A simple procedure is proposed for selective protein solubilization and trypsin digestion, followed by off-line liquid chromatography-matrix assisted laser desorption ionization mass spectrometry (LC-MALDI MS) analysis of Oenococcus oeni (O. oeni) bacterium. Peptides were identified from tryptic digests using sequencing by tandem mass spectrometry and database searches. Cytoplasmic and membrane related proteins (MRP) were identified in the O. oeni bacterium. MS/MS data analysis points out 13 peptides having one point mutation from 9 proteins. The major microheterogeneity was found for Zn-dependent alcohol dehydrogenase (Zn-ADH, Q04GE6) and 60 kDa chaperonin (GroEL, Q04E64) that are involved in methionine catabolism and post-translational protein folding, respectively. MS/MS data processing also leads to the identification of 34 unique phosphorylation sites from 19 phosphoproteins.