Stable, reproducible, and automated capillary zone electrophoresis–tandem mass spectrometry system with an electrokinetically pumped sheath–flow nanospray interface

•First automation of electrokinetically-pumped sheath–flow interface.•Eight analyses of Escherichia coli digest performed in 8 h.•An average of 1115 peptide IDs per run.

A PrinCE autosampler was coupled to a Q-Exactive mass spectrometer by an electrokinetically pumped sheath–flow nanospray interface to perform automated capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS). 20 ng aliquots of an Escherichia coli digest were injected to evaluate the system. Eight sequential injections over an 8-h period identified 1115 ± 70 (relative standard deviation, RSD = 6%) peptides and 270 ± 8 (RSD = 3%) proteins per run. The average RSDs of migration time, peak intensity, and peak area were 3%, 24% and 19%, respectively, for 340 peptides with high intensity. This is the first report of an automated CZE-ESI-MS/MS system using the electrokinetically pumped sheath–flow nanospray interface. The results demonstrate that this system is capable of reproducibly identifying over 1000 peptides from an E. coli tryptic digest in a 1-h analysis time.

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