title: MALDI TOF/TOF-Based Approach for the Identification of d- Amino Acids in Biologically Active Peptides and Proteins.
abstract: Several biologically active peptides contain a d- amino acid in a well-defined position, which is position 2 in all peptide epimers isolated to date from vertebrates and also some from invertebrates. The detection of such D- residues by standard analytical techniques is challenging. In tandem mass spectrometric (MS) analysis, although fragment masses are the same for all stereoisomers, peak intensities are known to depend on chirality. Here, we observe that the effect of a d- amino acid in the second N-terminal position on the fragmentation pattern in matrix assisted laser desorption time-of-flight spectrometry (MALDI-TOF/TOF MS) strongly depends on the peptide sequence. Stereosensitive fragmentation (SF) is correlated to a neighborhood effect, but the d- residue also exerts an overall effect influencing distant bonds. In a fingerprint analysis, multiple peaks can thus serve to identify the chirality of a sample in short time and potentially high throughput. Problematic variations between individual spots could be successfully suppressed by cospotting deuterated analogues of the epimers. By identifying the [d-Leu2] isomer of the predicted peptide GH-2 (gene derived bombininH) in skin secretions of the toad Bombina orientalis, we demonstrated the analytical power of SF-MALDI-TOF/TOF measurements. In conclusion, SF-MALDI-TOF/TOF MS combines high sensitivity, versatility, and the ability to complement other methods.
authors.affiliation: Centre for Physiology and Pharmacology, Medical University of Vienna , Schwarzspanierstraße 17, A-1090 Vienna, Austria.|School of Biomedical Sciences, The University of Queensland , Brisbane, QLD, 4072 Australia.
authors.forename: Johannes
authors.initials: J
authors.lastname: Koehbach
authors.affiliation: Centre for Physiology and Pharmacology, Medical University of Vienna , Schwarzspanierstraße 17, A-1090 Vienna, Austria.
authors.forename: Christian W
authors.initials: CW
authors.lastname: Gruber
authors.affiliation: Institute of Biological Chemistry, Department of Chemistry, University of Vienna , Währinger Straße 38, A-1090 Vienna, Austria.
authors.forename: Christian
authors.initials: C
authors.lastname: Becker
authors.affiliation: Chair of Bioinformatics, University of Natural Resources and Life Sciences , Muthgasse 18, A-1190 Vienna, Austria.
authors.forename: David P
authors.initials: DP
authors.lastname: Kreil
authors.affiliation: Institute of Biological Chemistry, Department of Chemistry, University of Vienna , Währinger Straße 38, A-1090 Vienna, Austria.|Chair of Bioinformatics, University of Natural Resources and Life Sciences , Muthgasse 18, A-1190 Vienna, Austria.
authors.forename: Alexander
authors.initials: A
authors.lastname: Jilek
mesh_terms: D000596:Amino Acids / Q000032:analysis*; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000032:analysis / Q000737:chemistry; D001001:Anura; D003903:Deuterium; D010455:Peptides / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D012867:Skin / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013237:Stereoisomerism
chemical_list: D000596:Amino Acids; D023181:Antimicrobial Cationic Peptides; D010455:Peptides; D011506:Proteins; C510197:bombinin H2; D003903:Deuterium
keywords: chirality; d- amino acid containing peptide; isotope label; peptidomics; post-translational modification; proteomics