PRESSURE-ASSISTED AND PRESSURE-PROGRAMMED CAPILLARY ELECTROPHORESIS ELECTROSPRAY IONIZATION TIME-OF-FLIGHT - MASS-SPECTROMETRY FOR THE ANALYSIS OF PEPTIDE MIXTURES/

Pressure assisting and pressure programming the inlet of the capillary electrophoresis instrument were used for the analysis of peptide mixt ures and protein digests using capillary electrophoresis/electrospray ionization - mass spectrometry (CE/ESI-MS). CE/ESI-MS of peptide mixtu res and tryptic digests of proteins was studied using three different types of capillary columns: (i) a freshly aminopropylsilane (APS)-trea ted column. (ii) an untreated column, and (iii) a degraded APS-treated column. To maintain a constant and adequate buffer flow toward the CE capillary outlet for stable CE and ESI operation, low pressure was ap plied to the inlet of the CE when an untreated or degraded APS capilla ry was used. By programming the inlet pressure, CE/ESI-MS analysis tim e was reduced to 1/3 of its original time. The utility of this techniq ue is demonstrated by CE/ESI-MS analysis of a hemoglobin variant (hemo globin-S) and its tryptic digests. Identification of the mutant peptid e in the tryptic digest of hemoglobin-S was achieved by collision-indu ced dissociation (CID) of the protein digests using CE/ESI time of fli ght - mass spectrometry (TOF-MS).