Rescuing discarded spectra: Full comprehensive analysis of a minimal proteome

In the present paper we have studied the proteome of Mycoplasma pneumoniae in order to find out why we have a high percentage of unassigned spectra when performing large-scale MS proteome analysis. The proteome of M. pneumonia cells, grown in defined media, was analyzed by MS. An initial search resulted in around 79% of the acquired spectra not having an assignment. The percentage of non-assigned spectra was reduced to 27% after re-analysis of the data with the PEAKS software, thereby increasing the proteome coverage of M. pneumoniae from the initial 60% to over 76%. To sum up, it is shown that combining a de novo and database search approach together with consideration of PTMs and amino acid substitutions, increases the percentage of assigned spectra.