title: Screening Method for the Discovery of Potential Bioactive Cysteine-Containing Peptides Using 3D Mass Mapping.
abstract: Animal venoms and toxins are a valuable source of bioactive peptides with pharmacologic relevance as potential drug leads. A large subset of biologically active peptides discovered up till now contain disulfide bridges that enhance stability and activity. To discover new members of this class of peptides, we developed a workflow screening specifically for those peptides that contain inter- and intra-molecular disulfide bonds by means of three-dimensional (3D) mass mapping. Two intrinsic properties of the sulfur atom, (1) its relatively large negative mass defect, and (2) its isotopic composition, allow for differentiation between cysteine-containing peptides and peptides lacking sulfur. High sulfur content in a peptide decreases the normalized nominal mass defect (NMD) and increases the 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. Graphical Abstract ᅟ.
authors.affiliation: Department of Biotechnology, Delft University of Technology, 2628 BC, Delft, The Netherlands.
authors.forename: Luuk N
authors.initials: LN
authors.lastname: van Oosten
authors.affiliation: Department of Biotechnology, Delft University of Technology, 2628 BC, Delft, The Netherlands.
authors.forename: Mervin
authors.initials: M
authors.lastname: Pieterse
authors.affiliation: Department of Biotechnology, Delft University of Technology, 2628 BC, Delft, The Netherlands.
authors.forename: Martijn W H
authors.initials: MW
authors.lastname: Pinkse
authors.affiliation: Department of Biotechnology, Delft University of Technology, 2628 BC, Delft, The Netherlands. peter.verhaert@med.lu.se.|Department of Biomedical Sciences, Antwerp University, 2610, Antwerp, Belgium. peter.verhaert@med.lu.se.|CEBMMS (Center of Excellence in Biological and Medical Mass Spectrometry), Department of Clinical Sciences, Lund University, 221 85, Lund, Sweden. peter.verhaert@med.lu.se.
authors.forename: Peter D E M
authors.initials: PD
authors.lastname: Verhaert
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001001:Anura / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003545:Cysteine / Q000032:analysis*; D021621:Imaging, Three-Dimensional / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods*; D010455:Peptides / Q000737:chemistry*
chemical_list: D010455:Peptides; D003545:Cysteine
keywords: 3D mass mapping; Animal toxin; Disulfide bridge containing peptides; MATLAB script; Orbitrap analysis