Integration of on-line protein digestion by trypsin in CZE by means of electrophoretically mediated microanalysis.
In this work, electrophoretically mediated microanalysis (EMMA) was applied to the in-capillary tryptic digestion of proteins for proteomic purposes. Compared with 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 a small amount of trypsin preparation. 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 CZE separation of peptides. In this set-up, a 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 BGE optimal for peptide separation (0.1 M phosphate buffer, pH 2.5). As the proteins differ in their isoelectric points, a sandwich type of injection was used. The analysed protein is thus injected between two trypsin zones, which ensures their mixing and digestion. The analysis of one protein comprising both the digestion and the peptide separation is then completed in 1 h using a commercial instrument for CE with no modifications.
Zeisbergerová
Marta
M
Department of Biochemistry, Faculty of Science, Masaryk University, Brno, Czech Republic.
Adámková
Andrea
A
Glatz
Zdenek
Z
D000818:Animals; D002364:Caseins / Q000737:chemistry; D045304:Cytochromes c / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods*; D007328:Insulin / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D014357:Trypsin / Q000737:chemistry*
D002364:Caseins; D007328:Insulin; D010446:Peptide Fragments; D011506:Proteins; D045304:Cytochromes c; D014357:Trypsin