High-Accuracy Mass Spectrometry Based Screening Method for the Discovery of Cysteine Containing Peptides in Animal Venoms and Toxins

Abstract

Venom and toxin samples derived from animal origins are a rich source of bioactive peptides. A high proportion of bioactive peptides that have been identified in venom contain one or more disulfide bridges, which are thought to stabilize tertiary structure, and therefore influence the peptides' specificity and activity. In this chapter, we describe a label-free mass spectrometry-based screening workflow specifically to detect peptides that contain inter- and intramolecular disulfide bonds, followed by elucidation of their primary structure. This method is based on the determination of the normalized isotope shift (NIS) and the normalized mass defect (NMD) of peptides, two parameters which are heavily influenced by the presence of sulfur in a peptide, where cysteines are the main contributing residues. Using ant defensive secretions as an example, we describe the initial fractionation of the venom on strong cation exchange followed by nanoflow HPLC and mass spectrometry. High resolution zoom scan spectra of high-abundance peptides are acquired, allowing an accurate determination of both monoisotopic and average mass, which are essential for calculation of NMD and NIS. Candidate peptides exhibiting relative low NMD and high NIS values are selected for targeted de novo sequencing. By fine-tuning the collision energy for optimal fragmentation of each selected precursor ions, the full sequence of several novel inter- and intramolecular disulfide bond containing ant defensive peptides can be established.

Machine learning approach for the prediction of the number of sulphur atoms in peptides using the theoretical aggregated isotope distribution

“…8 In case resolving power is lacking, it is still possible to detect the presence of sulphur by its effect on the profile of the third aggregated isotope peak. 9 Therefore, knowledge of the number of sulphur atoms can improve the identification of peptides and can be used for quality control in mass spectral experiments. 10 In a previous work the isotope fine structure extracted from MS1 spectra revealed the elemental composition of peptides, 11 proteins, 12,13 and metabolites 14,15 using Fourier-transform ion cyclotron resonance mass spectrometry.…”

“…The difference between the mentioned isotope variants is 0.0109 Da, which is a mass defect that can easily be resolved using modern mass spectrometers 8 . In case resolving power is lacking, it is still possible to detect the presence of sulphur by its effect on the profile of the third aggregated isotope peak 9 . Therefore, knowledge of the number of sulphur atoms can improve the identification of peptides and can be used for quality control in mass spectral experiments 10 …”