Effect of the mobile phase composition on the separation and detection of intact proteins by reversed-phase liquid chromatography–electrospray mass spectrometry

Abstract

Various buffers (ammonium acetate, ammonium formate, and ammonium hydrogencarbonate), acids (formic acid, acetic acid, heptafluorobutyric acid, and trifluoroacetic acid), and bases (ammonium hydroxide and morpholine) covering the range from 2 to 11.5 have been investigated for their performance in the separation of proteins by reversed-phase liquid chromatography (RPLC) and in their detection by electrospray mass spectrometry (ESI-MS). These additives were first tested for the detection of standard proteins by ESI-MS by flow-injection analysis (FIA). Those additives yielding the highest signals were employed for the separation of standard proteins by using three different reversed-phase columns: two C 18 columns (4.6 mm I.D. and 2.1 mm I.D.) and one perfusion column (2 mm I.D.). The sensitivity of the LC–MS system was evaluated with the column giving the best results and with those LC eluents enabling the LC separation of the proteins and also yielding the highest MS signals. For that purpose, calibration curves were compared for both LC–MS and FIA–MS. Formic acid was the additive yielding the highest responses in FIA–MS and trifluoroacetic acid (TFA) gave the best separation and recovery of the proteins. However, problems related to poor recovery of the proteins in the column when formic acid was used and the significant signal suppression observed in MS when TFA was employed, made neither of them suitable for the sensitive detection of the proteins in LC–MS.