With the publication of the complete genomes of organisms, a new scientific discipline, proteomics, has arisen. By comparing the proteinaceous composition (the proteome) of microorganisms, the contribution of differentially expressed proteins to specific microbial traits can be assessed. The proteome is visualised by 2D-gelelectrophoresis and proteins of interest can be identified on the genome by MALDI-TOF MS.
Two dimensional gel electrophoresis separates proteins first on their isoelectric point and in the second dimension on their molecular mass. Through hydration proteins are absorbed by dried gelstrips which carry an immobilized pH gradient. The hydrated first dimensional gel is subjected to a strong electric field. Acid proteins at the alkaline side of the gel will dissociate and become negatively charged. Because of the electric field these proteins will migrate to the positive pole (the acidic side of the gel). Proteins will reach a point where they become neutralised by the gel, lose their net charge and do not migrate further. The same scenario applies to basic proteins. With the currently available equipment it is possible to reproducably separate proteins which differ in only a single pH unit over 24 cm.
One of the current proteomics projects is the identification of proteins in Methanothermobacter thermoautotrophicus. In this project we study the differential expression of proteins as a function of the hydrogen concentration. The department uses Pharmacia IPGphor en Multiphor III electrophoresis equipment.
