Comprehensive Mapping of Cyclotides from Viola philippica by Using Mass Spectrometry-Based Strategy.

Cyclotides are plant cyclic peptides with exceptional stability and diverse bioactivity, making them promising candidates for biomedical applications. Therefore, the study of cyclotides has attracted increasing attention in recent years. However, the existing cyclotide detection methods face limitations in sensitivity, accuracy, and reliability. To address these challenges, we developed an integrated strategy using a combination of strong cation exchange chromatography techniques for removing interfering small molecules, Orbitrap Exploris 480 mass spectrometry (OEMS); this is a detection and database searching-based method for cyclotide verification, which greatly improved the sensitivity, accuracy, and reliability of cyclotide identification. This strategy was subsequently employed for cyclotide mapping in Viola with a minute amount of starting tissue, resulting the identification of 65 known and 18 potentially novel cyclotides, which is the largest dataset of cyclotides for Viola philippica. This strategy provided valuable insights into the cyclotide diversity and distribution in V. philippica, with potential applications in drug discovery and other biomedical fields.

High-throughput microfluidic chip with silica gel-C18 channels for cyclotide separation.

Over the past two decades, microfluidic-based separations have been used for the purification, isolation, and separation of biomolecules to overcome difficulties encountered by conventional chromatography-based methods including high cost, long processing times, sample volumes, and low separation efficiency. Cyclotides, or cyclic peptides used by some plant families as defense agents, have attracted the interest of scientists because of their biological activities varying from antimicrobial to anticancer properties. The separation process has a critical impact in terms of obtaining pure cyclotides for drug development strategies. Here, for the first time, a mimic of the high-performance liquid chromatography (HPLC) on microfluidic chip strategy was used to separate the cyclotides. In this regard, silica gel-C18 was synthesized and characterized by Fourier-transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (1H-NMR) and then filled inside the microchannel to prepare an HPLC C18 column-like structure inside the microchannel. Cyclotide extract was obtained from Viola ignobilis by a low voltage electric field extraction method and characterized by HPLC and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). The extract that contained vigno 1, 2, 3, 4, 5, and varv A cyclotides was added to the microchannel where distilled water was used as a mobile phase with 1 µL/min flow rate and then samples were collected in 2-min intervals until 10 min. Results show that cyclotides can be successfully separated from each other and collected from the microchannel at different periods of time. These findings demonstrate that the use of microfluidic channels has a high impact on the separation of cyclotides as a rapid, cost-effective, and simple method and the device can find widespread applications in drug discovery research.

Sequencing Orbitides by Acid-Mediated Ring Cleavage Followed by Tandem Mass Spectrometry.

Homodetic cyclic peptides have aroused interest because of their pharmacological potential. Sequencing cyclic peptides is difficult-Edman degradation is not possible as there is no N-terminus, NMR requires quantities that are hard to gather from native samples, and tandem mass spectrometry data are difficult to interpret due to the peptide ring opening at multiple points. Sequencing can be simplified by cleaving the peptide ring at a specific peptide bond. Partial acid hydrolysis is a possible solution, but to date sequencing by this method has only been demonstrated for linear peptides and cyclotides, which are larger cyclic peptides (∼30 amino acids) with three disulfide bonds. This study tests whether partial acid hydrolysis could be used to aid sequencing of Cys-less cyclic peptides with fewer than ten amino acid residues. We show that, with the right combination of temperature and acid, ring cleavage occurs and offers relatively simple MS/MS spectra amenable to sequencing. We describe how this method was used in our recent study in which we sequenced annomuricatin D (cyclo-GHSIFPPIP) from seeds of the soursop, Annona muricata. We found that orbitides can be linearized for MS/MS sequencing by incubation with 1.2 M HCl at 90 °C for 15-20 min. This fast, economical sequencing method will be useful to those studying small cyclic peptides lacking disulfide bonds, which are commonly found in many organisms, especially plants.

Gas-Phase Sequencing of Cyclotides: Introduction of Selective Ring Opening at Dehydroalanine via Ion/Ion Reaction.

