Electrospray Regime Controls Sensitivity and Quantitative Performance in Micro- and Nano-Flow CE-ESI-MS

The electrospray regime is a key determinant of ion formation during electrospray ionization (ESI), but it is often unreported and rarely verified in separation-coupled mass spectrometry (MS) workflows. In capillary electrophoresis (CE)-mass spectrometry (CE-ESI-MS), sheath-flow microflow ESI interfaces are widely used for metabolite analysis, whereas nanoflow ESI interfaces dominate peptide and protein measurements. Here, we benchmarked CE-ESI-MS performance in the cone-jet (CJ) and pulsating (P) regimes for trace-level metabolites, peptides, and proteome digests across microflow (μESI) and nanoflow (nanoESI) operation. In CE-μESI on a time-of-flight mass spectrometer, CJ increased metabolite sensitivity by up to ∼2-fold, improved signal stability, and shifted peptide ion generation to higher charge states relative to P. In CE-nanoESI proteomics on a timsTOF platform, CJ increased HeLa peptide and protein identifications by ∼50% and extended coverage to lower-abundance proteins while improving quantification completeness across technical replicates (i.e., a higher fraction of proteins quantified in all runs). For Xenopus laevis embryonic samples at single-cell-scale input, CJ significantly increased the number of detected metabolite features and improved recovery of functionally annotated protein groups. These results establish ESI regime as a controllable driver of sensitivity and quantitative performance in CE-ESI-MS and provide practical guidance for selecting ionization conditions for trace-limited analyses.