Characterization of Toxins within Crude Venoms by Combined Use of Fourier Transform Mass Spectrometry and Cloning

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.