ONLINE MICROREACTORS CAPILLARY ELECTROPHORESIS MASS-SPECTROMETRY FOR THE ANALYSIS OF PROTEINS AND PEPTIDES

We report the use of trypsin and carboxypeptidase Y-modified capillary microreactors (50 mu m i.d. and 20 nL volume per centimeter length) in combination with mass spectrometry for peptide molecular mass mapping of various peptides and proteins. Advantages of immobilized enzyme capillary microreactors include picoliter to nanoliter volume requirements, longer enzyme lifetimes, higher stability, and the ability to reuse enzymes conveniently, Additionally, extremely efficient sample handling modes are used, and the reaction products are easily separated from the enzyme reagents. Plasma desorption and matrix-assisted laser desorption/ionization were used for off-line analyses of digestion products, The use of MassMap for the identification of proteins is also discussed, Finally, a trypsin microreactor was integrated on-line with capillary electrophoresis/ion spray mass spectrometry for fast peptide mapping. Digestion of the oxidized insulin B-chain in an on-line trypsin microreactor and electromigration of aliquots from the capillary microreactor into the CE separation capillary allowed the entire peptide mapping procedure to be completed in <1 h. The online technique is especially well-suited for the characterization of minute quantities of proteins, because it transfers picoliter to nanoliter volumes of digestion products with minimum sample handling which could lead to losses or contamination.