ENHANCED SENSITIVITY FOR PEPTIDE-MAPPING WITH ELECTROSPRAY LIQUID-CHROMATOGRAPHY MASS-SPECTROMETRY IN THE PRESENCE OF SIGNAL SUPPRESSION DUE TO TRIFLUOROACETIC ACID-CONTAINING MOBILE PHASES

A method is described for improving the sensitivity of peptide mapping with electrospray liquid chromatography-mass spectrometry using trifl uoroacetic acid (TFA) containing HPLC mobile phases. The signal suppre ssing effects of TFA are shown to be due to the combined effect of ion -pairing and surface tension modifications. The post-column addition o f a propionic acid-2-propanol (75:25, v/v) in a 1:2 proportion with th e HPLC mobile phase counteracts the deleterious effects of TFA resulti ng in 10-100 x improvement of the signal-to-noise ratio. The system de scribed introduces total HPLC flow (plus additive) directly into the e lectrospray source without splitting. Using 2.1 mm I.D. HPLC columns, minimum detectable quantities are below 40 pmol total protein. As exam ples, separations of proteolytic enzyme digests of several proteins ar e shown using standard HPLC conditions, comparing results with and wit hout the addition of propionic acid. The application of the technique is shown in more depth in the identification of oxidative modification sites in glutamine synthetase. In this application, the enhanced sens itivity allowed location of a modified residue by comparison endoprote inase Lys C digest of native and oxidized forms of the protein without extensive sample preparation or concentration. A third application de monstrates the identification of glycosylation sites in an endoprotein ase Arg C digest of single-chain plasminogen activator through the use of in-source collisionally induced dissociation.