Vejayan et al. Journal of Venomous Animals and Toxins including Tropical Diseases 2014, 20:6 http://www.jvat.org/content/20/1/6 RESEARCH Open Access Comparative analysis of the venom proteome of four important Malaysian snake species Jaya Vejayan1*, Too Lay Khoon2 and Halijah Ibrahim2 Abstract Background: Naja kaouthia, Ophiophagus hannah, Bungarus fasciatus and Calloselasma rhodostoma are four venomous snakes indigenous to Malaysia. In the present study, their proteomic profile by two-dimensional gel electrophoresis (2-DE) have been separated and compared. Results: The 2-DE of venoms of the four species snake demonstrated complexity and obvious interspecies differences in proteome profiles. A total of 63 proteins were identified in the four species: C. rhodostoma – 26, N. kaouthia – 16, O. hannah – 15 and B. fasciatus – 6. Conclusions: Despite the identifications of major proteins in the four snake species, a large number of protein spots from the 2-DE were unidentified even though the spots displayed high-quality MALDI-TOF-MS spectra. Those identified included phospholipase A2 proteins in all four venoms, long neurotoxins in both cobra species and the C. rhodostoma venom found with the most varied types of peptidases, i.e. metalloproteinase kistomin, halystase and L-amino acid oxidase. Keywords: Mass spectrometry, Snake venoms, Proteomics, Two-dimensional gel electrophoresis Background previously known as Agkistrodon rhodostoma and Ancis- In Malaysia, more specifically Peninsular Malaysia, a very trodon rhodostoma), is rich in peptidases that exhibit rich snake fauna is present, consisting of approximately hemorrhagic activities and is capable of affecting blood co- 141 known species of land and sea snakes. Of these, only agulation, leading to a hemotoxic effect [4]. 16 species of land snakes and all 21 sea snake species are Since the early biochemical characterization of venomous [1]. This study focused on four of these venom- venoms, scientists have been unravelling the biological ous species, namely Naja kaouthia, Ophiophagus hannah, and pathological significance of their proteins and pep- Bungarus fasciatus and Calloselasma rhodostoma.The tides. The advances in mass spectrometry (MS) for pro- first three are of the Elapidae family while C. rhodostoma tein identification have revolutionized snake venom belongs to the Viperidae family. The venoms of N. proteomics, shedding light into the global complexity of kaouthia (monocled cobra, 1.5-2.0 meters long), O. han- venom proteomes and, subsequently, their pathological nah (king cobra, 3 to 4 meters long) and B. fasciatus activities. Using a multitude of proteomic approaches, (banded kraits, 1.6 meters long) are comprised mainly of venom proteomes have been analyzed in 55 snake gen- neurotoxins [2,3]. Other potent basic polypeptides – such era [3]. The application of proteomics, including the use as cardiotoxin, cytotoxin and cobramines – are also found of two-dimensional gel electrophoresis (2-DE) and MS abundantly in the venoms of elapids. The short-tempered, in studying venoms, has attracted considerable attention quick-to-attack cobra is one of the most frightening; while from researchers. The effective use of the 2-DE tech- kraits, though much more subdued, are also highly feared nique, coupled with MS, was first demonstrated in a for their toxic, frequently death-causing bites. The venom study conducted by Fox and colleagues [5] in their com- of C. rhodostoma (Malayan pit viper, 0.6-1 meters long, parative analysis of the venoms of Dispholidus typus, Crotalus atrox and Bothrops jararaca. The correlation * Correspondence: [email protected] between genetic, ecological and phylogeny factors with 1 Faculty of Industrial Sciences and Technology, University Malaysia Pahang, intraspecific variations in the composition of the Trimer- Lebuhraya Tun Razak, Kuantan, Pahang Darul Makmur 26300, Malaysia Full list of author information is available at the end of the article esurus stejnegeri venom was investigated by Creer and © 2014 Vejayan et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Vejayan et al. Journal of Venomous Animals and Toxins including Tropical Diseases 2014, 20:6 Page 2 of 9 http://www.jvat.org/content/20/1/6 colleagues [6] by employing matrix-assisted laser desorp- rehydration, the IPG strips were introduced with the ven- tion ionization-time-of-flight (MALDI-TOF)/MS and omous proteins (100 μg for silver staining and 300 μgfor isoelectric-focusing (IEF) technologies. To further assess Coomassie blue staining) via a sample-loading cup. Prior the extreme complexity of natural venoms, Li et al. [2] to this the venomous proteins had been dissolved in assessed the global venom proteomics profiles of Naja 100 μL of rehydration solution containing 8 M urea, 2% naja atra and Agkistrodon halys by a combination of (w/v) CHAPS, 