On-tissue Chemical Derivatization Research Articles

Mass Spectrometry Imaging of Amino Acids Enabled by Quaternized Pyridinium Salt MALDI Probe.

The distribution of small biomolecules, particularly amino acids (AAs), differs between normal cells and cancer cells. Imaging this distribution is crucial for gaining a deeper understanding of their physiological and pathological significance. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) provides accurate in situ visualization information. However, the analysis of AAs remains challenging due to the background interference by conventional MALDI matrices. On tissue chemical derivatization (OTCD) MSI serves as an important approach to resolve this issue. We designed, synthesized, and tested a series of pyridinium salt probes and screened out the 1-(4-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)phenyl)-2,4,6-triphenylpyridin-1-ium (DCT) probe with the highest reaction efficiency and the most effective detection. Moreover, a quantum chemistry calculation was executed to address mechanistic insight into the chemical nature of the novel probes. DCT was found to map 20 common AAs in normal mouse tissues for the first time, which allowed differentiation of AA distribution in normal, normal interstitium, tumor, and tumor interstitium regions and provided potential mechanistic insights for cancer research and other biomedical studies.

Spatial detection and consequences of nonrenal calcitriol production as assessed by targeted mass spectrometry imaging.

The immune benefits of vitamin D3 supplementation beyond calcium and phosphate maintenance are highly clinically debated. Kidney expression of CYP27B1 is the source of endocrine, circulating 1,25(OH)2D3 (active form of vitamin D) that maintains serum calcium and phosphate. 1,25(OH)2D3 may also be made by the CYP27B1 enzyme in nonrenal cells, like immune cells, in a process driven by cellular availability of 25(OH)D3 and inflammation. Due to the endocrine nature of 1,25(OH)2D3 in circulation, it is difficult to discern between these 2 sources. We recently created a regulatory deletion model of Cyp27b1 (M1/M21-DIKO) where mice have normal inflammatory-regulated Cyp27b1 expression in nonrenal tissues (unlike global Cyp27b1-KO) but no expression within the kidney. Here, utilizing on-tissue chemical derivatization and matrix assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI), we investigated the distribution of 1,25(OH)2D3 and 25(OH)D3 in the kidney, liver, spleen, and thymus. MALDI-MSI demonstrated increased 1,25(OH)2D3 in nonrenal tissues such as the spleen after vitamin D3 supplementation in M1/M21-DIKO mice. Additionally, from this, we found increased Il4 and decreased Tnfa in the spleen after vitamin D3 supplementation. Taken together, these data demonstrate nonrenal production of 1,25(OH)2D3 in vivo and provide a consequence of vitamin D3 supplementation and nonrenal 1,25(OH)2D3 production in cytokine changes.

Mobilized Electrospray Device for On-Tissue Chemical Derivatization in MALDI-MS Imaging.

Applying solutions of a matrix or derivatization agent via microdroplets is a common sample preparation technique for matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) experiments. 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. As demonstrated for on-tissue chemical derivatization (OTCD) with Girard's reagent T using a mimetic tissue model, the sprayer has the additional benefit of being able to investigate reaction acceleration in OTCD when comparing electrostatically charged spray to electrostatically neutral spray. Finally, MALDI-MSI of fatty aldehydes is successfully demonstrated in rat brain tissues using this device for both OTCD and matrix application.

