Expanded Coverage of Phytocompounds by Mass Spectrometry Imaging Using On-Tissue Chemical Derivatization by 4-APEBA

28 March 2023, Version 2
This content is a preprint and has not undergone peer review at the time of posting.

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.

Keywords

mass spectrometry imaging
MSI
matrix-assisted laser desorption/ionization
MALDI
MALDI-MSI
phytohormones
phytocompounds
soybean
poplar
4-APEBA
on-tissue chemical derivatization
OTCD

Supplementary materials

Title
Description
Actions
Title
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.
Actions
Title
Supporting Workbook
Description
Complete list of METASPACE annotations from multiple databases from the imaging results.
Actions

Comments

Comments are not moderated before they are posted, but they can be removed by the site moderators if they are found to be in contravention of our Commenting Policy [opens in a new tab] - please read this policy before you post. Comments should be used for scholarly discussion of the content in question. You can find more information about how to use the commenting feature here [opens in a new tab] .
This site is protected by reCAPTCHA and the Google Privacy Policy [opens in a new tab] and Terms of Service [opens in a new tab] apply.