Pressure-assisted and pressure-programmed capillary electrophoresis/electrospray ionization time of flight-mass spectrometry for the analysis of peptide mixtures.

Abstract

Pressure assisting and pressure programming the inlet of the capillary electrophoresis instrument were used for the analysis of peptide mixtures and protein digests using capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS). CE/ESI-MS of peptide mixtures and tryptic digests of proteins was studied using three different types of capillary columns: (i) a freshly aminopropylsilane (APS)-treated 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 applied to the inlet of the CE when an untreated or degraded APS capillary was used. By programming the inlet pressure, CE/ESI-MS analysis time was reduced to 1/3 of its original time. The utility of this technique is demonstrated by CE/ESI-MS analysis of a hemoglobin variant (hemoglobin-S) and its tryptic digests. Identification of the mutant peptide in the tryptic digest of hemoglobin-S was achieved by collision-induced dissociation (CID) of the protein digests using CE/ESI time of flight-mass spectrometry (TOF-MS).