title: Fragmentation follows structure: top-down mass spectrometry elucidates the topology of engineered cystine-knot miniproteins.
abstract: Over the last decades the field of pharmaceutically relevant peptides has enormously expanded. Among them, several peptide families exist that contain three or more disulfide bonds. In this context, elucidation of the disulfide patterns is extremely important as these motifs are often prerequisites for folding, stability, and activity. An example of this structure-determining pattern is a cystine knot which comprises three constrained disulfide bonds and represents a core element in a vast number of mechanically interlocked peptidic structures possessing different biological activities. Herein, we present our studies on disulfide pattern determination and structure elucidation of cystine-knot miniproteins derived from Momordica cochinchinensis peptide MCoTI-II, which act as potent inhibitors of human matriptase-1. A top-down mass spectrometric analysis of the oxidised and bioactive peptides is described. Following the detailed sequencing of the peptide backbone, interpretation of the MS(3) spectra allowed for the verification of the knotted topology of the examined miniproteins. Moreover, we found that the fragmentation pattern depends on the knottin's folding state, hence, tertiary structure, which to our knowledge has not been described for a top-down MS approach before.
authors.affiliation: Institute of Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Darmstadt, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Reinwarth
authors.affiliation: Institute of Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Darmstadt, Germany.
authors.forename: Olga
authors.initials: O
authors.lastname: Avrutina
authors.affiliation: AB Sciex Germany GmbH, Darmstadt, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Fabritz
authors.affiliation: Institute of Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Darmstadt, Germany.
authors.forename: Harald
authors.initials: H
authors.lastname: Kolmar
mesh_terms: D000595:Amino Acid Sequence; D047169:Cyclotides / Q000737:chemistry*; D057771:Cystine-Knot Miniproteins / Q000737:chemistry*; D004220:Disulfides / Q000032:analysis*; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D029775:Momordica / Q000737:chemistry*; D010940:Plant Proteins / Q000737:chemistry*; D011487:Protein Conformation
chemical_list: D047169:Cyclotides; D057771:Cystine-Knot Miniproteins; D004220:Disulfides; D010940:Plant Proteins; C409923:trypsin inhibitor MCoTI-II