PTM-driven differential peptide display: survey of peptides containing inter/intra-molecular disulfide bridges in...

Amphibian defensive skin secretions are complex species-specific mixtures of biologically active molecules, including many uncharacterized peptides. Many of these peptides are post-translationally modified and amongst the modifications discovered so far on amphibian defense peptides, disulfide bonds are quite frequently encountered. The presence of this PTM often complicates the MS-based sequencing. Here we demonstrate a method to target peptides containing inter/intra-molecular S-S bonds applying a PTM-driven differential display. Upon reduction of the disulfide bond both molecular mass and retention time of a peptide are altered. Assembling the LC-MS data by plotting the m/z data against retention time generates a peptide display and overlaying peptide displays of untreated and DTT-reduced material yields a differential display. From such an overlay, peptides originally carrying a disulfide bond are recognized due to the shift in both retention time and m/z values, whereas non cystine containing peptides remain unaltered in the differential display. The success of this approach is demonstrated by the visualization of the cystines-containing peptides in the skin secretion of Odorrana schmackeri, Phyllomedusa burmeisteri, Phyllomedusa rohdei, Kassina senegalensis, and Bombina variegata. The venoms from these different species yield complicated differential displays, showing interesting peptides, allowing one to target them for more detailed structural characterization.