Snake venoms
Venoms were obtained from wild Bothrops atrox specimens with no known litter relationships, captured in Manaus region (Amazonas State, Brazil), and maintained in the Herpetarium of the Gerência de Animais Peçonhentos-IMT-AM, Manaus. Classification of B. atrox snake specimens was done based on the total lenght of wild specimens according to Martins and Oliveira (1998) [26]. Therefore, three size classes were defined: juveniles (≤ 40 cm), sub-adults (> 40 – 70 cm), and adults (> 70 cm). Twenty days after the snakes were captured and before their first feeding in captivity, the venoms were manually milked by massaging the venom glands of specimens longer that 50 cm, and with the aid of a Pasteur pipette on snakes smaller than 50 cm. A single extraction was performed for each specimen. Three types of pooled venom samples were prepared, juvenile, comprising venoms of eight specimens, sub-adult, corresponding to twenty five specimens and adult, a mixture of venoms of twelve specimens. The pooled venom samples were centrifuged, filtered, lyophilized, weighted and stored at -20°C.
Two-dimensional gel electrophoresis
Freeze dried pooled venom samples (100 μg for silver stained gels and 300 μg for Coomassie blue stained gels) were solubilised in 370 μL of 2-DE sample buffer (7M urea, 2M thiourea, 1% DTT, 2% Triton X-100, 0.5% Pharmalyte 3–10) containing proteinase inhibitors (100 μM PMSF, 1 μM pepstatin A and 5 mM EDTA) and applied to 18 cm IPG gel strips (GE Healthcare, Upsala, Sweden) containing linear or non-linear 3–10 pH gradient by in-gel sample rehydration [27]. After 12 h of rehydration in the sample solution, isoelectric focusing (IEF) was carried out at 20°C using an IPGphor unit (GE Healthcare) in three successive steps: 500 Vh, 1000 Vh and 32000 Vh. For reduction and alkylation the IPG gel strips were soaked for 30 min in a solution containing 50 mM Tris pH 8.8, 6 M urea, 30% glycerol, 2% SDS and 125 mM DTT and for an additional 30 min in the same buffer containing 125 mM iodoacetamide and bromophenol blue instead of DTT. SDS-PAGE was performed on 10–20% T polyacrylamide gradient gels on a Protean II system (Bio Rad, Hercules, CA, USA) connected to a Multitemp II cooling bath (GE Healthcare). Electrophoresis was carried out at constant current (25 mAmp per gel) at 20°C until the dye front reached the lower end of the gel.
2-DE gels containing 100 μg of venom were silver stained [28] and submitted to image analysis. Alternatively, 2-DE gels containing 300 μg of venom were stained with 0.1% (w/v) Coomassie Blue in 40% (v/v) methanol, 10% (v/v) acetic acid and distained with the same solution without Coomassie Blue.
Image analysis
Silver stained gels were scanned with a SHARP JX-330 scanner (Tokyo, Japan) at 300 dpi resolution and the tiff images generated were analyzed with Image Master 2D Elite software (GE Healthcare). Spot detection and spot matching were performed in automated mode. Spot volumes were acquired without background subtraction and normalized using the total intensity of the detected spots.
Protein digestion
Coomassie stained spots were excised from the preparative gel and in-gel digested with trypsin as described in [15].
Peptide mass fingerprinting
Protein digests were first subjected to peptide mass fingerprinting by matrix-assisted laser desorption/ionization on a Bruker Reflex IV time-of-flight (MALDI-TOF) mass spectrometer equipped with Scout 384 ion source. Probes were prepared by dried-droplet method as described previously [29]. Briefly, 1 μL aliquot of the digest was mixed on the surface of AnchorChip™ 384/600 targets (Bruker Daltonics, Germany) with a saturated solution of matrix (α-cyano-4-hydroxycinnamic acid) in 1: 2 (v/v) solution of 2.5% aqueous TFA: acetonitrile. The mixture was allowed to dry at room temperature and the entire target was washed with 5 % formic acid.
Spots whose peptide mass fingerprints were similar, i.e. shared more than 75% of peaks with the relative intensity of more than 20 % within the mass accuracy of +/- 100 ppm, after removal of peaks of trypsin autolysis products and known keratin contaminants, were grouped as shown in Fig 1.
Sequencing by nanoelectrospray tandem mass spectrometry
If peptide mass fingerprinting did not identify the protein, peptides were extracted from the gel pieces with 5 % formic acid and acetonitrile and the extracts were pooled together and dried down in a vacuum centrifuge. The digests were taken up in 5 % formic acid and analyzed by nanoelectrospray tandem mass spectrometry on a hybrid quadrupole time-of-flight instrument QSTAR Pulsar i (MDS Sciex, Canada) as previously described [30,31].
Database searching
Peptide mass fingerprints were used for database searching using Mascot software (Matrix Science Ltd, UK) against the MSDB database (updated May 15, 2005; containing 2011425 protein sequence entries) downloaded from NCBI. Mass tolerance was set to 100 ppm, spectra were calibrated externally and no restrictions were imposed on protein molecular mass or phylogenetic lineage. Uninterpreted tandem mass spectra were first searched by Mascot against the above database to identify proteins with tryptic peptides identical to database entries. Precursor mass tolerance was set at 0.1 Da and fragment ion mass tolerance at 0.05 Da. Hits were considered significant if their protein score exceeded the threshold score calculated by Mascot software assuming p y-, b- and a- fragment ions [32] to corresponding m/z calculated from the peptide sequences. If still no identification was achieved, tandem mass spectra were interpreted de novo using BioAnalyst QS software as previously described [31]. Multiple sequence candidates were allowed per each interpreted tandem mass spectrum and partial peptide sequences were included into the search string. All candidate sequences were merged into a single search string and MS BLAST searches were performed against a non-redundant protein database (nrdb) at http://genetics.bwh.harvard.edu/msblast/webcite or http://dove.embl-heidelberg.de/Blast2/msblast.htmlwebcite under default settings. Parsing script operating at the MS BLAST web site was applied to identify and colour code statistically confident hits according to MS BLAST scoring scheme [20]. According to the selected MS BLAST threshold scores of statistical confidence, the expected rate of false positive identification was lower than 1 %.
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