Integration of on-line protein digestion by trypsin in capillary electrophoresis by means of electrophoretically mediated microanalysis
| Authors | |
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| Year of publication | 2009 |
| Type | Article in Periodical |
| Magazine / Source | Electrophoresis |
| MU Faculty or unit | |
| Citation | |
| Field | Biochemistry |
| Keywords | Proteomics; Tryptic digestion; Electrophoretically mediated microanalysis; EMMA; Partial filling technique; Sandwich injection |
| Description | In this work, electrophoretically mediated microanalysis (EMMA) was applied to the in-capillary tryptic digestion of proteins for proteomic purposes. Compared to classical in-solution tryptic digestion or the trypsin reactor commonly used for this purpose, the EMMA based method is rapid, can be automated and requires only small amounts of trypsin and protein preparations. Moreover, the protein digestion and the analysis of the resulting peptides are integrated into one procedure. A combination of the EMMA methodology with a partial filling technique was used in this study, since the pH optimum of the trypsin reaction differs strongly from the best pH for the CE separation of peptides. In this setup, part of the capillary is filled with the best buffer for the tryptic digestion (50 mM Tris-HCl buffer, pH 8.5) whereas the rest of the capillary is filled with the background electrolyte optimal for peptide separation (0.1 M phosphate buffer, pH 2.5). |
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