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

Aliphatic carboxylic acids, aldehydes, and ketones play diverse roles in microbial adaptation to their microenvironment, from excretion as toxins to adaptive metabolites for membrane fluidity. However, the spatial distribution of these molecules throughout biofilms and how microbes in these environments exchange these molecules remain elusive for many of these bioactive species due to inefficient molecular imaging strategies. Herein, we apply on-tissue chemical derivatization (OTCD) using 4-(2-((4-bromophenethyl)dimethylammonio)ethoxy)benzenaminium dibromide (4-APEBA) on a co-culture of a soil bacterium (Bacillus subtilis NCIB 3610) and fungus (Fusarium sp. DS 682) grown on agar as our model system. Using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), we spatially resolved more than 300 different metabolites containing carbonyl groups within this model system. Various spatial patterns are observable in these species, which indicate possible extracellular or intercellular processes of the metabolites and their up- or downregulation during microbial interaction. The unique chemistry of our approach allowed us to bring additional confidence in accurate carbonyl identification, especially when multiple isomeric candidates were possible, and this provided the ability to generate hypotheses about the potential role of some aliphatic carbonyls in this B. subtilis/Fusarium sp. interaction. The results shown here demonstrate the utility of 4-ABEBA-based OTCD MALDI-MSI in probing interkingdom interactions directly from microbial co-cultures, and these methods will enable future microbial interaction studies with expanded metabolic coverage.IMPORTANCEThe metabolic profiles within microbial biofilms and interkingdom interactions are extremely complex and serve a variety of functions, which include promoting colonization, growth, and survival within competitive and symbiotic environments. However, measuring and differentiating many of these molecules, especially in an in situ fashion, remains a significant analytical challenge. We demonstrate a chemical derivatization strategy that enabled highly sensitive, multiplexed mass spectrometry imaging of over 300 metabolites from a model microbial co-culture. Notably, this approach afforded us to visualize over two dozen classes of ketone-, aldehyde-, and carboxyl-containing molecules, which were previously undetectable from colonies grown on agar. We also demonstrate that this chemical derivatization strategy can enable the discrimination of isobaric and isomeric metabolites without the need for orthogonal separation (e.g., online chromatography or ion mobility). We anticipate that this approach will further enhance our knowledge of metabolic regulation within microbiomes and microbial systems used in bioengineering applications.

On-tissue chemical derivatization-enhanced spatially resolved lipidomics reveals abnormal metabolism in type 2 diabetic rat brain

Neurologic disorders are often accompanied by alterations in lipids and oxylipins in the brain. However, the complexity of the lipidome in the brain and its changes during brain damage caused by diabetes remain poorly understood. Herein, we developed an enhanced spatially resolved lipidomics approach with the assistance of on-tissue chemical derivatization to study lipid metabolism in the rat brain. This method enabled the spatially resolved analysis of 560 lipids and oxylipins in 19 brain microregions in coronal and sagittal sections and remarkably improved the coverage of lipidome detection. We applied this method to lipidomic studies of the diabetic rat brain and found that lipid dysregulation followed a microregion-specific pattern. Carnitines and glycerolipids were mainly elevated in the corpus callosum (midbrain) and pineal gland regions, respectively. In addition, most oxylipins, including fatty aldehydes and oxo fatty acids, were significantly upregulated in nine brain microregions. We produced a spatially resolved analysis of lipids and oxylipins, providing a novel analytical tool for brain metabolism research.

Development of an Efficient On-Tissue Epoxidation Reaction Mediated by Urea Hydrogen Peroxide for MALDI MS/MS Imaging of Lipid C═C Location Isomers.

Unsaturated lipids containing different numbers and locations of C═C bonds are significantly associated with a variety of cellular and metabolic functions. Although matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) has been used to visualize the spatial distribution patterns of various lipids in biological tissues, in situ identification, discrimination, and visualization of lipid C═C location isomers remain challenging. Herein, an efficient and fast on-tissue chemical derivatization (OTCD) approach was developed to pinpoint the locations of C═C bonds in complex lipids in situ via methyltrioxorhenium (MTO)-catalyzed epoxidation of C═C with a urea hydrogen peroxide (UHP)/hexafluoroisopropanol (HFIP) system. The efficiency of OTCD could reach 100% via one-step spray deposition of the solution mixture of MTO/UHP/HFIP at room temperature. The developed OTCD method provided rich structural information on lipid C═C location isomers, and their accurate spatial distribution patterns were resolved in mouse brain tissues. Tissue-specific distributions and changes of lipid C═C location isomers in the liver sections of obese ob/ob and diabetic db/db mice were further investigated, and their correlation in two animal models was revealed. The simplicity and high efficiency of the OTCD method developed for MALDI tandem MSI of lipid C═C location isomers possess great potential for functional spatial lipidomics.

