REFERENCE MAP OF THE LOW-MOLECULAR-MASS PROTEINS OF HAEMOPHILUS-INFLUENZAE

Analysis of the proteome of Haemophilus influenzae by two-dimensional polyacrylamide gel electrophoresis on conventional Tris-glycine gels d oes not usually result in efficient separation of the proteins in the 5-20 kDa range, which are mainly accumulated in the lower acidic and b asic regions. In order to improve the separation of the low molecular mass proteins, we used homogeneous Tricine gels of two urea concentrat ions in the second-dimensional separation. The Tricine gel systems all owed the efficient and reproducible separation of the proteins of the microorganism with masses between 5 and 20 kDa, however, no proteins w ith masses below 5 kDa could be visualized. Approximately 80 proteins migrating in the 5-25 kDa region were identified by matrix assisted la ser desorption/ionization - mass spectrometry, of which 40 identified for the first time. The digestion of the low mass proteins often produ ced only few peptides, which were insufficient for confident identific ation by mass spectrometry. Therefore, the identification was occasion ally achieved by a sequential digestion with two proteases, trypsin or endoproteinase Lys-C as first and carboxypeptidase P as second enzyme . The gel system described may be useful for the efficient separation of low molecular mass proteins from other organisms to construct stand ard maps.