title: Determination of disulfide linkages in antimicrobial peptides of the macin family by combination of top-down and bottom-up proteomics.
abstract: Macins are a distinct class of antimicrobial peptides (AMPs) produced by leeches and Hydra. Their function depends strongly on their three-dimensional structure. In order to support structural elucidation of these AMPs, the knowledge and proper assignment of disulfide bonds formed in these cysteine-rich peptides is a prerequisite. In this report, we outline an analytical strategy, encompassing a combination of top-down MS based analytics and sequence-dependent enzyme cleavage under native conditions followed by high mass accuracy and high resolution MS/MS analysis by LTQ-Orbitrap MS to assign disulfide linkages of three members of the macin family, namely neuromacin, theromacin, and hydramacin-1. The results revealed that the eight cysteine residues conserved in all three macins form the same four disulfide bonds, i.e. [C1:C6], [C2:C5], [C3:C7], and [C4:C8]. Theromacin, which possess two additional cysteine residues, forms a fifth disulfide bond.
  
  
  CONCLUSIONS
  Beside the high biological significance which is based on the inherent dependence of biological activity on the structural features of antimicrobial peptides (which holds true for entirely every protein), the presented analytical strategy will be of wide interest, as it widens the available toolbox for the analysis of this important posttranslational modification.
authors.affiliation: Institute for Experimental Medicine - Division of Systematic Proteome Research & Bioanalytics, Christian-Albrechts-Universität zu Kiel, 24105 Kiel, Germany.
authors.forename: Chien-Wen
authors.initials: CW
authors.lastname: Hung
authors.affiliation: Institute of Biochemistry, Christian-Albrechts-Universität zu Kiel, 24098 Kiel, Germany.
authors.forename: Sascha
authors.initials: S
authors.lastname: Jung
authors.affiliation: Institute of Biochemistry, Christian-Albrechts-Universität zu Kiel, 24098 Kiel, Germany.
authors.forename: Joachim
authors.initials: J
authors.lastname: Grötzinger
authors.affiliation: Institute of Zoology, Zoophysiology, Christian-Albrechts-Universität zu Kiel, 24098 Kiel, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Gelhaus
authors.affiliation: Institute of Zoology, Zoophysiology, Christian-Albrechts-Universität zu Kiel, 24098 Kiel, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Leippe
authors.affiliation: Institute for Experimental Medicine - Division of Systematic Proteome Research & Bioanalytics, Christian-Albrechts-Universität zu Kiel, 24105 Kiel, Germany. Electronic address: a.tholey@iem.uni-kiel.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: Tholey
mesh_terms: D000595:Amino Acid Sequence; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry* / Q000302:isolation & purification; D004220:Disulfides / Q000032:analysis*; D008970:Molecular Weight; D040901:Proteomics / Q000379:methods; D016415:Sequence Alignment; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D023181:Antimicrobial Cationic Peptides; D004220:Disulfides; C539006:hydramacin-1, Hydra magnipapillata; C000594596:neuromacin peptide, Hirudo medicinalis; C490018:theromacin, leech
keywords: Cleavage site; LC-MS; Posttranslational modification; Top-down; de-novo Sequencing