PROTEIN ANALYSIS

Proteins are responsible for maintaining all cellular functions and their production is governed by the genetic code. A disease may be the result of gene mutations that cause changes in the structure and activity of a protein. Therefore, characterising proteins and understanding their function is important for identifying novel drug targets and designing more effective medicines. Protein analysis is the study of the total protein content of a cell type or organism. It gives a better understanding of the function of genes and the proteins for which they code. This knowledge is of fundamental importance for the development of molecular medicine. Protein analysis is a complex process that requires multiple steps and advanced technologies. Two of the most important technologies are Mass Spectometry and 2D gel elctroforesis.

Mass Spectometry

Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS) is a relatively novel technique in which a co-precipitate of an UV-light absorbing matrix and a biomolecule is irradiated by a nanosecond laser pulse. Most of the laser energy is absorbed by the matrix, which prevents unwanted fragmentation of the biomolecule. The ionised biomolecules are accelerated in an electric field and enter the flight tube. During the flight in this tube, different molecules are separated according to their mass to charge ratio and reach the detector at different times. In this way each molecule yields a distinct signal. The method is used for detection and characterisation of biomolecules, such as proteins, peptides, oligosaccharides and oligonucleotides, with molecular masses between 400 and 350,000 Da. It is a very sensitive method, which allows the detection of low amounts of molecules.

Protein identification by this technique has the advantage of short measuring time (few minutes) and negligible sample consumption (less than 1 pmol) together with additional information on microheterogeneity (e.g. glycosylation) and presence of by-products. Although molecular biology has provided powerful techniques for DNA analysis, this is not yet reflected in protein analysis. Genome sequencing has yielded a wealth of information on predicted gene products, but for the majority of the expressed proteins no function is known. Proteomics is an important new field of study of protein properties (expression levels, interactions, post-translational modifications etc.) and thus can be described as functional genomics at the protein level. The mass accuracy of MALDI-TOF MS will be sufficient to characterise proteins (after tryptic digestion) from completely sequenced genomes.

(http://www-micrbiol.sci.kun.nl/tech/malditof.html)

2D Gel electrophoresis

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 immobilised 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.