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Dual-pH LC-ESI-MS/MS Expands Peptide Detectability via Wrong-Way-Round Ionization
Ola Svahn1, Jonatan Svahn2, Jan Astermark3
1School of Education and Environment, Division of Natural Sciences, Kristianstad University, SE-291 88Kristianstad, Sweden.
Analytical Chemistry
|July 21, 2026
Summary
Conventional acidic mobile phases for peptide analysis are challenged by basic conditions, revealing peptide-intrinsic properties enhance electrospray ionization (ESI) efficiency. Dual-pH liquid chromatography-tandem mass spectrometry (LC-MS/MS) improves sensitivity and reduces bias in peptide quantification.
Area of Science:
- Analytical Chemistry
- Biochemistry
- Mass Spectrometry
Background:
- Quantitative peptide analysis using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) typically employs acidic mobile phases.
- This acidic condition is believed to promote peptide protonation and stable precursor ion formation for optimal ionization.
- However, electrospray ionization (ESI) efficiency is increasingly recognized as being decoupled from bulk solution protonation equilibria.
Purpose of the Study:
- To systematically evaluate peptide ionization behavior under both acidic and basic mobile-phase conditions.
- To investigate the influence of mobile-phase pH on the electrospray ionization (ESI) efficiency of peptides.
- To determine factors governing peptide response in targeted LC-MS/MS assays.
Main Methods:
- A dual-pH liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflow was developed and applied.
- Human coagulation factor VIII (FVIII) was digested, and fourteen signature peptides were analyzed.
- Peptide quantification was performed using a fixed doubly charged precursor ion under both acidic (pH ≈ 2.8) and basic (pH ≈ 8.9) conditions.
Main Results:
- Several FVIII peptides exhibited enhanced signal intensity under basic mobile-phase conditions, even those predicted to be neutral or anionic.
- Peptide response was found to correlate with intrinsic peptide properties like hydrophobic surface activity and gas-phase proton affinity, not solely bulk protonation or isoelectric point.
- Basic mobile phases promoted a 'wrong-way-round' ionization regime, increasing selectivity and sensitivity for certain peptides by reducing unfavorable interactions with the aqueous phase.
Conclusions:
- Mobile-phase pH significantly impacts peptide ESI efficiency, with basic conditions sometimes yielding superior results.
- Peptide-intrinsic properties are critical determinants of ionization efficiency in LC-MS/MS.
- Incorporating dual-pH evaluation into peptide method development is a strategy to maximize sensitivity and minimize systematic bias in targeted LC-MS/MS assays.
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