The application of continuous flow-extractive desorption electrospray ionization (CF-EDESI), an ambient ionization source demonstrated previously for use with intact protein analysis, is expanded here for the coupling of reversed phase protein separations to mass spectrometry. This configuration allows the introduction of charging additives to enhance detection without affecting the chromatographic separation mechanism. Two demonstrations of the advantages of CF-EDESI are presented in this work. First, a proof-of- principle is presented to demonstrate the applicability of hyphenation of liquid chromatography (LC) to CF- EDESI. LC-CF-EDESI-MS has good sensitivity compared to LC-electrospray ionization (ESI)-mass spectrometry. Second, the supercharging mechanism investigated in CF-EDESI provides an insight into a highly debated supercharging process in ESI. The results indicate that the mechanism of protein charging seen in HPLC-CF-EDESI is different from supercharging phenomena in conventional ESI. The surface tension mechanism and binding mechanism may both contribute to protein supercharging in ESI.

Department of Chemistry and Biochemistry the University of Texas at Arlington Arlington Texas USA

Institute of Microbiology v v i Academy of Sciences of the Czech Republic Vídeňská 1083 14220 Prague Czech Republic

Institute of Microbiology v v i Academy of Sciences of the Czech Republic Vídeňská 1083 14220 Prague Czech Republic Regional Centre of Advanced Technologies and Materials Department of Analytical Chemistry Faculty of Science Palacky University 17 listopadu 12 771 46 Olomouc Czech Republic

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