Enhancing sensitivity of liquid chromatography–mass spectrometry of peptides and proteins using supercharging agents

Abstract

Trifluoroacetic acid (TFA) is often used as a mobile phase modifier to enhance reversed phase chromatographic performance. TFA adjusts solution pH and is an ion-pairing agent, but it is not typically suitable for electrospray ionization-mass spectrometry (ESI-MS) and liquid chromatography/MS (LC/MS) because of its significant signal suppression. Supercharging agents elevate peptide and protein charge states in ESI, increasing tandem MS (MS/MS) efficiency. Here, LC/MS protein supercharging was effected by adding agents to LC mobile phase solvents. Significantly, the ionization suppression generally observed with TFA was, for the most part, rescued by supercharging agents, with improved separation efficiency (higher number of theoretical plates) and lowered detection limits.

Urinary matrix effects in electrospray ionization mass spectrometry in the presence of DMSO

“…The classification of DMSO as a simple mobile phase additive or as a supercharging agent divides the opinion of researchers . It seems that DMSO is capable of supercharging intact proteins at high concentrations and thus increase CSs, but in the context of peptide ESI, DMSO actually contributes to an opposite effect …”

Quantitative Measurement of Melittin in Asian Honeybee Venom Using a New Method Including UPLC-QqTOF-MS

“…Common mobile phase additives include formic acid, trifluoroacetic acid (TFA), ammonium formate, and ammonium hydroxide, where formic acid and TFA are commonly used to enhance the ionization efficiency of peptides and proteins in the LC separation [43][44][45]. However, we found that TFA significantly suppressed the melittin signal, likely due to the effect of ion-pairing, which suppresses the ionization of melittin [46][47][48]. In contrast, no such signal suppression was observed for formic acid, and a better peak shape was obtained, in addition to shorter run time.…”

Technical Report: Targeted Proteomic Analysis Reveals Enrichment of Atypical Ubiquitin Chains in Contractile Murine Tissues

“…Importantly, we observed higher peptide ion intensities when using FA for most other peptides. There is a well-documented suppressive effect of TFA on electrospray ionisation, which can negatively affect assay sensitivity [45–47]. Our data therefore indicates that 5% FA is an ideal ion-pairing agent for ubiquitin peptide analysis by LC-MS/MS, as it balances ion suppression and improved chromatographic performance for these peptides.…”