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

Probing the entirety of any species metabolome is an analytical grand challenge, especially on 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 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.

On-Tissue Chemical Derivatization for Comprehensive Mapping of Brain Carboxyl and Aldehyde Metabolites by MALDI–MS Imaging

The visualization of small metabolites by MALDI mass spectrometry imaging in brain tissue sections is challenging due to low detection sensitivity and high background interference. We present an on-tissue chemical derivatization MALDI mass spectrometry imaging approach for the comprehensive mapping of carboxyls and aldehydes in brain tissue sections. In this approach, the AMPP (1-(4-(aminomethyl)phenyl)pyridin-1-ium chloride) derivatization reagent is used for the covalent charge-tagging of molecules containing carboxylic acid (in the presence of peptide coupling reagents) and aldehydes. This includes free fatty acids and the associated metabolites, fatty aldehydes, dipeptides, neurotoxic reactive aldehydes, amino acids, neurotransmitters and associated metabolites, as well as tricarboxylic acid cycle metabolites. We performed sensitive ultrahigh mass resolution MALDI-MS detection and imaging of various carboxyl- and aldehyde-containing endogenous metabolites simultaneously in rodent brain tissue sections. We verified the AMPP-derivatized metabolites by tandem MS for structural elucidation. This approach allowed us to image numerous aldehydes and carboxyls, including certain metabolites which had been undetectable in brain tissue sections. We also demonstrated the application of on-tissue derivatization to carboxyls and aldehydes in coronal brain tissue sections of a nonhuman primate Parkinson's disease model. Our methodology provides a powerful tool for the sensitive, simultaneous spatial molecular imaging of numerous aldehydes and carboxylic acids during pathological states, including neurodegeneration, in brain tissue.

On-tissue chemical derivatization of volatile metabolites for matrix-assisted laser desorption/ionization mass spectrometry imaging.

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. On-tissue chemical derivatization can dramatically reduce the volatility of analytes. Herein, a derivatization method is proposed utilizing N,N,N-trimethyl-2-(piperazin-1-yl)ethan-1-aminium iodide to chemically modify short-chain fatty acids in chicken cecum, ileum, and jejunum tissue sections before sample preparation for MSI visualization.

In situ droplet-based on-tissue chemical derivatization for lipid isomer characterization using LESA.

In this work, we present an in situ droplet-based derivatization method for fast tissue lipid profiling at multiple isomer levels. On-tissue derivatization for isomer characterization was achieved in a droplet delivered by the TriVersa NanoMate LESA pipette. The derivatized lipids were then extracted and analyzed by the automated chip-based liquid extraction surface analysis (LESA) mass spectrometry (MS) followed by tandem MS to produce diagnostic fragment ions to reveal the lipid isomer structures. Three reactions, i.e., mCPBA epoxidation, photocycloaddition catalyzed by the photocatalyst Ir[dF(CF3)ppy]2(dtbbpy)PF6, and Mn(II) lipid adduction, were applied using the droplet-based derivatization to provide lipid characterization at carbon-carbon double-bond positional isomer and sn-positional isomer levels. Relative quantitation of both types of lipid isomers was also achieved based on diagnostic ion intensities. This method provides the flexibility of performing multiple derivatizations at different spots in the same functional region of an organ for orthogonal lipid isomer analysis using a single tissue slide. Lipid isomers were profiled in the cortex, cerebellum, thalamus, hippocampus, and midbrain of the mouse brain and 24 double-bond positional isomers and 16 sn-positional isomers showed various distributions in those regions. This droplet-based derivatization of tissue lipids allows fast profiling of multi-level isomer identification and quantitation and has great potential in tissue lipid studies requiring rapid sample-to-result turnovers.

