Reversed-phase capillary high-performance liquid chromatography with on-line UV, fluorescence and electrospray ionization mass spectrometric detection in the analysis of peptides and proteins.

ABSTRACT

Analysis of peptide mixtures by reversed-phase capillary HPLC with gradient elution using three detectors in series UV (214 nm), fluorescence (lambda exc. = 280 nm, lambda emiss. = 356 nm), and electrospray ionization mass spectrometry (ES-MS) is reported. The chromatographic integrity of the system and the detection limits were evaluated. The effect of the mass spectrometer's acquisition rate on the total ion current (TIC) profile was also examined. The utility of fluorescence monitoring with UV and ES-MS detection was demonstrated in the analysis of proteolytic digests of proteins. The native fluorescence character of tryptophan-containing peptides provides selectivity in peptide mapping, while monitoring UV absorption at 214 nm affords detection of the peptide bond. Three tryptophan-containing tryptic peptides of bovine serum albumin were immediately located by fluorescence among many UV peaks and ES-MS provided molecular masses allowing the peptides to be identified.