The gas-phase linearization of cyclotides via site-selective ring opening at dehydroalanine residues and its application to cyclotide sequencing is presented. This strategy relies on the ability to incorporate dehydroalanine into macrocyclic peptide ions, which is easily accomplished through an ion/ion reaction. Triply protonated cyclotide cations are transformed into radical cations via ion/ion reaction with the sulfate radical anion. Subsequent activation of the cyclotide radical cation generates dehydroalanine at a single cysteine residue, which is easily identified by the odd-electron loss of ·SCH2CONH2. The presence of dehydroalanine in cyclotides provides a site-selective ring-opening pathway that, in turn, generates linear cyclotide analogues in the gas phase. Unlike cyclic variants, product ions derived from the linear peptides provide rich sequence information. The sequencing capability of this strategy is demonstrated with four known cyclotides found in Viola inconspicua, where, in each case, greater than 93% sequence coverage was observed. Furthermore, the utility of this method is highlighted by the partial de novo sequencing of an unknown cyclotide with much greater sequence coverage than that obtained with a conventional Glu-C digestion approach. This method is particularly well-suited for cyclotide species that are not abundant enough to characterize with traditional methods.

Microwave-assisted extraction of cyclotides from Viola ignobilis.

Cyclotides are an interesting family of circular plant peptides. Their unique three-dimensional structure, comprising a head-to-tail circular backbone chain and three disulfide bonds, confers them stability against thermal, chemical, and enzymatic degradation. Their unique stability under extreme conditions creates an idea about the possibility of using harsh extraction methods such as microwave-assisted extraction (MAE) without affecting their structures. MAE has been introduced as a potent extraction method for extraction of natural compounds, but it is seldom used for peptide and protein extraction. In this work, microwave irradiation was applied to the extraction of cyclotides. The procedure was performed in various steps using a microwave instrument under different conditions. High-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) results show stability of cyclotide structures on microwave radiation. The influential parameters, including time, temperature, and the ratio of solvents that are affecting the MAE potency, were optimized. Optimal conditions were obtained at 20 min of irradiation time, 1200 W of system power in 60 °C, and methanol/water at the ratio of 90:10 (v/v) as solvent. The comparison of MAE results with maceration extraction shows that there are similarities between cyclotide sequences and extraction yields.

Isolation and Characterization of Cyclotides from Brazilian Psychotria: Significance in Plant Defense and Co-occurrence with Antioxidant Alkaloids.

Plants from the genus Psychotria include species bearing cyclotides and/or alkaloids. The elucidation of factors affecting the metabolism of these molecules as well as their activities may help to understand their ecological function. In the present study, high concentrations of antioxidant indole alkaloids were found to co-occur with cyclotides in Psychotria leiocarpa and P. brachyceras. The concentrations of the major cyclotides and alkaloids in P. leiocarpa and P. brachyceras were monitored following herbivore- and pathogen-associated challenges, revealing a constitutive, phytoanticipin-like accumulation pattern. Psyleio A, the most abundant cyclotide found in the leaves of P. leiocarpa, and also found in P. brachyceras leaves, exhibited insecticidal activity against Helicoverpa armigera larvae. Addition of ethanol in the vehicle for peptide solubilization in larval feeding trials proved deleterious to insecticidal activity and resulted in increased rates of larval survival in treatments containing indole alkaloids. This suggests that plant alkaloids ingested by larvae might contribute to herbivore oxidative stress detoxification, corroborating, in a heterologous system with artificial oxidative stress stimulation, the antioxidant efficiency of Psychotria alkaloids previously observed in planta. Overall, the present study reports data for eight novel cyclotides, the identification of P. leiocarpa as a cyclotide-bearing species, and the absence of these peptides in P. umbellata.

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

Cyclotides are plant peptides comprising a circular backbone and three conserved disulfide bonds that confer them with exceptional stability. They were originally discovered in Oldenlandia affinis based on their use in traditional African medicine to accelerate labor. Recently, cyclotides have been identified in numerous plant species of the coffee, violet, cucurbit, pea, potato, and grass families. Their unique structural topology, high stability, and tolerance to sequence variation make them promising templates for the development of peptide-based pharmaceuticals. However, the mechanisms underlying their biological activities remain largely unknown; specifically, a receptor for a native cyclotide has not been reported hitherto. Using bioactivity-guided fractionation of an herbal peptide extract known to indigenous healers as "kalata-kalata," the cyclotide kalata B7 was found to induce strong contractility on human uterine smooth muscle cells. Radioligand displacement and second messenger-based reporter assays confirmed the oxytocin and vasopressin V1a receptors, members of the G protein-coupled receptor family, as molecular targets for this cyclotide. Furthermore, we show that cyclotides can serve as templates for the design of selective G protein-coupled receptor ligands by generating an oxytocin-like peptide with nanomolar affinity. This nonapeptide elicited dose-dependent contractions on human myometrium. These observations provide a proof of concept for the development of cyclotide-based peptide ligands.

