Characterization of toxins within crude venoms by combined use of Fourier transform mass spectrometry and cloning.
Résumé
The standard analytical procedure for screening the proteomic profile of a venom often relies on an appropriate combination of sample extraction, electrophoresis, reversed-phase high-performance liquid chromatography, mass spectrometry, and Edman degradation. We present in this study a new approach for venom screening based on Fourier transform mass spectrometry (FTMS) analysis directly on the crude venom. The venom chosen is a unique sample from Atractaspis irregularis, a species never studied at the molecular level previously. This snake belongs to the Atractaspidae family that is known to produce highly toxic venoms containing endothelin-like peptides called sarafotoxins (SRTXs). Nanoelectrospray-FTMS spectrum of the crude venom allowed the identification of 60 distinct compounds with molecular masses from 600 to 14,000 Da, which would have been impossible without the resolution of this kind of instrument. De novo sequencing within the entire venom confirmed the sequences of two new families of sarafotoxins, whose precursors had been cloned, and allowed the characterization of a third one. One particularly interesting point was that the propolypeptides appeared processed not in one unique compound, but rather in different length molecules ranging from 15 for the shorter to 30 amino acids for the longer. Moreover, our results clearly establish that in the case of A. irregularis only one copy of mature sarafotoxin emerges from each precursor, which is a totally different organization in comparison of other precursors of SRTXs.