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

Abstract

Probing the entirety of any species metabolome is an analytical grand challenge, especially on a cellular scale. Matrixassisted 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 the confident identification of several dozen elusive phytocompounds. Overall, this new OTCD method enabled the annotation of roughly 280 metabolites, with only a 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 by providing: (1) broad specificity toward 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 toward spatial hormonomics within plant samples, enhancing the 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.

Mass spectrometry imaging of natural carbonyl products directly from agar-based microbial interactions using 4-APEBA derivatization

“…4 ). This is because if 4-APEBA is used alone ( 35 ), then it can only derivatize aldehydes and ketones, and it will not derivatize carboxylic acids ( 21 ). EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) must be used as an activator for 4-APEBA to derivatize carboxylic acids (Fig.…”

“…Here, we provide a new MSI approach for mapping endogenous metabolites containing carbonyl groups from microbial systems. We applied our previously developed on-tissue chemical derivatization (OTCD) protocol to microbial cultures grown on agar as a proof of concept of this approach ( 21 ). There are a growing number of OTCD reagents and protocols that have been developed to increase the sensitivity and molecular coverage from mammalian and plant samples in MALDI-MSI ( 22 – 24 ).…”

“…Our approach uses 4-(2-((4-bromophenethyl)dimethylammonio)ethoxy)benzenaminium dibromide (4-APEBA), which adds a permanent positive charge to carbonyl analytes, making them amenable to positive ionization mode analysis in MSI, which is especially useful for analyzing agar-based microbial colonies. Additionally, the bromine in 4-APEBA introduces a unique isotopic pattern to derivatized molecules, which can be exploited for more confident analyte annotation ( 21 ). We used this approach to study the interaction of the soil microbes Bacillus subtilis NCIB 3610 and Fusarium sp.…”

Seeing is Believing: Developing Multimodal Metabolic Insights at the Molecular Level

“…Reactive matrices (L, M) can be used to react with specific functional groups on analytes of interest to form new compounds that are more easily ionized; they often result in the formation of inherently charged molecules. MALDI matrices are versatile for the ionization of biomolecules; hence, there is a significant overlap of usage of the matrices across biological classes. − …”

Identification of Plant Compounds from Mass Spectrometry Imaging (MSI)

“…A promising OTCD on plant tissue was reported in 2023 by Zemaitis et al to enhance the detection in MSI using MALDI [56]. The authors applied 4-(2-((4-bromophenethyl) dimethylammonio) ethoxy) benzenaminium bromide (4-APEBA) to derivatize molecules from soybean nodules and poplar roots.…”