On-tissue chemical derivatization enables spatiotemporal heterogeneity visualization of oxylipins in esophageal cancer xenograft via ambient mass spectrometry imaging

On-tissue chemical derivatization (OTCD) effectively enhances ionization efficiency of low abundant and poorly ionized functional molecules to improve detection sensitivity and coverage of mass spectrometry imaging (MSI). Combination OTCD and MSI provides a novel strategy for visualizing previously undisclosed metabolic heterogeneity in tumor. Herein, we present a method to visualize heterogeneous metabolism of oxylipins within tumor by coupling OTCD with airflow-assisted desorption electrospray ionization (AFADESI)-MSI. Taking Girard's P as a derivatization reagent, easily ionized hydrazide and quaternary amine groups were introduced into the structure of carbonyl metabolites via condensation reaction. Oxylipins, including 127 fatty aldehydes (FALs) and 71 oxo fatty acids (FAs), were detected and imaged in esophageal cancer xenograft with AFADESI-MSI after OTCD. Then t-distributed stochastic neighbor embedding and random forest were exploited to precisely locate the distribution of oxylipins in heterogeneous tumor tissue. With this method, we surprisingly found almost all FALs and oxo FAs significantly accumulated in the core region of tumor, and exhibited a gradual increase trend in tumor over time. These results reveal spatiotemporal heterogeneity of oxylipins in tumor progression, highlighting the value of OTCD combined with MSI to gain deeper insights into understanding tumor metabolism.

A sensitive on-tissue chemical derivatization-mass spectrometry imaging method for the quantitative visualization of helicid in mice

Helicid (4-formylphenyl-O-β-d-allopyranoside) is an aromatic phenolic glucoside. It is one of the main active constituents from the seeds of the Chinese herb Helicia nilagirica. Helicid has been reported to exert sedative, analgesic, hypnotic and antidepressant effects, and is currently used clinically to treat neurasthenic syndrome, vascular headaches and trigeminal neuralgia. On-tissue chemical derivatization (OTCD) combined with matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) was developed for the quantitative visualization of helicid in mice. The derivatization reagent, Girard’s reagent T (GT), was sprayed directly onto the tissue surface using an automated pneumatic TM-sprayer. The OTCD reaction was conducted in a closed container with 120 mL of 50% methanol with 0.4% TFA at 40 °C for 40 min. α-Cyano-4-hydroxycinnamic acid (2.22 μg/mm2) was used as the matrix, which was deposited onto the tissue surface. MALDI-MSI analysis was performed in the positive-ion mode with a spatial resolution of 200 μm. The sensitivity for the MALDI-MSI of helicid in tissue sections increased 1920 times after derivatization. The assay showed good linearity in the helicid concentration range of 0.32–32 ng/mm2. The method was used to investigate the spatiotemporal distribution of helicid in mice.

On-Tissue Chemical Oxidation Followed by Derivatization for Mass Spectrometry Imaging Enables Visualization of Primary and Secondary Hydroxyl-Containing Metabolites in Biological Tissues.

On-tissue chemical derivatization combined with mass spectrometry imaging (MSI) can effectively visualize low-abundance and poorly ionizable molecules in biological tissues. Owing to the lack of an effective chemical reaction environment on the tissue surface, the development of direct one-step derivatization reactions is challenging. Herein, we present a two-step reaction involving on-tissue chemical oxidation followed by derivatization combined with airflow-assisted desorption electrospray ionization-MSI, enabling the visualization of primary and secondary hydroxyl-containing metabolites (PSHMs) within the tissue sections. This method indirectly achieved on-tissue derivatization by combining two reactions. Hydroxyl was converted to carbonyl using chemical oxidants, and subsequently, carbonyl was derived using Girard's P reagent. Using this methodology, 169 PSHMs, including hydroxy fatty acids (OH-FAs), fatty alcohols (FOHs), and sterol lipids, were detected and imaged in the tissues of rat brain, kidney, and liver. Moreover, we found that the abundant PSHMs, fatty aldehydes, and oxo fatty acids were significantly dysregulated in the liver and kidney tissues of type 2 diabetic rats; in particular, OH-FAs and FOHs were remarkably up-regulated in the diabetic rat liver tissues. The aberrations of these oxidative metabolites provide insights into the understanding of the molecular pathological mechanism of diabetes. This study demonstrates a novel, two-step reaction strategy for on-tissue derivatization with the analysis of previously inaccessible molecules using MSI.

