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 trifluoroacetic acid (TFA) containing HPLC mobile phases. The signal suppressing effects of TFA are shown to be due to the combined effect of ion-pairing and surface tension modifications. The post-column addition of a propionic acid-2-propanol (75:25, v/v) in a 1:2 proportion with the HPLC mobile phase counteracts the deleterious effects of TFA resulting in 10-100 x improvement of the signal-to-noise ratio. The system described introduces total HPLC flow (plus additive) directly into the electrospray source without splitting. Using 2.1 mm I.D. HPLC columns, minimum detectable quantities are below 40 pmol total protein. As examples, separations of proteolytic enzyme digests of several proteins are shown using standard HPLC conditions, comparing results with and without 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 sensitivity allowed location of a modified residue by comparison endoproteinase Lys C digest of native and oxidized forms of the protein without extensive sample preparation or concentration. A third application demonstrates the identification of glycosylation sites in an endoproteinase Arg C digest of single-chain plasminogen activator through the use of in-source collisionally induced dissociation.

Signal enhancement for gradient reverse-phase high-performance liquid chromatography-electrospray ionization mass spectrometry analysis with trifluoroacetic and other strong acid modifiers by postcolumn addition of propionic acid and isopropanol.

Trifluoroacetic acid (TFA) and other volatile strong acids, used as modifiers in reverse-phase high-performance liquid chromatography, cause signal suppression for basic compounds when analyzed by electrospray ionization mass spectrometry (ESI-MS). Evidence is presented that signal suppression is caused by strong ion pairing between the TFA anion and the protonated sample cation of basic sample molecules. The ion-pairing process "masks" the protonated sample cations from the ESI-MS electric fields by rendering them "neutral. " Weakly basic molecules are not suppressed by this process. The TFA signal suppression effect is independent from the well-known spray problem that electrospray has with highly aqueous solutions that contain TFA. This previously reported spray problem is caused by the high conductivity and surface tension of aqueous TFA solutions. A practical method to enhance the signal for most basic analytes in the presence of signal-suppressing volatile strong acids has been developed. The method employs postcolumn addition of a solution of 75% propionic acid and 25% isopropanol in a ratio 1:2 to the column flow. Signal enhancement is typically 10-50 times for peptides and other small basic molecules. Thus, peptide maps that use ESI-MS for detection can be performed at lower levels, with conventional columns, without the need to use capillary chromatography or reduced mass spectral resolution to achieve satisfactory sensitivity. The method may be used with similar results for heptafluorobutyric acid and hydrochloric acid. A mechanism for TFA signal suppression and signal enhancement by the foregoing method, is proposed.