20 mM DTT (dithiothreitol), 0.5% (v/v) four different approaches. Nawarak et al. [7], on the IPG buffer, 0.002% (w/v) Bromophenol blue. Electrofocus- other hand, used 2-DE and MALDI-TOF MS to identify ing was carried out at 30 kVh using IPGphor (GE Health- moderate- to high-molecular-mass glycoproteins in N. care) at 20°C according to the manufacturer’s instruction. kaouthia venom, which had been previously fractionated Before the second dimensional electrophoresis, the IPG by binding with concanavalin A. These approaches have strips were equilibrated by two equilibration steps: reduc- also been utilized for the characterization of novel pro- tion buffer with 50 mM Tris/HCL, pH 8.8, 6 M urea, 30% teins that are yet to be inserted into protein databases. (v/v) glycerol, 2% (w/v) SDS, a trace of Bromophenol blue For instance, MALDI-TOF MS has been employed to and 1% (w/v) DTT on a rocking table for ten minutes; al- determine the molecular mass of purified proteins while kylation buffer with 50 mM Tris/HCL, pH 8.8, 6 M urea, 2-DE has been used to ascertain both the molecular 30% (v/v) glycerol, 2% (w/v) SDS, a trace of Bromophenol weights as well as pI values of isolated proteins [8-10]. blue and 2.5% (w/v) iodoacetamide for an additional ten Numerous additional investigations into venom pro- minutes. The equilibrated strips were loaded and run on teomes and subproteomes, using a wide array of prote- 15% polyacrylamide Laemmli gels (26 cm × 20 cm) omic strategies, have provided novel insights into venom using the Ettan Dalt II system (GE Healthcare) with a contents, their biological activities and the evolutionary programmable power control, initially 0.5 W per gel for relationships among snakes [3]. Nevertheless, as experi- 40 minutes, followed by 15 W per gel till the dye front enced by Li et al. [2], only 50% of the spots were con- reached the bottom of the gel. The separated gel proteins firmed to be venom proteins although approximately were visualized by Coomassie brilliant blue. 80% of the gel spots from 2-DE displayed high-quality MALDI-TOF MS spectra. Scarcity of venom sequence MALDI-TOF MS databases for the analysis of MS data has posed a chal- The Coomassie-stained gels were scanned using the Image lenge to all snake venom proteomic studies. Proteomics Scanner (Amersham Biosciences Limited, Sweden) and the tools provide enormous versatility in diverse applica- spots were detected, edited and annotated with ImageMas- W tions, ranging from unravelling the complexity of varies ter 2D Platinum software (Amersham Biosciences Lim- venoms to potentially identifying the minute differences ited, Sweden). The stained spots were selected, excised and between very closely related organisms [11]. The current dehydrated with 50 μL of acetonitrile for 15 minutes. To re- study aims to further underline the importance and hydrate, the supernatant was then replaced with 25 μLof challenges of proteomics in the study of snake venom by 25 mM NH4HCO3 for ten minutes. These steps of rehydra- profiling the venom of four snake species indigenous to tion followed by dehydration were repeated to give a total Malaysia. of three washes. Finally, the spots were dried for five mi- W nutes using a centrifugal evaporator (SpeedVac ,Thermo Methods Scientific, USA). Snake venoms All venoms used were from common venomous snakes In-Gel tryptic digestion in Malaysia, obtained from a local vendor, Bukit Bintang The dried spots were then re-suspended in 10 μLof Enterprise Sdn Bhd. The venoms were freeze-dried and 10 ng/μL trypsin in 25 mM NH4HCO3 and incubated stored at –20°C. for complete digestion at 37°C overnight. On comple- tion, the gel was once again dehydrated with the Protein content determination addition of acetonitrile. Finally, the gel piece was re- The protein content in the four venoms was estimated moved and the supernatant was dried down in the W using the dye-binding technique of Bradford [12] with SpeedVac for 20 minutes. bovine serum albumin (BSA) at 2.0 mg/mL concentra- tion, purchased from Thermo Scientific. Sample preparation for MS An equal amount of tryptic-digested samples was mixed Two-dimensional Gel electrophoresis (2-DE) together with a matrix solution consisting of α-cyano-4- Eighteen-centimeter IPG strips (GE Healthcare, Sweden) hydroxy cinnamic acid in acetonitrile acidified with tri- with a linear pH range of 3 to 10 were rehydrated over- fluroacetic acid (1 mg/mL). Thereafter, 0.4 μL of this night with 340 μL of rehydration solution. After mixture was spotted on the slide and air-dried. Vejayan et al. Journal of Venomous Animals and Toxins including Tropical Diseases 2014, 20:6 Page 3 of 9 http://www.jvat.org/content/20/1/6 Mass spectrometry snake venoms from Elapidae families using multidimen- MALDI-TOF mass spectra of peptide mixture were ob- sional chromatographic approaches.