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

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. Several supercharging agents were tested, including novel agents N,N,N'N’-tetraethylsulfamide (TES) and 3-methyl-2-oxazolidone (MOZ). This use of supercharging reagents could apply broadly to LC–MS mobile phases containing TFA, and especially in protein analysis and peptide mapping. The success of two new agents predicted to supercharge based on pK_BH+ < −1.7 confirms that the supercharging mechanism for increased positive ion charging requires low volatility, Brønsted bases weaker than H₂O. A mechanism for the suppression rescue is described that exploits the agents’ capacity to reduce solution phase ionization, hence decreasing trifluoroacetate anion concentrations in the droplet. The mechanism also addresses why signal improvement is not uniform across analytes.