the proteomic analysis provides the solid data to support the early observations that cobra venom contains abundant cardio- and neuro-toxins, and viper venom has high concentrations of haemotoxins and metalloproteinases. Furthermore, proteomic profiles offer a clear feature of protein distribution in both snake venoms, which is important for our understanding of envenomation mechanisms. In addition, the modifications of venomous proteins have been demonstrated by high resolution of 2DE techniques.
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