LARGE-SCALE IDENTIFICATION OF PROTEINS OF HAEMOPHILUS-INFLUENZAE BY AMINO-ACID-COMPOSITION ANALYSIS

Two-dimensional protein maps of microorganisms are useful tools for el ucidation and detection of target proteins, a process essential in the development of new pharmaceutical products. Mie applied amino acid co mposition analysis, following separation by two-dimensional gel electr ophoresis, for large-scale identification of proteins of Haemophilus i nfluenzae. N. influenzae is a bacterium of pharmaceutical interest of which the entire genome, comprising approximately 1700 open reading fr ames, has been sequenced. For amino acid analysis, we used both precol umn derivatization of amino acids followed by reversed-phase chromatog raphy of the derivatized residues and post-column derivatization of th e residues previously separated on an ion exchanger. The composition a nalyses derived from both methods allowed the identification of 110 pr otein spots. The proteins were identified using the AACompIdent softwa re on the ExPASy server accessible via tile World Wide Web with a succ ess rate of 52%. In some cases, introduction of the analysis data of 1 2 residues was sufficient for a correct identification. Proteins which contained an unusually high percentage of one residue could be unambi guously identified. Amino acid composition analysis proved to be an er ror-robust, efficient method for protein identification. The method ca n be practically established in every biochemical laboratory and, comp lementary to mass spectrometry, represents an important analytical too l for the mapping of the proteomes of organisms of interest.