Integrated High-Throughput Mass Spectrometry Framework for Multiplexed Substrate Screening in Enzyme Discovery and Development
- 1Pharmaceutical Analysis & Digital Technologies
- 2Merck & Co., Inc.
- 3Process Research & Development
Abstract
Biocatalysis continues to advance sustainable synthesis while relying on analytical technologies that can match the demands of high-throughput, miniaturized screening and complex assay formats. In particular, multiplexed substrate screening strategy accelerates enzyme discovery and the expansion of the enzyme toolbox, yet it demands greater selectivity, sensitivity, and throughput than conventional single-quadrupole LC-MS can provide. Here, an integrated high-throughput mass spectrometry framework enabling both quantitative targeted analysis and untargeted product discovery in biocatalytic reactions is introduced. The platform employs a tiered measurement strategy consisting of Acoustic Droplet Ejection-Multiple Reaction Monitoring Mass Spectrometry (ADE-MRM-MS) for targeted quantitation, Acoustic Droplet Ejection-Time-of-Flight Mass Spectrometry (ADE-TOF-MS) for untargeted product discovery through extracted ion chromatogram (XIC) analysis, and Acoustic Droplet Ejection-Quadrupole Time-of-Flight Tandem Mass Spectrometry (ADE-QTOF-MS/MS) for challenging selectivity scenarios involving near-isobaric compounds, closely related masses, and structural isomers. Application of this approach streamlined rapid discovery and evolution campaigns across pyrimidine nucleoside phosphorylases (PyNPs), transaminases (TAs), and nucleoside deoxyribosyltransferases (NDTs). Together, these results established the first unified ADE-MS framework for biocatalysis that delivers the speed, selectivity, and scalability required for next-generation biocatalyst screening and sustainable synthesis.
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BibTeX
@misc{li2026integrated,
title = {{Integrated High-Throughput Mass Spectrometry Framework for Multiplexed Substrate Screening in Enzyme Discovery and Development}},
author = {Wenxuan Li and Devin Makey and Vladimir Shchurik and Giulio Fittolani and Jennifer L. Kennemur and Richard S. Ayikpoe and Wai-Ling Cheung-Lee and Chunyu Wu and John A. McIntosh and Karla M. Camacho Soto and Rodell C. Barrientos and Erik L. Regalado and Emmanuel Appiah-Amponsah and Sasidhar N. Nirudodhi},
howpublished = {ChemRxiv},
year = {2026},
month = oct,
doi = {10.26434/chemrxiv.15010437/v1},
url = {https://chemrxiv.org/doi/full/10.26434/chemrxiv.15010437/v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Integrated High-Throughput Mass Spectrometry Framework for Multiplexed Substrate Screening in Enzyme Discovery and Development
AU - Wenxuan Li
AU - Devin Makey
AU - Vladimir Shchurik
AU - Giulio Fittolani
AU - Jennifer L. Kennemur
AU - Richard S. Ayikpoe
AU - Wai-Ling Cheung-Lee
AU - Chunyu Wu
AU - John A. McIntosh
AU - Karla M. Camacho Soto
AU - Rodell C. Barrientos
AU - Erik L. Regalado
AU - Emmanuel Appiah-Amponsah
AU - Sasidhar N. Nirudodhi
PY - 2026
DA - 2026/10/13
PB - ChemRxiv
DO - 10.26434/chemrxiv.15010437/v1
UR - https://chemrxiv.org/doi/full/10.26434/chemrxiv.15010437/v1
AB - Biocatalysis continues to advance sustainable synthesis while relying on analytical technologies that can match the demands of high-throughput, miniaturized screening and complex assay formats. In particular, multiplexed substrate screening strategy accelerates enzyme discovery and the expansion of the enzyme toolbox, yet it demands greater selectivity, sensitivity, and throughput than conventional single-quadrupole LC-MS can provide. Here, an integrated high-throughput mass spectrometry framework enabling both quantitative targeted analysis and untargeted product discovery in biocatalytic reactions is introduced. The platform employs a tiered measurement strategy consisting of Acoustic Droplet Ejection-Multiple Reaction Monitoring Mass Spectrometry (ADE-MRM-MS) for targeted quantitation, Acoustic Droplet Ejection-Time-of-Flight Mass Spectrometry (ADE-TOF-MS) for untargeted product discovery through extracted ion chromatogram (XIC) analysis, and Acoustic Droplet Ejection-Quadrupole Time-of-Flight Tandem Mass Spectrometry (ADE-QTOF-MS/MS) for challenging selectivity scenarios involving near-isobaric compounds, closely related masses, and structural isomers. Application of this approach streamlined rapid discovery and evolution campaigns across pyrimidine nucleoside phosphorylases (PyNPs), transaminases (TAs), and nucleoside deoxyribosyltransferases (NDTs). Together, these results established the first unified ADE-MS framework for biocatalysis that delivers the speed, selectivity, and scalability required for next-generation biocatalyst screening and sustainable synthesis.
ER -