Experimental

- Proteomic characterization of two snake venoms: Naja naja atra and Agkistrodon halys

Sephadex G-50, Immobiline DryStrips, ampholytes, TEMED (N,N,N′,N′-tetramethylethylenediamine) and ammonium persulphate were purchased from Amersham Biosciences. Trypsin was from Promega. All other chemicals were from Merck. Deionized water was prepared with a tandem Milli-Q system and was used for the preparation of all buffers.

 

Venom from Naja naja atra (Naja) from the cobra family and Agkistrodon halys (Agki) from the viper family were chosen as subjects for this study. Both snakes were purchased from Zhejiang Yiwu Snake Research Institute, Yiwu County, Zhejiang Province, China. The average length of the snake was 50 cm with an approximate age of 30 months. Snake venom was freshly drawn during the spring season and was frozen immediately at −80 °C until use.

 

For shotgun or SDS/PAGE analysis, venom was diluted directly in trypsin digestion buffer or PBS. For 2DE analysis, venom proteins were precipitated by pre-cooled 10% TCA (trichloroacetic acid)/acetone for 2 h at −20 °C, followed by centrifugation at 13000 g for 30 min. The precipitated proteins were washed once with acetone and twice with ethanol/ether (1:1, v/v), and finally dried by SpeedVac.

 

Venom samples (20 μg of protein) were reduced and denatured in SDS loading buffer and boiled for 3 min. The venomous proteins were resolved via SDS/PAGE using 15% polyacrylamide gels and a Hoefer electrophoresis device. The separated proteins were visualized by staining with Coomassie Brilliant Blue. Each lane on the gel was evenly excised to ten gel slices and was subjected to a complete tryptic digestion.

 

The precipitated venomous proteins were dissolved in 450 μl of rehydration solution containing 8 M urea, 2% (w/v) CHAPS, 20 mM DTT (dithiothreitol), 0.5% (v/v) IPG buffer, 0.002% (w/v) Bromophenol Blue. Commercial 24 cm IPG strips with a linear range of pH 3–10 were rehydrated overnight with 450 μl of venomous protein solution. Electrofocusing was carried out at 64 kV·h using IPGphor at 20 °C following the manufacturer's instructions. Before the second dimension, the IPG strips were equilibrated by two equilibration steps: reduction buffer with 50 mM Tris/HCl, pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, a trace of Bromophenol Blue and 1% (w/v) DTT on a rocking table for 15 min; alkylation buffer with 50 mM Tris/HCl, pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, a trace of Bromophenol Blue and 2.5% (w/v) iodoacetamide for an additional 15 min. The electrophoresed strips were loaded and run on 10% and 15% polyacrylamide Laemmli gels (26 cm×20 cm) for Naja and Agki respectively, using the Ettan DALT II system with a programmable power control, for 0.5 h at 0.5 W per gel, then at 15 W per gel until the dye front reached the bottom of the gel. The separated proteins were visualized by Coomassie Brilliant Blue staining.

 

Venom samples (100 mg) were loaded on to a 8.0 mm×250 mm Sephadex G-50 column pre-equilibrated with 10 mM Tris/HCl, pH 7.4, 10 mM DTT and 2 mM EDTA. Elution of venom was carried out using an equilibration buffer with a flow rate of 1.0 ml/min at room temperature (25 °C). Protein elution was monitored at 280 nm. The eluted fractions were collected at 200 μl/tube, and the fractions were analysed further by SDS/PAGE to check separation efficiency. Based upon results from SDS/PAGE, the fractions containing venomous proteins with a MM (molecular mass) less than 10 kDa were pooled for direct tryptic digestion, and the remaining fractions were pooled for 2DE analysis.

 

For direct digestion, venom samples or the pools from GF were diluted in a digestion solution containing 2 M urea, 50 mM ammonium bicarbonate and 1 mM CaCl₂. The tryptic digestion was carried out by the addition of the modified trypsin to a final substrate/trypsin ratio of 40:1 (w/w) and by incubation at 37 °C for 12 h. The enzymic digestion was stopped by acidification using 0.1% methanoic (formic) acid.

For in-gel digestion, gel slices from SDS/PAGE or gel spots from 2DE were carefully excised, and successively destained and dehydrated with 50% acetonitrile. The proteins were reduced with 10 mM DTT at 56 °C for 1 h and alkylated by 55 mM iodoacetamide in the dark at room temperature for 45 min in situ. Finally, the gel slices or spots were thoroughly washed with 25 mM ammonium bicarbonate in water/acetonitrile (1:1, v/v) solution and were completely dried in a SpeedVac. Proteins were digested in 25 μl of modified trypsin solution (10 ng/μl in 25 mM ammonium bicarbonate) by incubation overnight at 37 °C. The peptides were released with vigorous shaking and were extracted in 50 μl of 50% acetonitrile [containing 2.5% TFA (trifluoroacetic acid)].

Digested peptides were separated on reverse-phase (C₁₈) capillary columns [5 μm, 300 Å (1 Å=0.1 nm) particles, 0.15 mm×100 mm or 0.15 mm×150 mm; MicroTech] using an Agilent 1100 Capillary LC system. Flow was maintained at 100 μl/min, and elutants were analysed with a diode array detector. HPLC buffer A composed of 0.1% methanoic acid in water, and buffer B composed of 0.1% methanoic acid in acetonitrile, were used for peptide elution. A linear gradient of 2–80% buffer B was employed, but at different gradient periods, 360 min for separation of the shotgun digestion of total snake venom, and 70 min for the peptides generated from in-gel digestions.

 

Most digested peptides, obtained from shotgun digestion, the digestion of gel slices from SDS/PAGE, or digestion of pooled fractions (MMm/z range 200–1800 was scanned in 1.2 s, and the fragment amplitude was set at 1.15 V. The MSD ion-trap mass spectrometer was operated in a data-dependent mode for MS/MS for the most abundant ions.

For the digested peptides from 2DE, the samples were mainly analysed by MALDI–TOF MS. The digestions were mixed with a matrix solution consisting of α-cyano-4-hydroxycinnamic acid (12 mg/ml) in 50% acetonitrile with 0.1% TFA at ratio of 1:1. The suspensions were applied on to the target well, dried at room temperature and analysed by a Bruker AutoFlex MALDI–TOF MS. The mass spectrometer was operated under 19 kV accelerating voltage in the reflectron mode with a m/z range 600–4000. Some digestive products from 2DE spots were analysed with LC-MS/MS for further confirmation of amino acid sequence.

The ion spectra of peptides generated by MSD trap and mono-isotopic peptide masses obtained from MALDI–TOF MS were interpreted by utilizing the Mascot search engine (http://www.matrixscience.com/search_form_select.html; Matrix Science). The proposed peptide sequences were compared with the non-redundant databases of snake venomous proteins generated from data compiled at the NCBI (National Center for Biotechnology Information) and the EBI (European Bioinformatics Institute).