Multiplexed Enzyme Activity Microscopy by Semi-quantitative, Ratiometric Enzyme Activity MALDI Mass Spectrometry Imaging.
5 affiliations
- 1Institute of Radiology, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Charitéplatz 1, Berlin 10117, Germany.
- 2Institute of Chemistry, Humboldt-Universität zu Berlin, Brook-Taylor-Str. 2, Berlin 12489, Germany.
- 3Institute of Biochemistry, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Charitéplatz 1, Berlin 10117, Germany.
- 4Imaging Mass Spectrometry Unit; Berlin Institute of Health at Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, Berlin 13353, Germany.
- 5Berlin Institute of Health at Charité-Universitätsmedizin Berlin, Anna-Louisa-Karsch-Str. 2, Berlin 10178, Germany.
Abstract
Enzymes are often tightly regulated and exist in inactive forms. Therefore, rather than mere presence, it is the enzyme's activity that catalyzes cellular and tissue functions. However, multiplex activity mapping of enzymes in situ is particularly challenging. To address this, we introduce multiplexed enzyme activity MALDI-MSI (MEA-MSI), a ratiometric, semi-quantitative method that allows multiplex in situ visualization of enzyme activities in zinc-fixed tissue sections. To accomplish this, a library of 31 MEA-MSI substrates targeting proteases, kinases, histone acetyltransferases, and glycosyltransferases was synthesized. Each substrate is designed so that the masses of these substrates and their enzymatic products are distinct, enabling their simultaneous pixel-wise detection within tissue sections. A ratiometric calculation procedure was used to estimate the relative enzyme activity and compensate for tissue-dependent ionization bias, which is a general problem in MALDI-MSI. MEA-MSI was applied to healthy mouse liver, a murine hepatocellular carcinoma (HCC) model, and an incomplete microwave-ablated (iMWA) rabbit VX2 liver tumor model. The resulting activity maps revealed distinct regional enzyme activity patterns across tumor and normal tissues. Correlation and network analyses showed related enzyme activities and related groups, while unsupervised clustering and Uniform Manifold Approximation and Projection (UMAP) analysis reproduced histological patterns. Because MEA-MSI relies on enzymatic amplification, it is not restricted to the detection of highly abundant enzymes and enables multiplexed, spatially resolved readouts of enzyme activity in situ, providing a framework for comparative spatial enzymology.
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BibTeX
@article{hojjat2026multiplexed,
title = {{Multiplexed Enzyme Activity Microscopy by Semi-quantitative, Ratiometric Enzyme Activity MALDI Mass Spectrometry Imaging.}},
author = {Hamidreza Hojjat and Petra Henklein and Akvile Haeckel and Grit Nebrich and Sylwia Handzik and Pedro Augusto Dantas de Moraes and Jing Guo and Justus Ramtke and Yubei He and Lynn Jeanette Savic and Oliver Seitz and Oliver Klein and Eyk Schellenberger},
journal = {ACS measurement science au},
year = {2026},
month = aug,
doi = {10.1021/acsmeasuresciau.6c00070},
url = {https://europepmc.org/article/MED/42921441},
note = {CC BY},
}
RIS
TY - JOUR
TI - Multiplexed Enzyme Activity Microscopy by Semi-quantitative, Ratiometric Enzyme Activity MALDI Mass Spectrometry Imaging.
AU - Hamidreza Hojjat
AU - Petra Henklein
AU - Akvile Haeckel
AU - Grit Nebrich
AU - Sylwia Handzik
AU - Pedro Augusto Dantas de Moraes
AU - Jing Guo
AU - Justus Ramtke
AU - Yubei He
AU - Lynn Jeanette Savic
AU - Oliver Seitz
AU - Oliver Klein
AU - Eyk Schellenberger
PY - 2026
DA - 2026/08/12
JO - ACS measurement science au
DO - 10.1021/acsmeasuresciau.6c00070
UR - https://europepmc.org/article/MED/42921441
AB - Enzymes are often tightly regulated and exist in inactive forms. Therefore, rather than mere presence, it is the enzyme's activity that catalyzes cellular and tissue functions. However, multiplex activity mapping of enzymes in situ is particularly challenging. To address this, we introduce multiplexed enzyme activity MALDI-MSI (MEA-MSI), a ratiometric, semi-quantitative method that allows multiplex in situ visualization of enzyme activities in zinc-fixed tissue sections. To accomplish this, a library of 31 MEA-MSI substrates targeting proteases, kinases, histone acetyltransferases, and glycosyltransferases was synthesized. Each substrate is designed so that the masses of these substrates and their enzymatic products are distinct, enabling their simultaneous pixel-wise detection within tissue sections. A ratiometric calculation procedure was used to estimate the relative enzyme activity and compensate for tissue-dependent ionization bias, which is a general problem in MALDI-MSI. MEA-MSI was applied to healthy mouse liver, a murine hepatocellular carcinoma (HCC) model, and an incomplete microwave-ablated (iMWA) rabbit VX2 liver tumor model. The resulting activity maps revealed distinct regional enzyme activity patterns across tumor and normal tissues. Correlation and network analyses showed related enzyme activities and related groups, while unsupervised clustering and Uniform Manifold Approximation and Projection (UMAP) analysis reproduced histological patterns. Because MEA-MSI relies on enzymatic amplification, it is not restricted to the detection of highly abundant enzymes and enables multiplexed, spatially resolved readouts of enzyme activity in situ, providing a framework for comparative spatial enzymology.
SN - 2694-250X
ER -