Mass Spectrometric Analysis of Cyclotides from Clitoria ternatea: Xxx-Pro Bond Fragmentation as Convenient Diagnostic of Pro Residue Positioning.

Cyclotides, a class of macrocyclic plant peptides, characterized by a cyclic backbone and three inter-locking disulfide bonds, may be divided into two major structural subfamilies, Möbius and Bracelet, based on the presence or absence of a specific proline residue. The present study describes the suite of cyclotides obtained from Clitoria ternatea, characterized by LC-MS and MS/MS techniques. Notable variations in product ion distributions were observed in cyclotides belonging to different structural subfamilies based on the number and positions of proline residues. For instance, Cter M which is an abundant Möbius cyclotide in this plant containing three proline residues, displayed distinct b- and y- ion characteristics in the MS/MS spectra compared to Cliotide T1, another commonly identified cyclotide but belonging to the Bracelet subfamily having two proline residues. The distinct fragmentation pattern of prototypical cyclotides of each structural subfamily, determined by Xxx-Pro bond fragmentation, was used to rapidly identify and sequence a novel cyclotide ctr pep 30 from this plant.

Cyclotide host-defense tailored for species and environments in violets from the Canary Islands.

Cyclotides are cyclic peptides produced by plants. Due to their insecticidal properties, they are thought to be involved in host defense. Violets produce complex mixtures of cyclotides, that are characteristic for each species and variable in different environments. Herein, we utilized mass spectrometry (LC-MS, MALDI-MS), transcriptomics and biological assays to investigate the diversity, differences in cyclotide expression based on species and different environment, and antimicrobial activity of cyclotides found in violets from the Canary Islands. A wide range of different habitats can be found on these islands, from subtropical forests to dry volcano peaks at high altitudes. The islands are inhabited by the endemic Viola palmensis, V. cheiranthifolia, V. anagae and the common V. odorata. The number of cyclotides produced by a given species varied in plants from different environments. The highest diversity was noted in V. anagae which resides in subtropical forest and the lowest in V. cheiranthifolia from the Teide volcano. Transcriptome sequencing and LC-MS were used to identify 23 cyclotide sequences from V. anagae. Cyclotide extracts exhibited antifungal activities with the lowest minimal inhibitory concentrations noted for V. anagae (15.62 µg/ml against Fusarium culmorum). The analysis of the relative abundance of 30 selected cyclotides revealed patterns characteristic to both species and populations, which can be the result of genetic variability or environmental conditions in different habitats. The current study exemplifies how plants tailor their host defense peptides for various habitats, and the usefulness of cyclotides as markers for chemosystematics.

Transcriptomic profiling of the medicinal plant Clitoria ternatea: identification of potential genes in cyclotide biosynthesis.

Clitoria ternatea a perennial climber of the Fabaceae family, is well known for its agricultural and medical applications. It is also currently the only known member of the Fabaceae family that produces abundant amounts of the ultra-stable macrocyclic peptides, cyclotides, across all tissues. Cyclotides are a class of gene-encoded, disulphide-rich, macrocyclic peptides (26-37 residues) acting as defensive metabolites in several plant species. Previous transcriptomic studies have demonstrated the genetic origin of cyclotides from the Fabaceae plant family to be embedded in the albumin-1 genes, unlike its counterparts in other plant families. However, the complete mechanism of its biosynthesis and the repertoire of enzymes involved in cyclotide folding and processing remains to be understood. In this study, using RNA-Seq data and de novo transcriptome assembly of Clitoria ternatea, we have identified 71 precursor genes of cyclotides. Out of 71 unique cyclotide precursor genes obtained, 51 sequences display unique cyclotide domains, of which 26 are novel cyclotide sequences, arising from four individual tissues. MALDI-TOF mass spectrometry analysis of fractions from different tissue extracts, coupled with precursor protein sequences obtained from transcriptomic data, established the cyclotide diversity in this plant species. Special focus in this study has also been on identifying possible enzymes responsible for proper folding and processing of cyclotides in the cell. Transcriptomic mining for oxidative folding enzymes such as protein-disulphide isomerases (PDI), ER oxidoreductin-1 (ERO1) and peptidylprolyl cis-trans isomerases (PPIases)/cyclophilins, and their levels of expression are also reported. In particular, it was observed that the CtPDI genes formed plant-specific clusters among PDI genes as compared to those from other plant species. Collectively, this work provides insights into the biogenesis of the medicinally important cyclotides and establishes the expression of certain key enzymes participating in peptide biosynthesis. Also, several novel cyclotide sequences are reported and precursor sequences are analysed in detail. In the absence of a published reference genome, a comprehensive transcriptomics approach was adopted to provide an overview of diverse properties and constituents of C. ternatea.

