High-Pressure ESI with High-Temperature Pre- and Post-ESI Heating for High-Throughput Analysis of Hydrothermal and Gas-Phase Thermal Effects on Analytes

Abstract

We report a high-pressure electrospray ionization (ESI) source that incorporates a heated liquid transfer capillary, which functions as an online hydrothermal reactor, and a heated ion transport capillary for high-throughput analysis of heating effects on analytes and on-demand thermal dissociation. Both capillaries are resistively heated to elevated temperatures. The heated liquid transfer capillary is connected to the high-pressure ESI via a flow restrictor, and the back pressure provided by the flow restrictor, combined with the high-pressure operation of the ESI source, allows the liquid to be heated to 300 ℃ without boiling before being cooled to < 150 ℃ at the ESI emitter. The generated ions and charged droplets then pass through the ion transport capillary, which can be held at 20 ~ 400 ℃ before being released into the atmospheric pressure ambient for subsequent sampling by the mass spectrometer. The system has been applied to the hydrothermal acceleration of Asp-selective cleavage in ubiquitin, Pro-Pro bond-selective cleavage in bradykinin, and the formation of dipeptides of Gly-Gly and Ala-Ala from glycine and alanine. Post-ESI heating of the charged droplets and ions did not produce similar hydrothermal reactions; instead, it resulted in gas-phase thermal dissociation that differs from liquid-phase activation. The combination of liquid-phase and gas-phase activations can be used to tune the level of fragmentation and increase the abundance of fragments for tandem mass spectrometry, providing a potential tool for top-down proteomics.