On‐line buffer removal and fraction selection in gradient capillary high‐performance liquid chromatography prior to electrospray mass spectrometry of peptides and proteins

Abstract

The technique for on‐line removal of buffers and for fraction selection to eliminate source clogging and suppression of electrospray ionization by unwanted constituents consisted of: (i) selecting the initial mobile phase composition to retain analytes on top of a capillary high‐performance liquid chromatography (HPLC) column; (ii) washing the buffer isocratically to waste, using a two‐way micro dump valve; (iii) starting the gradient and switching the dump valve to introduce analytes into the source. Peptide and protein mixtures were prepared in 0.05–0.5 M phosphate and 0.55 mM tris‐based buffers, and water. After buffer removal, chromatograms and electrospray spectra were indistinguishable from those of aqueous controls, down to 1 pmol (acidic fibroblast growth factor) consumed, and up to 78 kDa (bovine transferrin) molecular weight. Aided by the dump valve, 100 fmol of angiotensin could be fractionated and identified on the slope of a 10 × 10⁶ fmol of leucine enkephalin HPLC peak. On‐line buffer removal and fraction selection eliminate the need for additional preparation steps than can lead to excessive sample losses.