REVERSED-PHASE CAPILLARY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH ONLINE UV, FLUORESCENCE AND ELECTROSPRAY-IONIZATION MASS-SPECTROMETRICDETECTION IN THE ANALYSIS OF PEPTIDES AND PROTEINS

Analysis of peptide mixtures by reversed-phase capillary HPLC with gra dient elution using three detectors in series: UV (214 nm), fluorescen ce (lambda(exc) = 280 nm, lambda(emiss.) = 356 nm), and electrospray i onization mass spectrometry (ES-MS) is reported. The chromatographic i ntegrity of the system and the detection limits were evaluated. The ef fect of the mass spectrometer's acquisition rate on the total ion curr ent (TIC) profile was also examined. The utility of fluorescence monit oring 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 pe ptide bond. Three tryptophan-containing tryptic peptides of bovine ser um albumin were immediately located by fluorescence among many UV peak s and ES-MS provided molecular masses allowing the peptides to be iden tified.