title: A comparative study of extraction methods reveals preferred solvents for cystine knot peptide isolation from Momordica cochinchinensis seeds.
abstract: 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.
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia. Electronic address: t.mahatmanto@imb.uq.edu.au.
authors.forename: Tunjung
authors.initials: T
authors.lastname: Mahatmanto
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia. Electronic address: a.poth@imb.uq.edu.au.
authors.forename: Aaron G
authors.initials: AG
authors.lastname: Poth
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia; School of Chemistry and Biochemistry & ARC Centre of Excellence in Plant Energy Biology, The University of Western Australia, Crawley, Perth, Western Australia 6009, Australia. Electronic address: joshua.mylne@uwa.edu.au.
authors.forename: Joshua S
authors.initials: JS
authors.lastname: Mylne
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia. Electronic address: d.craik@imb.uq.edu.au.
authors.forename: David J
authors.initials: DJ
authors.lastname: Craik
mesh_terms: D000595:Amino Acid Sequence; D047169:Cyclotides / Q000032:analysis / Q000302:isolation & purification*; D047168:Cystine Knot Motifs; D008969:Molecular Sequence Data; D029775:Momordica / Q000737:chemistry*; D010936:Plant Extracts / Q000737:chemistry / Q000302:isolation & purification*; D010940:Plant Proteins / Q000032:analysis / Q000302:isolation & purification; D012639:Seeds / Q000737:chemistry; D012997:Solvents / Q000737:chemistry
chemical_list: D047169:Cyclotides; D010936:Plant Extracts; D010940:Plant Proteins; D012997:Solvents
keywords: Cyclotides; Cystine knot peptides; Extraction methods; Momordica cochinchinensis seeds