What are Proteomic methods?

Proteomic methods

 

1. 2D protein electrophoresis

Nowadays the two-dimensional (2D) protein electrophoresis is a method of choice for protein separation. This method enables to distinguish up to 10 000 proteins. The method is based on two distinct physical and chemical features of proteins: first, the proteins are separated according to their isoelectric point (pI). Isoelectric point is such a pH value, where the overall protein charge equals to zero. This can be obtained by creating a pH gradient in the gel where protein is loaded and electric current is applied. Proteins then migrate towards cathode or anode according to their total charge up to the point where the gel pH equals pI of a given protein. After the separation in the first direction, common electrophoresis on a polyacrylamide gel (PAGE) is applied, but the electric current is applied perpendicular to the original orientation of electrodes. Proteins then migrate in the second direction through gel only according to their size. After both phases of 2D electrophoresis, it is necessary to visualize proteins by one of the staining or labeling methods (chemical or radioactive). The resulting "maps" of proteins can be compared for example in between the experimental and control sample or among the samples from patients with specific disease and their healthy controls and thus identify differentially expressed proteins that can be linked with the pathogenesis of the studied disease. The identity of differentially expressed proteins is verified after "cutting out" the area of the gel with given difference and subsequent analysis using mass spectrometry. 

Figure 2. 2D electrophoresis. After isoelectric focusing in pH gradient of the electric field the electrophoretic separation is used in polyacrylamide gel (SDS-PAGE….sodium dodecylsulphate - polyacrylamide gel electrophoresis). Proteins can be visualized after staining (on the picture in Coomasie blue).

2. Mass spectrometry (MS)

Mass spectrometry is a method that enables precise measurement of molecular weight of a broad spectrum of substances. As the studied substance has to be intact in gas phase, the use of mass spectrometry for protein analysis (but also of polysaccharides and oligonucleotides) was only enabled by development of "soft" ionization techniques of mass spectrometry, like matrix assisted laser detection of desorption/ionization (MALDI) and electrospray ionization (ESI). Protein identification is generally performed in two ways:

1. Protein is digested by trypsin or by other proteolytic enzyme to smaller peptides and their precise molecular weights are measured using MS. The spectrum of those molecular weights is then compared with theoretical spectra that are calculated from protein sequences from available databases (using bioinformatics tools).
2. Tandem MS enables to choose the peptide which is then fragmented by the collision with inert gas. The fragmentation pattern gives either full of partial information about protein sequence that is subjected to the search in databases.

Apart of protein identification, MS is a priceless tool for protein posttranslational modification analysis, because it enables to localize given modifications within the protein and also helps to find out the nature of such modification.

Source: http://biol.lf1.cuni.cz/ucebnice/en/proteomics.htm