Reporting a Transcript from Iranian Viola Tricolor, Which May Encode a Novel Cyclotide-Like Precursor: Molecular and in silico Studies.

The cyclotides are the largest known family of cyclic proteins, which are found in several plant families including Violaceae. They are circular bioactive peptides consisting of 28-37 amino acids, which possess a cyclic cystine knot (CCK) motif and could be useful in biotechnology and drug design as scaffolds for peptide-based drugs. This study describes our finding of a potentially novel gene transcript from the petals of the Iranian Viola tricolor (V. tricolor) flowers. This study is based on the cDNA screening method employed for isolation of cyclotide precursor genes and in silico analysis. Our study resulted in the finding of a novel cyclotide-like precursor from V. tricolor, which is documented in the NCBI by GenBank accession number: KP065812. The in silico analysis revealed that there are lots of similar sequences in many other plant families and they all exhibit some different features from previously discovered cyclotide precursors. The differences occur particularly in the main cyclotide domain that exists without the usual CCK structure. All of these hypothetical precursors have a conserved ER-signal sequence, a Cysteine (C)-rich sequence forming two zinc finger motifs and a cyclotide-like region containing several conserved elements including two highly conserved C residues. In conclusion, using the cDNA screening method we found a potentially new cyclotide-like precursor gene and in silico studies revealed its significant characteristics that may open up a new research line on the distribution and evolution of cyclotides.

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

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.

Circular proteins from Melicytus (Violaceae) refine the conserved protein and gene architecture of cyclotides.

Cyclotides are cyclic disulfide rich mini-proteins found in various Rubiaceae (coffee family), Violaceae (violet family) and Cucurbitaceae (squash family) plant species. Within the Violaceae, cyclotides have been found in numerous species of the genus Viola as well as species from two other genera, namely Hybanthus and Leonia. This is the first in-depth report of cyclotides in the genus Melicytus (Violaceae). We present the chromatographic profiles of extracts of eight Melicytus species and one Melicytus hybrid that were found to contain these circular peptides. We isolated and characterised five novel cyclotides (mra1 to mra5) from the aerial parts of a common New Zealand tree, Melicytus ramiflorus. All five peptides show the characteristics of the bracelet subfamily of cyclotides. Furthermore, we isolated 17 non-redundant cDNA clones from the leaves of Melicytus ramiflorus encoding cyclotide prepropeptides. This detailed report on the presence of cyclotides in several species of the genus Melicytus further strengthens our hypothesis that cyclotides are ubiquitous in Violaceae family plants and provides additional insight into the biochemical processing mechanisms that produce the cyclic protein backbone of this unique family of ultra-stable plant proteins.

Peptide quantification by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry: investigations of the cyclotide kalata B1 in biological fluids.

A rapid method has been developed for the quantification of the prototypic cyclotide kalata B1 in water and plasma utilizing matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry. The unusual structure of the cyclotides means that they do not ionise as readily as linear peptides and as a result of their low ionisation efficiency, traditional LC/MS analyses were not able to reach the levels of detection required for the quantification of cyclotides in plasma for pharmacokinetic studies. MALDI-TOF-MS analysis showed linearity (R2 > 0.99) in the concentration range 0.05-10 microg/mL with a limit of detection of 0.05 microg/mL (9 fmol) in plasma. This paper highlights the applicability of MALDI-TOF mass spectrometry for the rapid and sensitive quantification of peptides in biological samples without the need for extensive extraction procedures.

A comparative study of extraction methods reveals preferred solvents for cystine knot peptide isolation from Momordica cochinchinensis seeds.

