On a search for a software tool satisfying as many as possible of the criteria discussed above, one of the authors (TN) identified the software mMass to be the most suitable basis for further development. mMass is a portable and cross-platform open-source software for mass spectra processing, written mainly in Python [37] (link), which already offered a GUI, spectrum manipulation capabilities, the possibility to enter and save peptide sequences and do in-silico fragmentation and spectrum annotation. However, entering and fragmentation of peptides was limited to linear sequences and proteinogenic amino acids, and a user-editable monomer database as well as several fragmentation options important for cyclic peptides were missing. Since mMass is open-source, the software could be modified to meet all of the criteria described above.
First of all, a monomer library editor has been designed, giving the user the possibility to conveniently enter, edit and save monomers needed for the composition of peptide sequences. The library has been filled with all monomers compiled from the NORINE database [16] , facilitating its use for new users and allowing for easy addition of own variants. Furthermore, the monomer editor allows for the definition of possible neutral losses from individual monomers.
Secondly, the sequence organization and handling has been extended to enable the composition of peptides containing non-proteinogenic amino acids. For this purpose, an additional sequence editor has been designed that allows the convenient composition of peptides by typing or dragging and dropping monomers from the monomer library right into the editor. In addition, a peptide can be set as being linear or cyclic.
Finally, the fragmentation module has been improved to handle all possible known fragmentation pathways of cyclic peptides, to allow for the loss of custom neutrals, and to allow for multiple neutral losses from one fragment.
To assess the capabilities of the resulting software package, several cyanobacterial natural product MS2 spectra have been annotated, and the annotations have been compared to those made by NRP-Annotation.
First of all, a monomer library editor has been designed, giving the user the possibility to conveniently enter, edit and save monomers needed for the composition of peptide sequences. The library has been filled with all monomers compiled from the NORINE database [16] , facilitating its use for new users and allowing for easy addition of own variants. Furthermore, the monomer editor allows for the definition of possible neutral losses from individual monomers.
Secondly, the sequence organization and handling has been extended to enable the composition of peptides containing non-proteinogenic amino acids. For this purpose, an additional sequence editor has been designed that allows the convenient composition of peptides by typing or dragging and dropping monomers from the monomer library right into the editor. In addition, a peptide can be set as being linear or cyclic.
Finally, the fragmentation module has been improved to handle all possible known fragmentation pathways of cyclic peptides, to allow for the loss of custom neutrals, and to allow for multiple neutral losses from one fragment.
To assess the capabilities of the resulting software package, several cyanobacterial natural product MS2 spectra have been annotated, and the annotations have been compared to those made by NRP-Annotation.
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