High-pressure ESI with high-temperature pre- and post-ESI heating for high-throughput analysis of hydrothermal and gas-phase thermal effects on analytes

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 the high-throughput analysis of heating effects on analytes and on-demand thermal dissociation. Both the capillaries are resistively heated to elevated temperatures. The heated liquid transfer capillary is connected to the high-pressure ESI <i>via</i> 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 °C without boiling before being cooled to <150 °C at the ESI emitter. The generated ions and charged droplets then pass through the ion transport capillary, which can be held at 20-400 °C, before being released into the atmospheric pressure ambient for subsequent sampling by the mass spectrometer. The system is applied to the hydrothermal acceleration of Asp-selective cleavage in ubiquitin, the Pro-Pro bond-selective cleavage in bradykinin, and the formation of dipeptides of Gly-Gly and Ala-Ala from glycine and alanine, respectively. The post-ESI heating of the charged droplets and ions did not produce similar hydrothermal reactions; instead, it resulted in a gas-phase thermal dissociation that differs from the 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.