MCoTI-I and MCoTI-II (short for Momordica cochinchinensis Trypsin Inhibitor-I and -II, respectively) are attractive candidates for developing novel intracellular-targeting drugs because both are exceptionally stable and can internalize into cells. These seed-derived cystine knot peptides are examples of how natural product discovery efforts can lead to biomedical applications. However, discovery efforts are sometimes hampered by the limited availability of seed materials, highlighting the need for efficient extraction methods. In this study, we assessed five extraction methods using M. cochinchinensis seeds, a source of well-characterized cystine knot peptides. The most efficient extraction of nine known cystine knot peptides was achieved by a method based on acetonitrile/water/formic acid (25:24:1), followed by methods based on sodium acetate (20 mM, pH 5.0), ammonium bicarbonate (5 mM, pH 8.0), and boiling water. On average, the yields obtained by these four methods were more than 250-fold higher than that obtained using dichloromethane/methanol (1:1) extraction, a previously applied standard method. Extraction using acetonitrile/water/formic acid (25:24:1) yielded the highest number of reconstructed masses within the majority of plant-derived cystine knot peptide mass range but only accounted for around 50% of the total number of masses, indicating that any single method may result in under-sampling. Applying acetonitrile/water/formic acid (25:24:1), boiling water, and ammonium bicarbonate (5 mM, pH 8.0) extractions either successively or discretely significantly increased the sampling number. Overall, acetonitrile/water/formic acid (25:24:1) can facilitate efficient extraction of cystine-knot peptides from M. cochinchinensis seeds but for discovery purposes the use of a combination of extraction methods is recommended where practical.

Analysis of cyclotides in Viola ignobilis by Nano liquid chromatography fourier transform mass spectrometry.

Cyclotides are macrocyclic knotted peptides originating from plants. They are extremely stable and have a range of bioactivities including anti-HIV and insecticidal activity. Given the stability of the cyclotide framework, there is interest in using these peptides as scaffolds in drug design. In the current study, we have shown that nano-LC Fourier transform mass spectrometry (FTMS) is an effective method of analyzing cyclotides in plants. In addition, we have used this technique to find cyclotides in a novel species, Viola ignobilis (Violaceae plant family), which was collected from the West Azerbaijan province of Iran. Varv peptide A, cycloviolacin B2, and cycloviolacin O8 were found in this species. This study provides a novel method for directly analyzing cyclotide sequences without enzymatic digestion and further information regarding the distribution of cyclotides in plant species.

A liquid chromatography-electrospray ionization-mass spectrometry method for quantification of cyclotides in plants avoiding sorption during sample preparation.

Cyclotides are plant-produced, bioactive, cyclic mini-proteins with interesting pharmaceutical and agricultural applications. A reverse phase liquid chromatography electrospray ionization mass spectrometry (RP-LC-ESI-MS) method for analysis of cyclotides in plant materials with a minimum of sample pre-treatment is presented. Three exemplary cyclotides (kalata B1, kalata B2 and cycloviolacin O2) were used as reference substances for the method development. Linearity (r(2)>0.99) was achieved in the concentration range 0.05-10 mg/L and the limit of detection was 1.7-4.0 µg/L. The present study is the first to demonstrate that cyclotides dissolved in water sorb to glass vials, but the addition of 15% of acetonitrile or 40 mg/L of bovine serum albumin is sufficient to keep the cyclotides in solution. Cyclotides were extracted from candied violets, violet tea, and the plants Oldenlandia affinis and Viola odorata using 70% methanol containing 0.1% formic acid (v/v). The plant content was determined to be 23.5-14,200 µg/g (dry weight). The highest content of cyclotide was found in wild Danish V. odorata, and it is the highest content of cyclotide in a plant reported hitherto. Candied violets contained 0.00-8.66 µg/g (dry weight), while no cyclotides were detected in commercial violet tea.

Analysis and classification of circular proteins in CyBase.

CyBase is a database dedicated to the study of the sequences and three-dimensional structures of ribosomally synthesized, backbone cyclized proteins, and their synthetic variants. This article describes CyBase data and tools that are useful in the analysis of circular proteins. Circular proteins have now been discovered in organisms from all kingdoms of life, and given the current rate of discovery they could soon number in the thousands. Presently CyBase manages 427 protein sequences, 106 nucleic acid sequences, and 49 protein three-dimensional structures from 44 different species. Circular proteins are grouped into distinct classes according to their origin and sequence similarities. These classes include trypsin inhibitors, bacterial proteins, mushroom toxins, cyclotides, and cyclic defensins from primates. Several protein classification types are used in CyBase to designate proteins extracted from natural resources (wild type and precursor) or engineered (modified wild type, grafted, mutant, cyclic permutant, and acyclic permutant). CyBase has tools for the analysis of mass spectrum fingerprints of cyclic peptides, and assists in the discovery of new circular proteins. Some of the developments detailed here have been made specifically for the largest class of circular proteins, the cyclotides, but could be adapted for other classes of cyclic proteins. The cyclotide-specific tools include two-dimensional representations of domains and alternative displays of alignments for precursor sequences. This alignment prompted us to propose a revision of the cydclotide precursor organization, in which the repeated regions now include a small C-terminal region, which appears to have a significant role in the biosynthesis of mature cyclotides.