Characterizing circular peptides in mixtures: sequence fragment assembly of cyclotides from a violet plant by MALDI-TOF/TOF mass spectrometry

Abstract

Cyclotides are a very abundant class of plant peptides that display significant sequence variability around a conserved cystine-knot motif and a head-to-tail cyclized backbone conferring them with remarkable stability. Their intrinsic bioactivities combined with tools of peptide engineering make cyclotides an interesting template for the design of novel agrochemicals and pharmaceuticals. However, laborious isolation and purification prior to de novo sequencing limits their discovery and hence their use as scaffolds for peptide-based drug development. Here we extend the knowledge about their sequence diversity by analysing the cyclotide content of a violet species native to Western Asia and the Caucasus region. Using an experimental approach, which was named sequence fragment assembly by MALDI-TOF/TOF, it was possible to characterize 13 cyclotides from Viola ignobilis, whereof ten (vigno 1–10) display previously unknown sequences. Amino acid sequencing of various enzymatic digests of cyclotides allowed the accurate assembly and alignment of smaller fragments to elucidate their primary structure, even when analysing mixtures containing multiple peptides. As a model to further dissect the combinatorial nature of the cyclotide scaffold, we employed in vitro oxidative refolding of representative vigno cyclotides and confirmed the high dependency of folding yield on the inter-cysteine loop sequences. Overall this work highlights the immense structural diversity and plasticity of the unique cyclotide framework. The presented approach for the sequence analysis of peptide mixtures facilitates and accelerates the discovery of novel plant cyclotides.Electronic supplementary materialThe online version of this article (doi:10.1007/s00726-012-1376-x) contains supplementary material, which is available to authorized users.

Oxytocic plant cyclotides as templates for peptide G protein-coupled receptor ligand design

“…Analysis of peptides was performed on a MALDI-TOF/TOF 4800 Analyzer (AB Sciex). MS and tandem MS experiments were carried out as described previously (8). Before MALDI analysis, samples were desalted using C 18 ZipTips (Millipore).…”

“…The peptides responsible for the contractility effects (5) raised interest because they survived boiling, presumably as a result of their unique 3D structure, which was elucidated in 1995 (6). Since then, several plant species of the coffee (Rubiaceae) (7), violet (Violaceae) (8), legume (Fabaceae) (9), potato (Solanaceae) (10) and grass (Poaceae) families (11) have been identified to produce cyclotides. Currently, ∼300 sequences have been reported (12), and the predicted number of >50,000 cyclotides in Rubiaceae alone (7) suggests them to be one of the largest peptide classes within the plant kingdom.…”

Sequence analysis of gene encoding cyclotides in three species of Violaceae and determination of their anti-microbial activities

“…In the current study, we screened V. odorata, V. ignobilis [31] and V. occulta for tracing the cyclotides. Recent studies have proven that cyclotides expression varies in different tissues, and that within the same tissues, their expression also varies with seasonal and environmental changes [32].…”

Structure-activity relationships and development of disulfide-rich cyclic peptides as pharmaceutical templates

“…Recently, Gruber's laboratory described the characterization of cyclotides from Viola ignobilis (Hashempour et al, 2013), a species of the violet family from Iran, which was recently reported to contain cyclotides (Hashempour et al, 2011). The analysis involved the use of MALDI-TOF MS and tandem MS with a selection of complementary enzymatic digests to attain full peptide sequence coverage by assembling and aligning peptide sequences.…”

“…Typically, this multiplicity means that subsequent purification steps are necessary before sequence characterization, and this may be problematic as each purification step results in sample loss. Gruber and colleagues (Hashempour et al, 2013) sought to develop a protocol to perform MALDI-TOF/TOF experiments for characterization of cyclotide sequences using semipure cyclotide fractions containing two or more cyclotides. This protocol involved a combination of single or combined enzyme digestions (to acquire fragments encompassing entire peptide sequences) with tandem MS.…”