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

Area of Science:

• Analytical Chemistry
• Biochemistry
• Mass Spectrometry

Background:

• Electrospray ionization (ESI) is crucial for ion formation in mass spectrometry (MS), but its regime is often poorly defined in workflows.
• Capillary electrophoresis-mass spectrometry (CE-ESI-MS) utilizes different ESI interfaces (microflow for metabolites, nanoflow for peptides/proteins).

Purpose of the Study:

• To benchmark the performance of cone-jet (CJ) and pulsating (P) ESI regimes in CE-ESI-MS for trace-level analyses.
• To evaluate the impact of CJ and P regimes across microflow (μESI) and nanoflow (nanoESI) interfaces.

Main Methods:

• Comparative analysis of CJ and P ESI regimes using time-of-flight and timsTOF mass spectrometers.
• Application to trace metabolites, peptides, and proteome digests, including single-cell-scale samples.

Main Results:

• In CE-μESI, CJ improved metabolite sensitivity (up to 2-fold), signal stability, and peptide charge state compared to P.
• In CE-nanoESI proteomics, CJ increased peptide/protein identifications (~50%), identified more low-abundance proteins, and improved quantification completeness.
• CJ enhanced metabolite feature detection and protein group recovery in Xenopus laevis samples.

Conclusions:

• The ESI regime is a controllable factor influencing sensitivity and quantitative performance in CE-ESI-MS.
• Selecting the optimal ESI regime, particularly CJ, is critical for trace-limited analyses and provides practical guidance.