Abstract
Probing the entirety of any species metabolome is an analytical grand challenge, especially at a cellular scale. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a common spatial metabolomics assay, but this technique has limited molecular coverage for several reasons. To expand the application space of spatial metabolomics, we developed an on-tissue chemical derivatization (OTCD) workflow using 4-APEBA for confident identification of several dozen elusive phytocompounds. Overall, this new OTCD method enabled the annotation of roughly 280 metabolites, with only 10% overlap in metabolic coverage when compared to analog negative ion mode MALDI-MSI on serial sections. We demonstrate that 4-APEBA outperforms other derivatization agents providing: (1) broad specificity towards carbonyls, (2) low background, and (3) introduction of bromine isotopes. Notably, the latter two attributes also facilitate more confidence in our bioinformatics for data processing. The workflow detailed here trailblazes a path towards spatial hormonomics within plant samples, enhancing detection of carboxylates, aldehydes, and plausibly other carbonyls. As such, several phytohormones, which have various roles within stress responses and cellular communication can now be spatially profiled, as demonstrated in poplar root and soybean root nodule.
Supplementary materials
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Supporting Information
Description
Extended Materials and Methods:
Figure.S1: Additional ion images from derivatization performed using a 50% MeOH solution for EDC
and 4-ABEPA.
Figure.S2: NOR vs DHB: MALDI matrix effect on sensitivity and molecular coverage of 4-APEBA in-situ derivatization.
Figure.S3: Comparison of spatial patterns of metabolites with and without in-situ derivatization.
Figure.S4: Ion images of in-situ derivatized exemplary natural product standards.
Figure.S5: MALDI-CID-MS/MS of derivatized standards.
Figure.S6: Measurement of derivatized abscisate within METASPACE imaged from poplar roots.
Figure.S7: Ion images within MALDI-MSI analyses using NEDC and 4-APEBA and evaluation of MSM.
Figure.S8: Comparative plot of MALDI-MSI analyses using NEDC and 4-APEBA.
Figure.S9: 1H-NMR spectrum of synthesized APEBA.
Figure.S10: 1H-NMR spectrum of synthesized BTA.
Table.S1: Variables tested for on tissue chemical derivatization.
Table.S2: A compiled list of all standards tested within the dried droplet derivatization experiments.
Table.S3: Characteristic neutral losses within Figure.S5.
Table.S4: Comparison of classes of molecules from 4-APEBA and NEDC analyses.
References.
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Supporting Workbook
Description
Complete list of METASPACE annotations from multiple databases from the imaging results.
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