Dual-pH LC-ESI-MS/MS Expands Peptide Detectability via Wrong-Way-Round Ionization

Quantitative LC-ESI-MS/MS methods for peptides are conventionally developed and performed under acidic mobile-phase conditions to favor protonation and stable precursor formation. However, despite this long-standing practice, an increasing number of studies indicate that electrospray ionization (ESI) efficiency is often decoupled from bulk-solution protonation equilibria. Here, we systematically evaluate peptide-dependent ionization behavior under acidic (pH ≈ 2.8) and basic (pH ≈ 8.9) conditions using a dual-pH LC-MS/MS workflow applied to human coagulation factor VIII (FVIII). Fourteen FVIII signature peptides were generated by tryptic digestion and quantified using a fixed doubly charged precursor to ensure methodological consistency. Despite being predicted to be neutral or strongly anionic in bulk solution at pH 8.9, several FVIII peptides produced their highest signal under basic conditions, including peptides that were weakly detected or undetectable using acidic workflows. Comparison of the observed responses with estimated solution-phase charge shows that neither bulk protonation nor isoelectric point reliably predicts ESI performance. Instead, the data indicate that peptide response reflects peptide-intrinsic properties, notably hydrophobic surface activity and gas-phase proton affinity, which together govern access to the electrospray droplet interface and stabilization of charge during desolvation. Basic mobile phases selectively enhanced ESI+ response for peptides that interacted less favorably with the bulk aqueous phase, revealing a robust "wrong-way-round" ionization regime in which reduced solution-phase control leads to greater selectivity and higher sensitivity. Incorporation of dual-pH evaluation during peptide method development therefore provides a simple strategy for maximizing sensitivity and avoiding systematic bias in targeted LC-MS/MS assays.