Two-dimensional gel electrophoresis in combination with mass spectrometry has already been applied successfully to study beer proteome. Due to the abundance of protein Z in beer samples, prefractionation techniques might help to improve beer proteome coverage. Proteins from four lager beers of different origins were separated by two-dimensional electrophoresis (2-DE) followed by tandem mass spectrometric analysis. Initially 52 proteins mostly from Hordeum vulgare (22 proteins) and Saccharomyces species (25 proteins) were identified. Preparative isoelectric focusing by OFFGEL Fractionator was applied prior to 2-DE to improve its resolution power. As a result of this combined approach, a total of 70 beer proteins from Hordeum vulgare (30 proteins), from Saccharomyces species (31 proteins), and from other sources (9 proteins) were identified. Of these, 37 proteins have not been previously reported in beer samples.