CYCLONE—A Utility for De Novo Sequencing of Microbial Cyclic Peptides

Abstract

We have developed a de novo sequencing software tool (CYCLONE) and applied it for determination of cyclic peptides. The program uses a non-redundant database of 312 nonribosomal building blocks identified to date in bacteria and fungi (more than 230 additional residues in the database list were isobaric). The software was used to fully characterize the tandem mass spectrum of several cyclic peptides and provide sequence tags. The general strategy of the script was based on fragment ion pre-characterization to accomplish unambiguous b-ion series assignments. Showcase examples were a cyclic tetradepsipeptide beauverolide, a cyclic hexadepsipeptide roseotoxin A, a lasso-like hexapeptide pseudacyclin A, and a cyclic undecapeptide cyclosporin A. The extent of ion scrambling in smaller peptides was as low as 5 % of total ion current; this demonstrated the feasibility of CYCLONE de novo sequencing. The robustness of the script was also tested against database sets of various sizes and isotope-containing data. It can be downloaded from the http://ms.biomed.cas.cz/MSTools/ website. ᅟ

CycloBranch: De Novo Sequencing of Nonribosomal Peptides from Accurate Product Ion Mass Spectra

“…The sequence has been reported among peptide sequence candidates at the positions 1-2 of a result list of NRP sequence candidates. Beauverolide can also be easily identified using D 33 by Cyclone if the number of (four) monomers in the peptide was predicted [23].…”

“…All monomers in the database were annotated by reference numbers publicly available through the chemical databases ChemSpider, PubChem, Protein Data Bank (PDB), and Norine. Three databases of building blocks were used for software evaluation-a database D 19 of proteinogenic amino acids (19 building blocks where leucine and isoleucine were not distinguished), a database D 33 (all building blocks involved in 11 testing peptides), or D 287 (the complete nonredundant nonribosomal database) [23]. Data processing was performed on a desktop personal computer with the processor Intel Core i7-3770 (3.4GHz), 8 GB RAM, and OS Windows 7 (64-bit).…”

“…In cyclic peptides up to k possible ring primary ring opening sites exist for a cyclic NRP with k building blocks. The initial ring cleavage may create up to k different linear ions i-j b k (i-j stands for positions of building blocks between which the ring is primarily opened) [23,26]. For example, the ions b k may undergo a further fragmentation, so the spectrum of a cyclic peptide usually represents the superposition of up to k spectra corresponding to different linear sequences [10].…”

“…In our previous work, we introduced a hypertext preprocessor (PHP) script Cyclone for identification and de novo sequencing of cyclic NRPs [23]. It involved a simple text interface and required manual conversion of NRP sequence candidates from one PHP script to another.…”

Exploitation of the Ornithine Effect Enhances Characterization of Stapled and Cyclic Peptides

“…Two of the most recent algorithms are CYCLONE and CycloBranch, which were developed to characterize cyclic peptides via a de novo strategy [48,49]. An in-house algorithm was developed in C# to assign fragment ions of cyclic peptides based on interpretation of 193 nm UVPD mass spectra.…”