Comprehensive Spectral Library from the Pathogenic Bacterium Streptococcus pneumoniae with Focus on Phosphoproteins

To understand bacterial reactions
to environmental stress or infection-related
processes, it is necessary to identify the involved proteins. In mass
spectrometry-based proteomics, the method of choice for spectra-to-peptide-match
is database search, but in recent times, spectral libraries have come
into focus. Here, we built a mass spectral library from Streptococcus pneumoniae D39, reflecting 76% of the
theoretical proteome of the organism. Besides the proteins themselves,
posttranslational protein modifications especially reveal central
hubs of regulation in bacterial pathogenesis. Here, for example, phosphorylation
events are involved in the signal transduction and regulation of virulence.
Although there have been major advances in phosphoproteomics, identification
of this modification is still challenging. To enhance the number of
phosphorylated peptides, which can be reproducibly detected, a comprehensive
mass spectral library of the S. pneumoniae D39 phosphoproteome has been compiled in addition to the comprehensive
total proteome mass spectral library. The phosphopeptide library was
manually validated, and the data quality was additionally proven by
analyses of synthetic phosphorylated peptides. In total, 128 phosphorylated
proteins were revealed, of which many are involved in glycolysis,
purine metabolism, protein biosynthesis, and virulence. The publicly
available, thoroughly validated spectral libraries are an excellent
resource to improve and speed up future investigations on the proteome
and phosphoproteome of pneumococci.