Mapping of Fatty Aldehydes in the Diabetic Rat Brain Using On-Tissue Chemical Derivatization and Air-Flow-Assisted Desorption Electrospray Ionization-Mass Spectrometry Imaging.

Fatty aldehydes (FALs) are involved in various biological processes, and their abnormal metabolism is related to the occurrence and development of neurological diseases. Because of their low ionization efficiency, methods for in situ detection and mass spectrometry imaging (MSI) analysis of FALs remain underreported. On-tissue chemical tagging of hardly ionizable target analytes with easily ionized moieties can improve ionization efficiency and detection sensitivity in MSI experiments. In this study, an on-tissue chemical derivatization-air-flow-assisted desorption electrospray ionization-MSI method was developed to visualize FALs in the rat brain. The method showed high sensitivity and specificity, allowing the use of in situ high-resolution MS3 to identify FALs. The methodology was applied to investigate the region-specific distribution of FALs in the brains of control and diabetic encephalopathy (DE) rats. In DE rats, FALs were found to be significantly enriched in various brain regions, especially in the cerebral cortex, hippocampus, and amygdala. Thus, increased FAL levels and oxidative stress occurred in a region-dependent manner, which may contribute to cognitive function deficits in DE. In summary, we provide a novel method for the in situ detection of FALs in biological tissues as well as new insights into the potential pathogenesis of DE.

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

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.

Spatial localization of β-unsaturated aldehyde markers in murine diabetic kidney tissue by mass spectrometry imaging

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease. Limitations in current diagnosis and screening methods have sparked a search for more specific and conclusive biomarkers. Hyperglycemic conditions generate a plethora of harmful molecules in circulation and within tissues. Oxidative stress generates reactive α-dicarbonyls and β-unsaturated hydroxyhexenals, which react with proteins to form advanced glycation end products. Mass spectrometry imaging (MSI) enables the detection and spatial localization of molecules in biological tissue sections. Here, for the first time, the localization and semiquantitative analysis of “reactive aldehydes” (RAs) 4-hydroxyhexenal (4-HHE), 4-hydroxynonenal (4-HNE), and 4-oxo-2-nonenal (4-ONE) in the kidney tissues of a diabetic mouse model is presented. Ionization efficiency was enhanced through on-tissue chemical derivatization (OTCD) using Girard’s reagent T (GT), forming positively charged hydrazone derivatives. MSI analysis was performed using matrix-assisted laser desorption ionization (MALDI) coupled with Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR). RA levels were elevated in diabetic kidney tissues compared to lean controls and localized throughout the kidney sections at a spatial resolution of 100 µm. This was confirmed by liquid extraction surface analysis–MSI (LESA-MSI) and liquid chromatography–mass spectrometry (LC–MS). This method identified β-unsaturated aldehydes as “potential” biomarkers of DN and demonstrated the capability of OTCD-MSI for detection and localization of poorly ionizable molecules by adapting existing chemical derivatization methods. Untargeted exploratory distribution analysis of some precursor lipids was also assessed using MALDI-FT-ICR-MSI.Graphical abstract

On-tissue pyrene-1-boronic acid labeling assisted MALDI imaging of catecholamines in porcine adrenal gland

