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Reference map of the low molecular mass proteins of Haemophilus influenzae
Author(s) -
Fountoulakis Michael,
Juranville JeanFrançois,
Röder Daniel,
Evers Stefan,
Berndt Peter,
Langen Hanno
Publication year - 1998
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150191046
Subject(s) - chromatography , molecular mass , mass spectrometry , tricine , proteome , chemistry , trypsin , bottom up proteomics , gel electrophoresis , polyacrylamide gel electrophoresis , two dimensional gel electrophoresis , biochemistry , protein mass spectrometry , proteomics , electrospray ionization , enzyme , gene
Abstract Analysis of the proteome of Haemophilus influenzae by two‐dimensional polyacrylamide gel electrophoresis on conventional Tris‐glycine gels does not usually result in efficient separation of the proteins in the 5–20 kDa range, which are mainly accumulated in the lower acidic and basic regions. In order to improve the separation of the low molecular mass proteins, we used homogeneous Tricine gels of two urea concentrations in the second‐dimensional separation. The Tricine gel systems allowed the efficient and reproducible separation of the proteins of the microorganism with masses between 5 and 20 kDa, however, no proteins with masses below 5 kDa could be visualized. Approximately 80 proteins migrating in the 5–25 kDa region were identified by matrix assisted laser desorption/ionization ‐ mass spectrometry, of which 40 identified for the first time. The digestion of the low mass proteins often produced only few peptides, which were insufficient for confident identification by mass spectrometry. Therefore, the identification was occasionally 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 standard maps.