Rescuing discarded spectra: Full comprehensive analysis of a minimal proteome.

A common problem encountered when performing large-scale mass spectrometry proteome analysis is the loss of information due to the high percentage of unassigned spectra. To determine the causes behind this loss we have analyzed the proteome of one of the smallest living bacteria that can be grown axenically, Mycoplasma pneumoniae (729 ORFs). M. pneumoniae cultures were grown in defined media, and their proteomes were analyzed by mass spectrometry. An initial database search with a curated M. pneumoniae protein database left around 78% of the acquired spectra without an assignment. After re-analysis of the data with the PEAKS software and a larger protein database, the percentage of non-assigned spectra was reduced to 27%, thereby increasing the proteome coverage of M. pneumoniae from the initial 60% to over 76%. Nonetheless, 33413 of spectra with assigned amino acid sequences, could not be mapped in any NCBInr database protein sequence. Approximately, 1% of these unassigned peptides corresponded to post-translational modifications and 4% to protein variants. We found that deamidation of Asn affected preferentially to integral membrane proteins and that about 1% of the peptides had repetitions of the same aromatic/hydrophobic amino acid at the N-terminus, or Arg/Lys at the C-terminus. Thus, in an ideal system, we maximized the level of assignment to 73% of the spectra (51453 out of 70040 initial acquired spectra).