Primary Structural Analysis of Cyclotides

Cyclotides are topologically complex molecules owing to the unique combination of a head-to-tail cyclized backbone and a knotted arrangement of disulfide bonds. The so-called cyclic cystine knot imparts exceptional stability to this family of peptides but brings with it a great many analytical challenges. Standard peptide sequencing approaches such as Edman degradation are not useful for studying the native peptides owing to the blocked (cyclic) termini. Over the past 20 years the number of cyclotides appearing in the literature has expanded exponentially (4 in 1994; similar to 50 in 2004; >280 in 2014) from reported discoveries in the Violaceae and Rubiaceae to reports in the Cucurbitaceae, Fabaceae, Solanaceae, and Apocynaceae. The cystine knot and the site of cyclization are conserved, but significant sequence variation occurs in the loop regions, the backbone segments between the disulfide bonds. This variation may be as subtle as a single amino acid to variation in both loop size and composition. The gene architecture varies between plant families and the majority of cyclotide-containing plant species identified to date have not been the target of genome sequencing. For these reasons, standard proteomic approaches to peptide/protein discovery are not well suited to cyclotide sequencing. The most fruitful approach to cyclotide primary structure analysis has been the application of tandem mass spectrometry. This chapter will examine the first attempts to sequence cyclotides, review the tools and techniques that have been employed to characterize cyclotides, present the challenges and complexities with their analysis, and discuss the future prospects for cyclotide discovery.