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PLoS One. 2014 Oct 10;9(10):e108626. doi: 10.1371/journal.pone.0108626. eCollection 2014.

Fragmentation follows structure: top-down mass spectrometry elucidates the topology of engineered cystine-knot miniproteins.

Author information

1
Institute of Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Darmstadt, Germany.
2
AB Sciex Germany GmbH, Darmstadt, Germany.

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.

PMID:
25303319
PMCID:
PMC4193770
DOI:
10.1371/journal.pone.0108626
[Indexed for MEDLINE]
Free PMC Article

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