The Use of Ammonium Formate as a Mobile Phase Modifier for Reversed-Phase LC-MS/MS
University of Georgia
Wednesday, March 28, 2012 - 11:15am
Chemistry, Room 400
Current mass spectrometry based proteomics faces limitations of dynamic range due to the large concentration range displayed by most biological samples. One way to overcome dynamic range limitations is by improving separation methodologies, specifically online reversed-phase liquid chromatography. While trifluoroacetic acid (TFA) yields peptide separations far superior to formic acid, ion pairing creates signal suppression in electrospray ionization (ESI) mass spectrometry. Formic acid exhibits significantly less signal suppression, but displays substantial band broadening and poor sample load tolerance of basic nature peptides. An alternative mobile phase modifier is the combination of formic acid and ammonium formate to improve peptide separations with minimal losses in ionization efficiency. This work examines the superior separation metrics displayed by formic acid/ammonium formate modified mobile phases over formic acid alone. Standard tryptic digest peptides were used for comparative analysis to measure improvements in peak capacity and sample load tolerance. The compatibility of ammonium formate for use in a proteomic application is also examined, analyzing canine prostate carcinoma soluble proteins. The improved separations achieved with ammonium formate produced significant increases in peptide identifications leading to improved protein sequence coverage and number of protein identifications.