Large‐scale identification of proteins of Haemophilus influenzae by amino acid composition analysis

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Abstract
Two-dimensional protein maps of microorganisms are useful tools for elucidation and detection of target proteins, a process essential in the development of new pharmaceutical products. We applied amino acid composition analysis, following separation by two-dimensional gel electrophoresis, for large-scale identification of proteins of Haemophilus influenzae. H. influenzae is a bacterium of pharmaceutical interest of which the entire genome, comprising approximately 1700 open reading frames, has been sequenced. For amino acid analysis, we used both precolumn derivatization of amino acids followed by reversed-phase chromatography of the derivatized residues and post-column derivatization of the residues previously separated on an ion exchanger. The composition analyses derived from both methods allowed the identification of 110 protein spots. The proteins were identified using the AACompIdent software on the ExPASy server accessible via the World Wide Web with a success rate of 52%. In some cases, introduction of the analysis data of 12 residues was sufficient for a correct identification. Proteins which contained an unusually high percentage of one residue could be unambiguously identified. Amino acid composition analysis proved to be an error-robust, efficient method for protein identification. The method can be practically established in every biochemical laboratory and, complementary to mass spectrometry, represents an important analytical tool for the mapping of the proteomes of organisms of interest.