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 ᅟElectronic supplementary materialThe online version of this article (doi:10.1007/s13361-015-1282-z) contains supplementary material, which is available to authorized users.
Highlights
The venoms and toxins of animals are a major source of biologically active peptides and currently over 5000 of these peptides are recorded in the UniProt protein database [1, 2]
By making a three-dimensional (3D) plot of nominal mass defect (NMD), normalized isotopic shift (NIS), and monoisotopic mass, peptides containing sulfur are differentiated from non-sulfur containing peptides, and those can be further targeted for extensive MSn based analysis and full primary structure elucidation. We demonstrate this workflow via the analysis of peptides in the secretions produced by the granular skin glands of two anuran amphibian species, Odorrana schmackeri and Bombina variegata
Our experimental determination of NMD and NIS values of unknown peptides is based on high resolution MS and zoom-scans
Summary
The venoms and toxins of animals are a major source of biologically active peptides and currently over 5000 of these peptides are recorded in the UniProt protein database [1, 2]. We expand the mass mapping approach to select for cysteine-containing peptides prior to de novo sequencing, based on NMD, NIS, and overall mass. We developed a data-dependent analysis workflow to experimentally determine the monoisotopic and average masses of unknown peptides, in order to calculate NIS and NMD values.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callMore From: Journal of the American Society for Mass Spectrometry
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
