Exploring the Diversity of Cysteine-Rich Natural Product Peptides via MS/MS Fingerprint Ions

Abstract

Natural product extracts present inherently complex matrices in which the identification of novel bioactive peptide species is challenged by low-abundance masses and significant structural and sequence diversity. Additionally, discovery efforts often result in the re-identification of known compounds, where modifications derived in vivo or during sample handling may obscure true sequence identity. Herein, we identify mass spectral (MS²) "fingerprint" ions characteristic of cyclotides, a diverse and biologically active family of botanical cysteine-rich peptides, based on regions of high sequence homology. We couple mass shift analysis with MS² spectral fingerprint ions cross referenced with CyBase-a cyclotide database-to discern unique mass species in Viola communis extracts from mass species that are likely already characterized and those with common modifications. The approach is extended to a related class of cysteine-rich peptides, the trypsin inhibitors, using the characterized botanical species Lagenaria siceraria. Coupling the observation of highly abundant MS² ions with mass shift analysis, we identify a new set of small, highly disulfide-bound cysteine-rich L. siceraria peptides.

Figure 1.

Fingerprint ions derived from the Glu-C digestion and b/y fragmentation of the prototypical cyclotides cyO2 (top, bracelet) and kalata B2 (bottom, Möbius). Fragment ion color-coding aligns to the sequence colors; below the fragment ion m/z is the chance of finding that ion in the MS² of a non-cyclotide (% occurrence), followed by the chance of finding that ion in combination with those preceding it towards the nearest terminus (% combined) in a non-cyclotide. Loop regions between cysteine residues are numbered above and below sequences in grey and allow for facile sequence comparison among cyclotide sequences; cysteine connectivites, conserved among all cyclotides, are represented by black bars above the cyO2 sequence. Structures of cyO2 (PDB 2KNM) and kalata B2 (PDB 1PT4) modeled in PyMol (Schrödinger) are shown to the left of the corresponding sequences, where the Glu cleavage site for linearization is represented by a blue star.

Figure 2.

(A) Total ion chromatograms (TICs) of V. communis harvests. (B-H) MS² spectra of V. communis reduced, alkylated, and Glu-C digested material. Common fingerprint ions are labeled; the bracelet aromatic peak often differentiates masses and is bolded in green in B-G.

Figure 3.

Sequences and neutral, disulfide-intact (NI) molecular weights (Da) of Lagenaria siceraria known trypsin inhibitors LLTI-I, II, and III (top) and previously uncharacterized Lagenaria siceraria cysteine-rich peptides LSCRP-I, II, and III (bottom). Bolded, blue Glu residue (E) at the LLTI-I N-terminus is modified to a pyroglutamic acid. Regions where sequences diverge are highlighted in blue.

Figure 4.

MS² spectra of L. siceraria known trypsin inhibitors (A-C) and novel cysteine-rich sequences (D-F); fingerprint ions common to each type of CRP are labeled.