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 x 1.0 mm I.D. microbore-columns packed with 2.3 microns highly crosslinked, octadecylated poly(styrene-divinylbenzene) particles. Proteins were eluted at temperatures of 80-90 degrees 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.