To implement the 2-DE database of serogroup A Neisseria meningitidis (MenA) and improve its potential of investigation in bacterial biology, cell extracts were separated by tricine-SDS-PAGE and 131 novel proteins were identified by microLC-ESI-IT-MS/MS. These identifications extended to 404, the number of MenA gene expression products characterized at the proteome level, approximately covering 20% of the total ORFs predicted from genome sequence. This technical approach was particularly useful in ascertaining expression of ribosomal as well as hypothetical proteins. Particular attention was paid to functional characterization of hypothetical proteins by means of software analyses and database searches.