Evolution has presented Nature with a tremendously diverse number of proteins and peptides in snake venom with equally diverse biological functions. With traditional analysis of snake venom, the primary focus has been on understanding the structural as well as functional aspects of individual proteins and peptides [18]. These studies have been successful in providing important information regarding specific components of venom as well as venom-induced pathologies. Nevertheless, these studies fall short in global identification of venom proteins, which is necessary for our understanding of the complexity of venom toxicity as well as elucidating the major biochemical differences among snake families. More than 40 years ago, researchers conducted electrophoresis experiments to globally isolate venom proteins [19]. Interestingly, what they noticed with starch electrophoresis was very similar to our proteomic observations: that on 2DE, Naja venom proteins appeared to be basic and Agki venom was mainly composed of acidic proteins. The limitations that existed at the time precluded more detailed analysis that is now possible with current technology. Advances in proteomic techniques have given researchers an edge in protein identification and characterization. Recently these methods have been used in several venom studies. Rioux et al. [11] separated the proteomes from Laticauda colubrine and Vipera russelli with 2DE and analysed the venom proteins by microsequencing of their N-terminal sequences and internal fragments. Using LC-MS, Fry et al. [14] measured the peptides from eight snake venoms and claimed that a hitherto unsuspected diversity of toxins was present in all lineages. Nawarak et al. [15] delineated the proteomes of eight snake venoms from elapidae and viperidae using multidimensional chromatographic approaches. The venom proteins from the elapidae family displayed similar distribution patterns on 2DE, whereby most protein spots had high pIs and low MMs, and the proteins obtained from the viperidae family were found in the neutral pH range with MMs of 30–45 kDa. Obviously, these investigations have revealed substantial information for the studies of venomous proteins; however, these data are short in convincingly demonstrating a complete proteomic profile for any species of snake venom. To our knowledge, this study is the first to document a complete proteomic profile of snake venom. Since the proteins and peptides in snake venom span a wide range of molecular size as well as biochemical properties, proteomic analysis with a single approach could certainly result in loss of proteins in separation as well as identification.
In spite of great advancements in recent years, proteomic analysis is still considered to be a prototype technique. To date, there is no unique method that can universally be utilized for all proteomic analyses [20]. In the present study, we have employed a combination strategy to profile the venom proteomes. This strategy includes three different combinations: (i) separation of proteins using HPLC and electrophoresis, (ii) digestion of proteins with trypsin, in-gel or aqueous, and (iii) identification of protein by ion-trap MS, tandem MS and MALDI–TOF MS. Using these combinations, it is reasonable that a comprehensive profile for venom proteomes can be established. In Naja venom, 31 proteins were detected by all of the proteomic approaches, whereas only 14 Agki proteins were found in the same way. There are a few proteins uniquely identified by a single approach. For instance, in Naja venom, 13 proteins were only detected by shotgun-LC-MS/MS, two by 1DE-LC-MS/MS and eight by GF-LC-MS/MS; in Agki venom, none was detected only by shotgun-LC-MS/MS, 15 by 1DE-LC-MS/MS and 12 by GF-LC-MS/MS. Moreover, the detection coverage of venomous proteins is compensated by each approach. GF-LC-MS/MS played well in the detection of cardiotoxins, but missed the signals of phospholipase A2, and shotgun-LC-MS/MS and 1DE-LC-MS/MS performed sensitive identification for other large sizes of proteins. In the present study, most identified proteins were identified by at least two approaches; thus the combination strategy for proteomic analysis not only enhances the detection rate, but also ensures the accuracy of the measurements.
