Abstract
Snake venom proteomes/peptidomes are highly complex and maintenance of their integrity within the gland lumen is crucial for the expression of toxin activities. There has been considerable progress in the field of venom proteomics, however, peptidomics does not progress as fast, because of the lack of comprehensive venom sequence databases for analysis of MS data. Therefore, in many cases venom peptides have to be sequenced manually by MS/MS analysis or Edman degradation. This is critical for rare snake species, as is the case of Bothrops cotiara (BC) and B. fonsecai (BF), which are regarded as near threatened with extinction. In this study we conducted a comprehensive analysis of the venom peptidomes of BC, BF, and B. jararaca (BJ) using a combination of solid-phase extraction and reversed-phase HPLC to fractionate the peptides, followed by nano-liquid chromatography-tandem MS (LC-MS/MS) or direct infusion electrospray ionization-(ESI)-MS/MS or MALDI-MS/MS analyses. We detected marked differences in the venom peptidomes and identified peptides ranging from 7 to 39 residues in length by de novo sequencing. Forty-four unique sequences were manually identified, out of which 30 are new peptides, including 17 bradykinin-potentiating peptides, three poly-histidine-poly-glycine peptides and interestingly, 10 L-amino acid oxidase fragments. Some of the new bradykinin-potentiating peptides display significant bradykinin potentiating activity. Automated database search revealed fragments from several toxins in the peptidomes, mainly from l-amino acid oxidase, and allowed the determination of the peptide bond specificity of proteinases and amino acid occurrences for the P4-P4' sites. We also demonstrate that the venom lyophilization/resolubilization process greatly increases the complexity of the peptidome because of the imbalance caused to the venom proteome and the consequent activity of proteinases on venom components. The use of proteinase inhibitors clearly showed different outcomes in the peptidome characterization and suggested that degradomic-peptidomic analysis of snake venoms is highly sensitive to the conditions of sampling procedures.
Highlights
Snake venoms are the products of specialized secretory glands located above the upper jawbone in venomous snakes
Bothrops cotiara (BC), B. fonsecai (BF), and B. jararaca (BJ) venoms showed clearly distinct RPHPLC profiles (Fig. 1) and no peptide was simultaneously found by de novo sequencing in the three venom species (Table I)
Other LAAO fragments have been found in the venoms of BC, BF and BJ (Table I), as previously described in the venoms of B. moojeni [31, 57] and B. insularis [58]
Summary
Venoms—Lyophilized venom from BC, BF and B. jararaca (BJ) was from Instituto Butantan (Sao Paulo, Brazil). Venom solutions were centrifuged at 10,000 ϫ g for 10 min at room temperature and fractionated in Sep-pak C18 cartridges previously conditioned with methanol and 0.1% TFA (Waters, Milford, MA) according to Menin et al [31]. De novo Peptide Sequencing from MALDI Q-TOF MS/MS—The RP-HPLC fractions were dissolved in 20 l water/ACN/formic acid (49.8/49.8/0.5) and 1.5 l were spotted onto the MALDI sample plate, dried and mixed with 1.5 l of a saturated solution of ␣-cyano-4hydroxycinnamic acid (Sigma, St. Louis, MO) in 0.1% TFA in H20/ACN (70:30). De novo Peptide Sequencing from ESI Q-TOF MS/MS—The RPHPLC fractions were dissolved in 20 l water/ACN/formic acid (49.8/49.8/0.5) and directly infused by a syringe pump at 500 nL/ min in the Q-TOF Ultima API (Waters) with a nano-ESI ionization source in the positive ionization mode. All assays were performed in triplicate under conditions such that the results varied Ͻ 10%
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