FOCUS: MASS SPECTROMETRY-BASED STRATEGIES FOR NEUROPROTEOMICS AND PEPTIDOMICS: RESEARCH ARTICLE Screening Method for the Discovery of Potential Bioactive Cysteine-Containing Peptides Using 3D Mass Mapping

Animal venoms and toxins are a valuable source of bioactive peptides with pharmacologic relevance as potential drug leads. A large subsetofbiologically active peptides discovered up till now contain disulfide bridges that enhance stability and activity.Todiscover newmembers of thisclass of peptides,wedeveloped a workflow screening specifically for those peptides that contain inter- and intra-molecular disul- fide bonds by means of three-dimensional (3D) mass mapping. Two intrinsic proper- ties of the sulfur atom, (1) its relatively large negative mass defect, and (2) its isotopic composition, allow for differentiation between cysteine-containing peptides and pep- tides lacking sulfur. High sulfur content in a peptide decreases the normalized nominal mass defect (NMD) and increasesthe normalized isotopic shift (NIS). Hence in a 3D plot of mass, NIS, and NMD, peptides with sulfur appear in this plot with a distinct spatial localization compared with peptides that lack sulfur. In this study we investigated the skin secretion of two frog species; Odorrana schmackeri and Bombina variegata. Peptides from the crude skin secretions were separated by nanoflow LC, and of all eluting peptides high resolution zoom scans were acquired in order to accurately determine both monoisotopic mass and average mass. Both the NMD and the NIS were calculated from the experimental data using an in-house developed MATLAB script. Candidate peptides exhibiting a low NMD and high NIS values were selected for targeted de novo sequencing, and this resulted in the identification of several novel inter- and intra-molecular disulfide bond containing peptides.