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

Ovesen, Rikke Gleerup; Göransson, Ulf; Hansen, Steen Honoré; Nielsen, John; Hansen, Hans Christian Bruun

doi:10.1016/j.chroma.2011.08.095

Cited by 17 publications

(21 citation statements)

References 27 publications

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“…Judging from LC-MS analyses, the abundance of petunia cyclotides in source plant material was within the range previously reported for cyclotides in V. odorata and O. affinis (52), with Cter A in wet leaf material estimated to be 30.0 g/g, whereas Cter K and Cter M were present in wet root material at 2.3 and 7.6 g/g, respectively.…”

Section: Detection and Sequencing Of Cyclotides And Acyclotides Frommentioning

confidence: 72%

Cyclotides Associate with Leaf Vasculature and Are the Products of a Novel Precursor in Petunia (Solanaceae)

Poth

Mylne

Grassl

et al. 2012

Journal of Biological Chemistry

145

139

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Background: Cyclotides are defense-related cyclic plant peptides. Results: Petunia cyclotides are encoded by novel cyclotide genes and occur in a discrete pattern in leaf architecture. Conclusion: Novel cyclotides exist in the Solanaceae and are abundant in vascular tissues. Significance: Cyclotide localization is consistent with an anti-herbivory role. Novel Solanaceae genes provide opportunities for expressing designer cyclic peptides in major crop species.

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Section: Detection and Sequencing Of Cyclotides And Acyclotides Frommentioning

confidence: 72%

Cyclotides Associate with Leaf Vasculature and Are the Products of a Novel Precursor in Petunia (Solanaceae)

Poth

Mylne

Grassl

et al. 2012

Journal of Biological Chemistry

145

139

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“…The development of selective and robust methods for the analysis of cyclotides in plasma and tissues is highly important for the continued investigation of the usefulness of these compounds in drug development. The currently published methods focus mainly on the analysis of cyclotides from plant extracts or are not sensitive enough to be optimal for peptide quantification in samples from in vivo experiments . The principles of quantitative analysis of peptides in biological matrices has been reviewed by van den Broek et al in 2008 and highlights the specific considerations that must be taken when analysing peptides with LC‐MS systems …”

Section: Introductionmentioning

confidence: 99%

Improved method for quantitative analysis of the cyclotide kalata B1 in plasma and brain homogenate

et al. 2016

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This study provides a new method for quantifying the cyclotide kalata B1 in both plasma and brain homogenate. Cyclotides are ultra‐stable peptides with three disulfide bonds that are interesting from a drug development perspective as they can be used as scaffolds. In this study we describe a new validated LC‐MS/MS method with high sensitivity and specificity for kalata B1. The limit of quantification was 2 ng/mL in plasma and 5 ng/gmL in brain homogenate. The method was linear in the range 2–10,000 ng/mL for plasma and 5–2000 ng/g for brain. Liquid Chromatographic separation was performed on a HyPurity C18 column, 50 × 4.6 mm, 3 µm particle size. The method had inter‐ and intra‐day precision and accuracy levels <15% and 12% respectively. Applying the method to in vivo plasma samples and brain homogenate samples from equilibrium dialysis yielded satisfying results and was able to describe the plasma pharmacokinetics and brain tissue binding of kalata B1. The described method is quick, reproducible and well suited to quantifying kalata B1 in biological matrices.

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“…To date, more than 300 different cyclotides have been isolated from about 40 plant species and six different plant families (Violaceae, Solanaceae, Rubiaceae, Fabaceae, Poaceae, and Cucurbitaceae) according to Cybase, the database of cyclic proteins (http://www.cybase.org.au). This unique family of cyclic peptides is defined by three disulfide bonds arranged in a cyclic cystine knot (CCK) motif (Figure ), making these peptides highly resistant to enzymatic, thermal, and chemical degradation . Cyclotides have recently attracted attention in drug development because of this stability and the possibilities for generating diverse sequences and activities in loops between cysteines .…”

Section: Introductionmentioning

confidence: 99%

“…Cyclotides can be divided into two major subfamilies, Möbius and bracelet, depending on the presence/absence of a cis ‐Pro peptide linkage (Figure ) . Plants containing cyclotides express them as complex mixtures . Thus, purification of individual cyclotides is, in many cases, not straightforward, requiring multiple steps of chromatography .…”

Section: Introductionmentioning

confidence: 99%

Single‐step purification of cyclotides using affinity chromatography

et al. 2017

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Cyclotides are considered promising scaffolds for drug development owing to their inherent host defence activities and highly stable structure, defined by the cyclic cystine knot. These proteins are expressed as complex mixtures in plants. Although several methods have been developed for their isolation and analysis, purification of cyclotides is still a lengthy process. Here, we describe the use of affinity chromatography for the purification of cyclotides using polyclonal IgG antibodies raised in rabbits against cycloviolacin O2 and immobilized on NHS-activated Sepharose columns. Cycloviolacin O2 was used as a model substance to evaluate the chromatographic principle, first as a pure compound and then in combination with other cyclotides, that is, bracelet cyclotide cycloviolacin O19 and Möbius cyclotide kalata B1, and in a plant extract. We demonstrate that single-step purification of cyclotides by affinity chromatography is possible but cross reactivity may occur between homologue cyclotides of the bracelet subfamily.

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

Cited by 17 publications

References 27 publications

Cyclotides Associate with Leaf Vasculature and Are the Products of a Novel Precursor in Petunia (Solanaceae)

Cyclotides Associate with Leaf Vasculature and Are the Products of a Novel Precursor in Petunia (Solanaceae)

Improved method for quantitative analysis of the cyclotide kalata B1 in plasma and brain homogenate

Single‐step purification of cyclotides using affinity chromatography

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