title: Online Monitoring of Enzymatic Reactions Using Time-Resolved Desorption Electrospray Ionization Mass Spectrometry.
abstract: Electrospray ionization mass spectrometry (ESI-MS) is powerful for determining enzymatic reaction kinetics because of its soft ionization nature. However, it is limited to use ESI-favored solvents containing volatile buffers (e.g., ammonium acetate). In addition, lack of a quenching step for online ESI-MS reaction monitoring might introduce inaccuracy, due to the possible acceleration of reaction in the sprayed microdroplets. To overcome these issues, this study presents a new approach for online measuring enzymatic reaction kinetics using desorption electrospray ionization mass spectrometry (DESI-MS). By using DESI-MS, enzymatic reaction products in a buffered aqueous media (e.g., a solution containing Tris buffer or high concentration of inorganic salts) could be directly detected. Furthermore, by adjusting the pH and solvent composition of the DESI spray, reaction can be online quenched to avoid the postionization reaction event, leading to fast and accurate measurement of kinetic constants. Reaction time control can be obtained simply by adjusting the injection flow rates of enzyme and substrate solutions. Enzymatic reactions examined in this study include hydrolysis of 2-nitrophenyl-β-D-galactopyranoside by β-galactosidase and hydrolysis of acetylcholine by acetylcholinesterase. Derived Michaelis-Menten constants Kmfor these two reactions were determined to be 214 μM and 172 μM, respectively, which are in good agreement with the values of 300 μM and 230 μM reported in literature, validating the DESI-MS approach. Furthermore, this time-resolved DESI-MS also allowed us to determine Kmand turnover number kcatfor trypsin digestion of angiotensin II (Kmand kcatare determined to be 6.4 mM and 1.3 s-1, respectively).
authors.affiliation: Center for Intelligent Chemical Instrumentation, Department of Chemistry and Biochemistry, Edison Biotechnology Institute, Ohio University , Athens, Ohio 45701, United States.
authors.forename: Si
authors.initials: S
authors.lastname: Cheng
authors.affiliation: Center for Intelligent Chemical Instrumentation, Department of Chemistry and Biochemistry, Edison Biotechnology Institute, Ohio University , Athens, Ohio 45701, United States.|Department of Chemistry, College of Science, Agricultural University of Hebei , Baoding 071001, China.
authors.forename: Qiuhua
authors.initials: Q
authors.lastname: Wu
authors.affiliation: Center for Intelligent Chemical Instrumentation, Department of Chemistry and Biochemistry, Edison Biotechnology Institute, Ohio University , Athens, Ohio 45701, United States.
authors.forename: He
authors.initials: H
authors.lastname: Xiao
authors.affiliation: Center for Intelligent Chemical Instrumentation, Department of Chemistry and Biochemistry, Edison Biotechnology Institute, Ohio University , Athens, Ohio 45701, United States.
authors.forename: Hao
authors.identifier: 0000-0001-8090-8593
authors.initials: H
authors.lastname: Chen
mesh_terms: D000109:Acetylcholine / Q000032:analysis; D000110:Acetylcholinesterase / Q000378:metabolism*; D005960:Glucosides / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D001616:beta-Galactosidase / Q000378:metabolism*
chemical_list: D005960:Glucosides; C018616:2-nitrophenyl-beta-D-glucopyranoside; D000110:Acetylcholinesterase; D001616:beta-Galactosidase; D000109:Acetylcholine