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. 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

Michael Reinwarth  1 , Olga Avrutina  1 , Sebastian Fabritz  2 , Harald Kolmar  1
Affiliations

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

Michael Reinwarth et al. PLoS One. .

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.

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Conflict of interest statement

Competing Interests: S. Fabritz is associated with a commercial company, AB Sciex Germany GmbH. There are no patents, products in development or marketed products to declare. All other authors declare no competing interests. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Structure of synthetic open-chain MCoTI, structural overview and fragment ion formation.
(A) 3D structure of synthetic open-chain cystine knot oMCoTI (pdb: 2IT8).15 Active loop is shown in red. (B) A 6 Å close-up on the disulfide-tightened core of MCoTI. For both (A) and (B) disulfides are shown in yellow. (C) Overview of the generation of peptidic fragment ions upon CID. (D) Sequences of the parent MCoTI wild type (wt) and the miniproteins (1–3) used in this study. Positions with altered amino acid residues (regarding the wild type sequence) are marked red.
Figure 2
Figure 2. MS2 of MCoTI peptide 1.
(A) CID of the doubly charged ion of reduced peptide 1. (B) CID of the doubly charged ion of folded miniprotein. (C) CID of the five-fold charged ion of folded miniprotein.
Figure 3
Figure 3. MS3 of the CID-obtained major fragments of MCoTI 1.
(A) MS3 of c10 of the doubly charged ions of unfolded peptide. (B) MS3 of c16y19 of the doubly-charged ions of folded miniprotein. (C) MS3 of y14 of the doubly charged ions of unfolded peptide. (D) MS3 of y24 of the five-fold charged ions of folded miniprotein. Arrows above the spectra indicate intensity amplifications.
Figure 4
Figure 4. MS3 of y14-32 (A) and y14+32 (B) and the resulting combinatorial interpretation.
In red are inexistent peaks to provide evidence on the respective Ptc or Dha cleavage. Arrows above the spectra indicate intensity amplifications.
Figure 5
Figure 5. Spectra and assigned structures of the bridged fragments.
(A) MS3 of the bridged fragment of 1. (B) MS3 of the bridged fragment of 2. Arrows above the spectra indicate intensity amplifications.
Figure 6
Figure 6. Optimization of the collision energy towards a maximum formation of fragment c10y26 of 2.
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