To generate a database of snake venom proteins, we collected all snake-venom-related data from NCBI and EBI. Interestingly, protein searches based upon mass signals from either MALDI–TOF-MS or LC-MS/MS consistently retrieve species-specific information. As summarized in Table 2, 91.9±6.1% of identified proteins from Naja (except for 2DE-MALDI–TOF-MS data) belong to the cobra species and 95.1±2.8% of the Agki proteins are from the viper species. For instance, phospholipase A2 is commonly present in both venoms. Verifying biological sources, 96% of the identified phospholipase A2 from Agki venom in the present study matches well with the enzymes from vipers listed in the database, and 94% of the proteins in Naja venom closely matched cobra proteins. These results demonstrate that the accuracy of protein identification based upon mass signals from the digestive peptides is robust and accurate. Furthermore, it is worthwhile to point out that proteomic analysis provides a set of fundamental data, such as how many proteins can be expressed in a certain species or a specific period; however, it does not answer all of the questions related to venom proteins, especially for their biological reactivity. For example, four functional domains, preprosequence, metalloproteinase, disintegrin and cysteine-rich, may exist in snake venom metalloproteinase [21]. Herein the proteomic analysis has identified the peptide fragments from preprosequence, metalloproteinase and disintegrin by MS. Nevertheless, these data cannot give a certain answer as to whether all of the domains are in one compact molecule or are in free forms.
On 2DE images (Figure 4), totals of 190 and 169 protein spots are detected by Coomassie Blue staining from Naja or Agki respectively. Approximately half of these spots, 54% for Naja and 50% for Agki, were identified as venom proteins by MALDI–TOF-MS. However, the unique proteins are only 16 from Naja and 15 from Agki venom. Therefore it is reasonable to assume that these proteins must undergo a series of post-translational modifications in the snake venom gland. Comparing the apparent mass sizes of these modified proteins with the theoretically predicted values, 24% (24/100) of Naja proteins and 61% (52/85) of Agki proteins appear to be significantly reduced in size. This is logical due to the fact that Agki venom contains abundant proteinases, both metalloproteinases and serine proteases. In contrast with Agki, Naja has fewer proteases which may account for fewer degraded protein products. Interestingly, several venom proteins displayed significant increases in their apparent MMs. For instance, 27 gel spots, which represent almost 14% of the detected spots on 2DE from Naja venom, were confirmed to be the same protein, HMK (haemorrhagic metalloproteinase kaouthiagin; GI number 32469675), with a theoretical MM of 44 kDa. The image analysis revealed that 89% (24 out of 27) of this protein appeared to have higher MMs, ranging from 55 to more than 100 kDa on 2DE. Moreover, the similar conclusion was drawn from 1DE-LC-MS/MS data. Not only HMK, but also several venomous proteins, natrin (GI number 32492059), actin (GI number 113307), 5′-END (GI number 11024643), and nerve growth factor β (GI number 11275218) in Naja, and halysetin metalloproteinase (GI number 15076875), coagulation factor IX-binding protein (GI number 33638229), phospholipase A2 (GI number 27151651) and L-amino acid oxidase (GI number 15887054) in Agki, have been observed to significantly increase the apparent masses. Obviously, modifications of venomous proteins are a common phenomenon in snake venoms. Of a number of mechanisms inducing chemical modification of protein, glycosylation is one of the most probable causes. Upon HMK amino acid sequence analysis by NetOglyc 3.0 and NetNGlyc 1.0 [22,23], one N-glycosylation site (112–115) is predicted, which just matches the observation of Ito et al. [24] that native HMK treated with peptide N-glycosidase resulted in the apparent MM decreasing. According to glycosylation prediction, all of the proteins with increased apparent masses on 2DE contain at least one N-glycosylation consensus sequence. However, further biochemical experiments must be conducted to confirm the predictions. By searching the literature, L-amino acid oxidase [25], phospholipase A2 [26], coagulation factor IX [27] and other snake venom proteins [28] have been reported as having glycosylation modifications in venom.
In conclusion, by combining four different proteomic approaches, more than 100 proteins or peptides have been identified in snake venoms of Naja and Agki. This comprehensive proteomic analysis reveals global expression of venomous proteins from Naja and Agki, defines the fundamentally differential proteomes from the two species and demonstrates a series of chemical modifications in the venomous proteins. These data certainly contribute to the current knowledge of venom proteins, and will benefit the studies on the mechanisms of snake envenomation.
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