Poster Presentation 10th Australian Peptide Conference 2013

ISD high-throughput platform for the sequencing of animal peptide toxins  (#113)

Loic Quinton 1 , Michel Degueldre 1 , Eric Opsomer 1 , Nicolas Vandewalle 1 , Edwin De Pauw
  1. University of Liege, Liege, Belgium

Animal venoms are complex mixtures, comprising a wide range of biologically active reticulated peptides that target with high selectivity and efficacy a variety of membrane receptors. Assuming the fact that each of the 170,000 venomous species reported can produce more than 250 bioactive toxins, at least 40,000,000 bioactive peptides/proteins may be discovered. Following this idea, a FP7 European Project called VENOMICS (http://venomics.eu/) was proposed in November 2011. Its goal is to construct a bank of 10,000 toxin sequences generated from the study of 200 selected venomous species. This bank will be exploited to discover new peptides that can potentially be used as human drugs. High-throughput toxin sequencing represents one of the main bottlenecks of the project.

This work shows the strategy designed to generate high-throughput accurate sequence tags from crude venoms. The sequencing strategy rests on a chromatographic purification of the venoms followed by MALDI-In-Source Decay fragmentation. ISD has already been demonstrated efficient for toxin sequencing 1 , and especially when using 1,5-DAN as a reducing matrix. Even if this technique requires simple peptide mixtures, reached by LC-MALDI, ISD yields to sequence tags that cover more than 70% of the full toxins, even for high molecular mass toxins (>6500 Da). Additionally, ISD spectra are simpler to interpret than classical MS/MS ones. Indeed, when using 1,5-DAN, ISD generates intense singly charged c-type ions that can be easily exploited to sequence unknown peptides. This property permitted the development of in-house software, dedicated to the automated spectra interpretation. The software firstly treats the spectra with a smart smoothing, then normalizes the peaks and finally analyzes the spectra to propose relevant sequence tags. A score is calculated for each tag, on the basis of the intensity of the fragments and on the difference between the measured amino-acid masses and the theoretical ones.

  1. Quinton, L., et al., New method for characterizing highly disulfide-bridged peptides in complex mixtures: Application to toxin identification from crude venoms, 2007, J. Proteome Res., 6(8), 3216-3223.