Lee Laboratory
for Biological Mass Spectrometry
Department of Chemistry
Iowa State University
NSF-CMI: On-tissue chemical derivatization for untargeted spatial metabololmics (renewal project)
Project Objective
Spatial metabolomics using mass spectrometry imaging (MSI) is a powerful tool for understanding metabolism with cellular resolution. One critical bottleneck is limited compound coverage due to the low ionization efficiency of certain classes of compounds and the inherently small sampling size. As a result, typical MSI studies can visualize only abundant ions under given MS conditions. On-tissue chemical derivatization (OTCD) has become popular to overcome this limitation by converting poorly ionizing compounds to permanently charged or highly ionizable compounds, thereby dramatically increasing their signals. However, OTCD has only been applied to targeted imaging of selected metabolites. The Lee group is exploring OTCD-MSI as a tool for untargeted spatial metabolomics by combining multiple OTCD on sequential tissue sections to increase overall metabolite coverage and annotation confidence.
Built upon previous success in implementing OTCD to METASPACE, a free web-based metabolite annotation platform for spatial metabolomics, the proposed research will further advance untargeted spatial metabolomics. First, bioinformatics tools will be developed to improve metabolite annotations in OTCD-based untargeted spatial metabolomics by using uniquely labeled reagents and functional group search algorithms. Second, in-source gas-phase reactions will be developed to assist in determining molecular structures. Carbon-carbon double bond positions of lipids will be determined using ozone gas and the number of labile hydrogens in OTCD-derivatized metabolites using D2O vapor. Finally, microdroplet reaction acceleration in OTCD will be investigated to better understand the underlying phenomena and further improve OTCD reaction efficiency.
Published Outcome (through prior NSF-CMI funding)
High-throughput metabolite annotation with METASPACE
: Anal. Chem. 2022, 94(25), 8983-8991.
On-tissue chemical derivatization is a valuable tool for expanding compound coverage in untargeted metabolomic studies with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). Applying multiple derivatization agents in parallel increases metabolite coverage even further but results in large and more complex datasets that can be challenging to analyze.In collaboration with Theodore Alexandrov at EMBL, OTCD is implemented to METASPACE, a web-based open MSI metabolite annotation tool kit (https://metaspace2020.eu/). It allows rapid and robust untargeted metabolite annotation for large OTCD-MSI datasets with false discovery rate filtering.
In-source gas phase hydrogen deuterium exchange to improve metabolite annotation
: Anal. Chem. 2022, 94(32), 11129–11133
Highly efficient hydrogen–deuterium exchange (HDX) is developed for mass spectrometry imaging (MSI) with low-vacuum matrix-assisted laser desorption/ionization (MALDI). A HDX efficiency of 73–85% is achieved by introducing D2O vapor into a heated MALDI source in combination with a deuterium-labeled matrix, which allows correct determination of the number of possible H/D exchanges for up to 17 labile hydrogens. This provides valuable orthogonal information to supplement m/z, allowing for increased confidence in metabolite identification while retaining the spatial information MSI supplies.
MS imaging of volatile metabolites with OTCD
: J. Mass Spectrom., 2023, 58(5):e4918
Mass spectrometry imaging (MSI) of volatile metabolites is challenging, especially in matrix-assisted laser desorption/ionization (MALDI). Most MALDI ion sources operate in vacuum, which leads to the vaporization of volatile metabolites during analysis. In addition, tissue samples are often dried during sample preparation, leading to the loss of volatile metabolites even for other MSI techniques. In this study, we adopted on-tissue chemical derivatization to dramatically reduce the volatility of analytes, and applied to short-chain fatty acids in chicken cecum, ileum, and jejunum tissue sections.
Mobilized Electrospray Device for OTCD in MALDI-MS Imaging
: ", J. Am. Soc. Mass Spectrom, 2024, 35 (2), 205-213
Mobilized nebulizer sprayers are commonly used to create a homogeneous matrix or reagent layer across large surfaces. Electrospray devices have also been used to produce microdroplets for the same purpose but are rarely used for large tissues due to their immobility. Herein, we present a movable electrospray device that can be used for large tissue sample preparation through a simple modification to an automatic commercial nebulizer device. The sprayer has the additional benefit of being able to investigate reaction acceleration in OTCD when comparing electrostatically charged spray to electrostatically neutral spray.
Other publications supported by this funding.
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Forsman, T. T.; Dueñas, M. E.; Lee, Y. J., On-tissue boronic acid derivatization for the analysis of vicinal diol metabolites in maize with MALDI-MS imaging. J. Mass Spectrom. 2021, 56 (3), e4709.
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O’Neill, K. C.; Dueñas, M. E.; Larson, E.; Forsman, T. T.; Lee, Y.-J., Enhancing Metabolite Coverage for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Through Multiple On-Tissue Chemical Derivatizations. In Mass Spectrometry Imaging of Small Molecules: Methods and Protocols, Lee, Y.-J., Ed. Springer US: New York, NY, 2022; pp 197-213.
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O’Neill, K. C.; Lee, Y. J., Visualizing Genotypic and Developmental Differences of Free Amino Acids in Maize Roots With Mass Spectrometry Imaging. Frontiers in Plant Science 2020, 11 (639).