A stationary phase with a positively charged surface allows for minimizing formic acid concentration in the mobile...

The default choice of mobile phase acidifier for bottom-up LC-MS proteomic analyses is 0.10% formic acid because of its decent acidity, decent ion pairing ability, and low suppression of electrospray ionization. In recent years, state-of-the-art columns have been designed specifically to provide efficient separation even when using an MS-friendly mobile phase of low ionic strength. Despite this, no attempts have been made to improve the sensitivity of the MS-based analytical methods by reducing the amount of formic acid in the mobile phase. In this study, we evaluated the effect of reduced formic acid concentration in the mobile phase on the chromatographic behavior and MS response of peptides when separated using columns packed with a C₁₈ stationary phase with a positively charged surface. Using 0.01% formic acid in the mobile phase maintained excellent chromatographic performance and increased MS signal response compared to the standard of 0.10%. The enhanced MS response translated to about 50% improved peptide identifications depending on the complexity and amount of sample injected. The increased retention of peptides at a reduced formic acid concentration was directly proportional to the number of acidic residues in the peptide sequence. The study was carried out by covering a spectrum of protein samples with varied complexity using analytical flow, micro-, and nanoflow regimes to expand the applicability in routine practice.