Využití elektroforeticky zprostředkované mikroanalýzy v proteomice pro on-line tryptické štěpení proteinů
| Title in English | Electrophoretically mediated microanalysis as a tool in proteomic research |
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| Authors | |
| Year of publication | 2008 |
| Type | Article in Proceedings |
| Conference | XXI. biochemický sjezd ČSBMB a SSBMB |
| MU Faculty or unit | |
| Citation | |
| Field | Biochemistry |
| Keywords | kapilární elektroforéza; EMMA; proteomika |
| Description | Proteomics is a rapidly developing field of biochemical research which aims to characterize complex mixtures of proteins extracted from cells, tissues or organisms. The predominant method of proteom analysis involves the proteins separation by two-dimensional electrophoresis (2D-PAGE) following by protein spots visualization and proteolytical digestion. However 2D-PAGE has serious limitations. Although it is able to resolve thousands of proteins in a single run, 2D-PAGE is also relatively labour-intensive, time-consuming and has both sensitivity and dynamic range limits. In this consequence new high efficient separation methods are looked for especially their direct combinations with MS or even MS/MS. Capillary electrophoresis (CE) could be the other method of the choice for this purpose since the CE analyses are rapid, can be automated, and require only small amounts of samples. Moreover CE based systems using the multi-capillaries formats or the microfabricated multiple-channel chips have a great potential for high-throughput analyses. The electrophoretically mediated microanalysis (EMMA) methodology is probably the best candidate for this purpose because its main feature is full automatization combined with very low sample consumption. The reason is that this CE modification introduced by Bao and Regnier in 1992 uses the capillary not only as a separation medium but also as a reaction chamber for the enzymatic and even non-enzymatic reactions. This work aims to apply the EMMA methodology for on-capillary protein digestion in proteomic research with planned possibility of MS connection. The combination of the EMMA methodology with a partial filling technique was used in this study since the pH optimum of trypsin reaction strongly differs from the pH best for the CE separation of peptides. In this set-up the part of the capillary is filled with the buffer best for the tryptic digestion (Tris-HCl buffer pH 8.5) whereas the rest of the capillary with the background electrolyte optimal for peptides separation and certain detection system (phosphate or formate buffer pH 2.5). As the proteins differ in pI the sandwich type of injection was used. The analysed protein is thus injected between two trypsin zones that should ensure their mixing and digestion. The analysis of one protein comprising both the digestion and the peptides separation is thus finished in 1 hour. Compared to in-solution tryptic digestion or trypsin reactors commonly used for this purpose, the EMMA digestion is rapid, can be automated and relatively easily connected with MS detection, and requires only small amount of protein sample. |
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