The Use of Ammonium Formate as a Mobile-Phase Modifier for LC-MS/MS Analysis of Tryptic Digests

Abstract

A major challenge facing current mass spectrometry (MS)-based proteomics research is the large concentration range displayed in biological systems, which far exceeds the dynamic range of commonly available mass spectrometers. One approach to overcome this limitation is to improve online reversed-phase liquid chromatography (RP-LC) separation methodologies. LC mobile-phase modifiers are used to improve peak shape and increase sample load tolerance. Trifluoroacetic acid (TFA) is a commonly used mobile-phase modifier, as it produces peptide separations that are far superior to other additives. However, TFA leads to signal suppression when incorporated with electrospray ionization (ESI), and thus, other modifiers, such as formic acid (FA), are used for LC-MS applications. FA exhibits significantly less signal suppression, but is not as effective of a modifier as TFA. An alternative mobile-phase modifier is the combination of FA and ammonium formate (AF), which has been shown to improve peptide separations. The ESI-MS compatibility of this modifier has not been investigated, particularly for proteomic applications. This work compares the separation metrics of mobile phases modified with FA and FA/AF and explores the use of FA/AF for the LC-MS analysis of tryptic digests. Standard tryptic-digest peptides were used for comparative analysis of peak capacity and sample load tolerance. The compatibility of FA/AF in proteomic applications was examined with the analysis of soluble proteins from canine prostate carcinoma tissue. Overall, the use of FA/AF improved online RP-LC separations and led to significant increases in peptide identifications with improved protein sequence coverage.

Development of a reversed phase liquid chromatographic method for analysis of pyridoxal‐5′‐phosphate and its impurities

“…It consisted of a 0.1% solution of formic acid which is compatible with MS. This method is preferable to this suggested by Mohamed‐Ahmed since the latter contains trifluoroacetic acid which is known to suppress the MS signal .…”

A Rapid LC–MS/MS Quantification of Peramivir Using A Simple and Inexpensive Sample Precipitation: Application to Pk

“…Duchateau et al [21] evaluated the effect of different volatile buffers, including ammonium formate, ammonium acetate and ammonium bicarbonate, on LC-MS/MS analysis and found that ammonium formate gave the highest signal-to-noise ratio but at the expense of poor resolution. Ammonium formate combined with formic acid has been reported to have higher ionic strength (7.4 vs 1.9 mM) than formic acid alone [22][23][24] and to enhance sensitivity and improve peptide separations. Such modification of the additives had no influence on chromatographic resolution in our assay (Figure 2).…”

Recent update on methodologies for extraction and analysis of soybean seed proteins

“…For better separation of peptides, selection of the proper mobile phase is critical. Johnson et al . used a combination of ammonium formate and formic acid to improve reverse phase LC separation, which increased the sensitivity for peptide detection.…”

“…For better separation of peptides, selection of the proper mobile phase is critical. Johnson et al 73 used a combination of ammonium formate and formic acid to improve reverse phase LC separation, which increased the sensitivity for peptide detection. In another study, Hahne et al 74 showed that low-concentration dimethyl sulfoxide (DMSO) addition resulted in a significant improvement in the sensitivity (up to tenfold) of peptides by LC/ESI-MS. Kim et al 75 purified the adipogenesis inhibitory peptide from black soybean protein hydrolysate using consecutive methods including ultrafiltration, gel filtration chromatography and reverse phase HPLC.…”