Evaluation of volatile eluents and electrolytes for high-performance liquid chromatography–electrospray ionization mass spectrometry and capillary electrophoresis–electrospray ionization mass spectrometry of proteins: I. Liquid chromatography

Abstract

Proteins ranging in molecular mass from 14 000 to 80 000 were analyzed by reversed-phase high-performance liquid chromatography–electrospray mass spectrometry (RP-HPLC–ESI-MS) using 60×1.0 mm I.D. microbore-columns packed with 2.3 μm highly crosslinked, octadecylated poly(styrene–divinylbenzene) particles. Proteins were eluted at temperatures of 80–90°C with gradients of acetonitrile in 0.10–0.50% aqueous solutions of trifluoroacetic acid, formic acid or acetic acid. Substitution of trifluoroacetic acid, the most commonly used mobile phase additive for RP-HPLC, by formic acid resulted in a 35–160-fold improvement in analyte detectability at the cost of an only 32–104% increase in peak width at half height of eluting chromatographic peaks. The lower limits of detection for carbonic anhydrase ( M r 29 022.7) in full scan and selected ion monitoring mode were 37 and 2.3 fmol, respectively. Measurement of protein masses by RP-HPLC–ESI-MS was accurate and highly reproducible with maximum mass deviations of 0.025% and relative standard deviations of less than 0.011%. Calibration plots of peak area versus concentration allowed the reliable quantitation of proteins in a concentration range of 0.010–1.0 mg/ml. Finally, the optimized method was applied to the separation, identification and quantification of proteins in real samples such as commercial protein preparations, monoclonal antibody fragments, allergen extracts and whey drinks.