Application of EMMA methodology for on-capillary protein digestion in proteomic research
| Authors | |
|---|---|
| Year of publication | 2008 |
| Type | Article in Proceedings |
| Conference | Book of Abstracts 22nd International Symposium on MicroScale Bioseparations and Methods for Systems Biology |
| MU Faculty or unit | |
| Citation | |
| Field | Biochemistry |
| Keywords | capillary electrophoresis; EMMA; protein digestion; proteomics |
| Description | Sixteen years ago a new application for the evaluation of enzymatic reactions in capillary electrophoresis was proposed and developed by Bao and Regnier. In this method called electrophoretically mediated microanalysis (EMMA), the capillary is used not only as a separation medium but also as a reaction chamber. Since its discovery the EMMA methodology has been utilized in a number of biochemical systems - for assays of enzyme activities, determinations substrates, Michaelis constants, inhibitors and inhibition constants etc. In this work, the EMMA methodology was applied for the on-capillary tryptic digestion of proteins for proteomic reason. 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 trypsine 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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