Molecular Networking and On-Tissue Chemical Derivatization for Enhanced Identification and Visualization of Steroid Glycosides by MALDI Mass Spectrometry Imaging

Abstract

Spatial metabolomics describes the spatially resolved analysis of interconnected pathways, biochemical reactions, and transport processes of small molecules in the spatial context of tissues and cells. However, a broad range of metabolite classes (e.g., steroids) show low intrinsic ionization efficiencies in mass spectrometry imaging (MSI) experiments, thus restricting the spatial characterization of metabolic networks. Additionally, decomposing complex metabolite networks into chemical compound classes and molecular annotations remains a major bottleneck due to the absence of repository-scaled databases. Here, we describe a multimodal mass-spectrometry-based method combining computational metabolome mining tools and high-resolution on-tissue chemical derivatization (OTCD) MSI for the spatially resolved analysis of metabolic networks at the low micrometer scale. Applied to plant toxin sequestration in Danaus plexippus as a model system, we first utilized liquid chromatography (LC)–MS-based molecular networking in combination with artificial intelligence (AI)-driven chemical characterization to facilitate the structural elucidation and molecular identification of 32 different steroidal glycosides for the host-plant Asclepias curassavica. These comprehensive metabolite annotations guided the subsequent matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) analysis of cardiac-glycoside sequestration in D. plexippus. We developed a spatial-context-preserving OTCD protocol, which improved cardiac glycoside ion yields by at least 1 order of magnitude compared to results with untreated samples. To illustrate the potential of this method, we visualized previously inaccessible (sub)cellular distributions (2 and 5 μm pixel size) of steroidal glycosides in D. plexippus, thereby providing a novel insight into the sequestration of toxic metabolites and guiding future metabolomics research of other complex sample systems.

Lewis Acidic Metal–Organic Framework Assisted Ambient Liquid Extraction Mass Spectrometry Imaging for Enhancing the Coverage of Poorly Ionizable Lipids in Brain Tissue

“…Moreover, the calculated equilibrium distribution coefficient (k L ) on Zr 6 OTf-BTB (0.08 L•mg −1 ) is also much higher than those on Zr 6 -BTB (0.04 L•mg −1 ) with low Lewis acidity and the other Lewis acidic material TiO 2 @SiO 2 (0.05 L•mg −1 ), indicating higher binding strength between phospholipids and Zr 6 OTf-BTB. The measured high adsorption capacity and high distribution coefficient indicate that Zr 6 OTf-BTB has the potential to adsorb high-abundance phospholipids from complex biological samples. Then, the adsorption kinetics of lipids on different materials was investigated from adsorption time curves obtained by dSPE experiments with varied time.…”

“…Mass spectrometry imaging (MSI) is an effective multiplexed and label-free analytical technique for the visualization and characterization of metabolites, lipids, proteins, and drugs in complex samples. − However, in biological samples, low-abundance or poorly ionizable substances such as sphingolipids and glycolipids were difficult to be imaged because of the strong ion suppression from endogenous compounds with high abundance and ionization efficiency, such as phosphatidylcholine (PC). − This largely hindered MSI from providing more spatiochemical information for biological and clinical studies.…”

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

“…12−15 This approach enables visualization of the spatial distribution of many biological compounds and molecular networks in microbial, plant, and mammalian cells. 16 Specifically, OTCD enhances the detection sensitivity by introducing a charged moiety or a readily ionizable functional group into the analyte. Concurrently, this derivatization process increases masses of the metabolites toward more sensitive regions, bypassing issues related to discrimination of metabolites from complex background spectral features (i.e., isobaric separation) and low mass transmission limitations of high-resolution mass spectrometers, such as Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) instruments.…”

“…Recently, on-tissue chemical derivatization (OTCD) coupled with MALDI-MSI has emerged as a powerful approach to overcome sensitivity and other mass analyzer limitations. – This approach enables visualization of the spatial distribution of many biological compounds and molecular networks in microbial, plant, and mammalian cells . Specifically, OTCD enhances the detection sensitivity by introducing a charged moiety or a readily ionizable functional group into the analyte.…”

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

“…[12][13][14][15] This approach enables visualization of the spatial distribution of many biological compounds and molecular networks in microbial, plant, and mammalian cells. 16 Specifically, OTCD enhances the detection sensitivity by introducing a charged moiety or a readily ionizable functional group to the analyte. Concurrently, this derivatization process increases masses of the metabolites toward more sensitive regions, bypassing issues related to discrimination of metabolites from complex background spectral features (i.e., isobaric separation) and low mass transmission limitations of high-resolution mass spectrometers, such as Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) instruments.…”