Publication Date
2026
Document Type
Dissertation/Thesis
First Advisor
Ryzhov, Victor
Degree Name
M.S. (Master of Science)
Legacy Department
Department of Chemistry and Biochemistry
Abstract
Paper spray mass spectrometry (PS-MS) is a novel ambient ionization method that allows chemical and biological samples to be rapidly analyzed with little preparation being required. In this paper, PS-MS was set up and streamlined as an acetylcholinesterase (AChE)-monitoring platform in addition to screening an existing and newly produced AChE-inhibitors. The detection of the substrate, acetylcholine (m/z 146), and the AChE catalysis product, choline (m/z 104) was performed without applying chromatographic separation or derivatization to the dried paper substrates. Assay conditions were optimized in a systematic way, to develop a robust MS-compatible enzymatic workflow.
Calibration curves for both, acetylcholine and choline were found to be good in linearity as choline exhibited a linear response over the range of 15 ng–1050 ng (R² = 0.955), whereas acetylcholine showed linear behavior from 10 ng–500 ng (R² = 0.923). Thus, allowing the reaction progress to be quantified and the analytical parameters to be calculated, including, LOD 0.38 ng for choline, and 0.78 ng for acetylcholine, as well as LOQ of 1.2 ng and 2.4 ng respectively. The time-course experiments also established that product formation generally leveled off after about 24 or 30 min thus defining the time scale to use as the maximum limit of the inhibition experiment. The PS-MS test was able to identify the inhibition of AChE activity in a dose-dependent manner with Huperzine A as a model inhibitor, confirming that the method can detect any form of inhibition with the highest level of sensitivity. The newly synthesized inhibitors A-F were tested at constant concentration, and the inhibitory effects of the newly synthesized inhibitors were measured by time-dependent product formation and the logistic dose-response curve. Out of them, compound F was the most potent at 5.26 µM IC50, then compound E (IC50 in the 35 µM), the rest of the compounds demonstrated low inhibition. These compounds were compared to Huperzine A whose level of performance was shown to be at the nanomolar range. Lastly, an otherwise isotopically labeled analogue of a well-known internal standard (ACh-d4) was determined to be optimal in terms of normalization used to conduct quantitative PS-MS analysis, at 50 µM.
Comprehensively, this study makes PS-MS high-throughput, low-sample-consuming, and preparation-reduced method of real-time AChE activity monitoring and inhibitor screening. The simplicity and scalability of the assay, along with conceptual multi-sample PS-MS holder designs described herein demonstrate a high potential of high-throughput enzyme among other biochemical studies and biochemical applications.
Recommended Citation
Gautam, Diksha, "Application of Paper Spray Mass Spectrometry for Screening Acetylcholinesterase Inhibitors" (2026). Graduate Research Theses & Dissertations. 8204.
https://huskiecommons.lib.niu.edu/allgraduate-thesesdissertations/8204
Extent
85 pages
Language
en
Publisher
Northern Illinois University
Rights Statement
In Copyright
Rights Statement 2
NIU theses are protected by copyright. They may be viewed from Huskie Commons for any purpose, but reproduction or distribution in any format is prohibited without the written permission of the authors.
Media Type
Text