In this study, an on-tissue chemical labeling - matrix assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) method was developed for visualization of the distribution of three catecholamine (CA) compounds (dopamine, epinephrine and norepinephrine) in porcine adrenal gland. Commercially available pyrene-1-boronic acid (PBA) was employed as an effective in situ derivatizing reagent dissolved in acetonitrile containing 0.1% pyridine for the chemical labeling and the matrix coating. Without extra matrix coating, the tissue section was directly analyzed by MALDI-MS. The detection specificity and sensitivity were greatly improved with the on-tissue PBA labeling and successful imaging of the three CAs in porcine adrenal gland was achieved. Compared with previously reported methods for MALDI-MSI of the CAs, the analytical strategy proposed in the study provided a robust, easy-to-use and low-cost on-tissue chemical derivatization method that facilitated simultaneous molecular imaging of the three compounds.

Rapid and Automatic Annotation of Multiple On-Tissue Chemical Modifications in Mass Spectrometry Imaging with Metaspace

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 this work, we present a pipeline to provide rigorous annotations for on-tissue derivatized MSI data using Metaspace. To test and validate the pipeline, maize roots were used as a model system to obtain MSI datasets after chemical derivatization with four different reagents, Girard's T and P for carbonyl groups, coniferyl aldehyde for primary amines, and 2-picolylamine for carboxylic acids. Using this pipeline helped us annotate 631 unique metabolites from the CornCyc/BraChem database compared to 256 in the underivatized dataset, yet, at the same time, shortening the processing time compared to manual processing and providing robust and systematic scoring and annotation. We have also developed a method to remove false derivatized annotations, which can clean 5-25% of false derivatized annotations from the derivatized data, depending on the reagent. Taken together, our pipeline facilitates the use of broadly targeted spatial metabolomics using multiple derivatization reagents.

Enhancing Metabolite Coverage for Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Through Multiple On-Tissue Chemical Derivatizations.

The ability to study and visualize metabolites on a cellular and sub-cellular level is important for gaining insights into biological pathways and metabolism of multicellular organisms. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is a powerful analytical tool for metabolomics experiments due to its high sensitivity and small sampling size. The spatial resolution in MALDI-MSI is mainly limited by the number of molecules available in a small sampling size. When the sampling size is low enough to achieve cellular or subcellular spatial resolution, signal intensity is sacrificed making poorly ionized metabolites difficult to detect. To overcome this limitation, on-tissue chemical derivatization reactions have been used to enhance the desorption/ionization efficiency of selected classes of compounds by adding a functional group with a permanent positive charge or one that can be easily ionized. By utilizing several chemical derivatizations in parallel, metabolite coverage can be drastically improved. This chapter outlines methodology for sample preparation and data analysis for on-tissue chemical derivatization using various derivatization reagents.

Enhanced On-Tissue Chemical Derivatization with Hydrogel Assistance for Mass Spectrometry Imaging.

The improvement of on-tissue chemical derivatization for mass spectrometry imaging (MSI) of low-abundance and/or poorly ionizable functional molecules in biological tissue without delocalization is challenging. Here, we developed a novel hydrogel-assisted chemical derivatization (HCD) approach coupled with airflow-assisted desorption electrospray ionization (AFADESI)-MSI, allowing for enhanced visualization of inaccessible molecules in biological tissues. The derivatization reagent Girard's P (GP) reagent was creatively packaged into a hydrogel to form HCD blocks that have reactivity to carbonyl compounds as well as the feasibility of "cover/uncover" contact mode with tissue sections. The HCD blocks provided a favorable liquid microenvironment for the derivatization reaction and reduced matrix effects from derivatization reagents and tissue without obvious molecular migration, thus improving the derivatization efficiency. With this methodology, unusual carbonyl metabolites, including 166 fatty aldehydes (FALs) and 100 oxo fatty acids (FAs), were detected and visualized in rat brain, kidney, and liver tissue. This study provides a new approach to enhance chemical labeling for in situ tissue submetabolome profiling and improves our knowledge of the molecular histology and complex metabolism of biological tissues.