mMass as a Software Tool for the Annotation of Cyclic Peptide Tandem Mass Spectra Timo H. J. Niedermeyer1, Martin Strohalm2 1 Institute of Pharmacy, EMA-University, Friedrich-Ludwig-Jahn-Str. 17, 17489 Greifswald, Germany, and Cyano Biotech GmbH, Magnusstr. 11, 12489 Berlin, Germany 2 Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i., Vídeňská 1083, 142 20 Prague, Czech Republic Introduction Natural or synthetic cyclic peptides often possess pronounced bioactivity. Their mass spectrometric characterization is difficult due to the predominant occurrence of non-proteinogenic monomers and the complex fragmentation patterns observed. Even though several software tools for cyclic peptide tandem mass spectra annotation have been published, these tools are still unable to annotate a majority of the signals observed in experimentally obtained mass spectra. They are thus not suitable for extensive mass spectrometric characterization of these compounds. The lack of an advanced and user-friendly software tool has motivated us to extend the fragmentation module of a freely available open-source software, mMass (http://www.mmass.org), to allow for cyclic peptide tandem mass spectra annotation and interpretation. The resulting software has been tested on several cyanobacterial and other naturally occurring peptides and has been found to be superior to other tools currently available concerning both usability and annotation extensiveness. Thus it is highly useful for accelerating the structure confirmation and elucidation of cyclic as well as linear peptides and depsipeptides. Main Features and Implemented Fragmentation Pathways • off-line tool with advanced spectrum manipulation and processing abilities (e.g. support for various data formats, baseline correction, peak picking etc.) • ability to save spectra, sequences and interpretation results for future use • monomer database and editor for easy monomer and sequence management • matching of calculated theoretically possible fragments to and annotation of experimental data • backbone fragmentation pathways after initial break-up to a b-like ion resulting in a-, b-, c-, x-, y-, and z-like fragment ions (Figure 1) • alternative initial a/x-like ring opening resulting in +CO fragment ions (Figure 2) • sequence scrambling • neutral losses of e.g. H2O and NH3 as well as custom side chain losses from individual monomers • multiple neutral losses from one fragment • formation of b+H2O fragment ions (Figure 3) Figure 1 Backbone fragmentation of a cyclic peptide taking Microcystin LF as an example. Figure 3 Proposed mechanism for the formation of observed b+H2O ions of Cryptophycin-1. Microcystin LF Cryptophycin-1 NRP-Annotation 23 32 mMass* 23 32 mMass** 95 80 Figure 1 Proposed pathway for the formation of an observed N-formyl fragment ion of microcystin LF after initial protonation of D-alanine. Figure 5 Tandem mass spectra of Microcystin LF (A) and cryptophycin-1 (B). Signals discussed are indicated with a star (filled star: not annotated by NRP-Annotation). Table 1 Percentage of the ion intensity annotated using NRP-Annotation or mMass. * options chosen reflecting the fragments NRP-Annotation can calculate ** all available options chosen Case Study I – Microcystin LF The fragmentation behavior of microcystins, cyclic heptapeptides found in several cyanobacterial genera, has been very well studied. Thus microcystin LF (Figure 4) has been used to examine the accuracy and comprehensiveness of our fragmentation algorithms and to compare the results to the software NRP-Annotation. The tandem mass spectrum of microcystin LF (Figure 5) shows several significant peaks that had not been assigned by NRP-annotation, but were annotated by mMass. • m/z 478.2644 annotated as c4[7|1][1-4] (Δ 3.3 ppm) • m/z 561.3017 annotated as c5[6|7][1-5] (Δ 2.5 ppm) • m/z 559.3127 annotated as z5[2|3][3-7] - C9H10O, at (Δ 0.2 ppm) • m/z 693.3816 annotated as z5[2|3][3-7] (Δ 3.1 ppm) * • m/z 852.4