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Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a fast-growing technique for visualization of the spatial distribution of the small molecular and macromolecular biomolecules in tissue sections. Challenges in MALDI-MSI, such as poor sensitivity for some classes of molecules or limited specificity, for instance re...
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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 an...
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... There are multiple components of MSI sample preparation that need to be considered, from sample collection to surface pretreatment ahead of investigation. 71) A typical MALDI-MSI workflow highlighting the importance of tissue preparation is shown in Fig. 5. For tissue preparation, many procedures can be used, ranging from tissue harvest to data acquisition. ...
... For tissue preparation, many procedures can be used, ranging from tissue harvest to data acquisition. 71) Washes, on-tissue enzymatic digestion, on-tissue chemical derivatization (OTCD), and the utilization of internal standard are possibilities in addition to the primary experimental workflow, depending on tissue type, targeted analyte species, and experimental goals. 71) Internal standards can be employed for normalization and comparative quantification to counteract the effects of ion suppression in diverse tissue sections. ...
... 71) Washes, on-tissue enzymatic digestion, on-tissue chemical derivatization (OTCD), and the utilization of internal standard are possibilities in addition to the primary experimental workflow, depending on tissue type, targeted analyte species, and experimental goals. 71) Internal standards can be employed for normalization and comparative quantification to counteract the effects of ion suppression in diverse tissue sections. This produces findings that are more highly comparable between different tissues and pixels. ...
Among the most typical posttranslational modifications is glycosylation, which often involves the covalent binding of an oligosaccharide (glycan) to either an asparagine (N-linked) or a serine/threonine (O-linked) residue. Studies imply that the N-glycan portion of a glycoprotein (GP) could serve as a particular disease biomarker rather than the protein itself because N-linked glycans have been widely recognized to evolve with the advancement of tumors and other diseases. N-glycans found on protein asparagine sites have been especially significant. Since N-glycans play clearly defined functions in the folding of proteins, cellular transport, and transmission of signals, modifications to them have been linked to several illnesses. However, because these N-glycans’ production is not template driven, they have a substantial morphological range, rendering it difficult to distinguish the species that are most relevant to biology and medicine using standard techniques. Mass spectrometry (MS) techniques have emerged as effective analytical tools for investigating the role of glycosylation in health and illness. This is due to developments in MS equipment, data collection, and sample handling techniques. By recording the spatial dimension of a glycan’s distribution in situ, mass spectrometry imaging (MSI) builds atop existing methods while offering added knowledge concerning the structure and functionality of biomolecules. In this review article, we address the current development of glycan MSI, starting with the most used tissue imaging techniques and ionization sources before proceeding on to a discussion on applications and concluding with implications for clinical research.
... This process enables visualization of the relative abundance and spatial distribution of each detected molecule. MALDI MSI has been used for metabolomic imaging in samples such as kidney, brain, and liver (Djambazova et al., 2020;Kaya et al., 2020;Zhou et al., 2021;He et al., 2022). Immunofluorescence (IF) is the gold standard for labeling cell types/states. ...
... desorption for specific molecular classes and method polarities. Factors to consider when choosing a matrix include the molecular class of interest (e.g., lipids, small molecules, proteins, glycans), ionization polarity (i.e., negative or positive ion mode), matrix volatility, MALDI laser wavelength, minimal background signal, and, in this case, the ability to gently remove the matrix for fluorescence experiments (Zhou et al., 2021). Three common matrix selections for MALDI MSI of lipids in positive mode are CHCA, DAN, and DHB, although DAN is capable of ionizing in positive and negative ion modes. ...
Proper neurological function relies on the cellular and molecular microenvironment of the brain, with perturbations of this environment leading to neurological disorders. However, studying the microenvironments of neurological tissue has proven difficult because of its inherent complexity. Both the cell type and metabolomic underpinnings of the cell have crucial functional roles, thus making multimodal characterization methods key to acquiring a holistic view of the brain’s microenvironment. This study investigates methods for combining matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) and immunofluorescence (IF) microscopy to enable the concurrent investigation of cell types and lipid profiles on the same sample. In brief, 1,5-diaminonaphthalene (DAN), α-cyano-4-hydroxy-cinnamic acid (CHCA), and 2,5-dihydroxybenzoic acid (DHB) were tested in addition to instrument-specific parameters for compatibility with IF. Alternatively, the effects of IF protocols on MALDI MSI were also tested, showing significant signal loss with all tested permutations. Ultimately, the use of CHCA for MALDI MSI resulted in the best IF images, while the use of DAN gave the lowest quality IF images. Overall, increasing the laser power and number of shots per laser burst resulted in the most tissue ablation. However, optimized parameter settings allowed for minimal tissue ablation while maintaining sufficient MALDI MSI signal.
... 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)(23)(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. ...
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.
IMPORTANCE
The 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.
... 5) Novel MALDI matrices have been reported to detect lipids such as diacylglycerols (DAGs), ceramides, and sphingomyelins for 3-aminophthalhydrazide, and cholesterol and polyamines for 1,1′-binaphthyl-2,2′-diamine high sensitively. 6,7) Almost all steroids and some DAGs could not be detected directly and high sensitively by MALDI/MS although it provides high sensitivity in the analysis of phosphatidylcholines. The derivatization method for the steroids 8,9) and application of other ionization method would be considered as the critical steps in the analysis. ...
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) has been widely used for analyses of biomolecules and industrial materials. Surface-assisted laser desorption/ionization (SALDI) is studied to complement the ionization ability for the MALDI/MS. In this study, lab-made mist chemical vapor deposition (mist CVD) system was used to produce metal films as ionization assistance materials for SALDI/MS. The system could give Ag film from inexpensive silver trifluoroacetate solution rapidly and simply under atmospheric pressure. Phosphatidylcholines could be detected high sensitively and diacylglycerols (DAGs) could not be detected in MALDI/MS. In the SALDI/MS and the MS imaging with Ag film by mist CVD, both the phosphatidylcholines and the DAGs could be detected and the localized images. In the Ag film-SALDI/MS of lipids, not only Ag-adducted ions but also Na- and K-adducted ions were detected. The Ag film formed by the mist CVD to act as an ionization-assistance material and a cationization agent in SALDI would be useful in MS imaging of biological tissue sections.
... Recently, on-tissue chemical derivatization (OTCD) coupled with MALDI-MSI has emerged as a powerful approach to overcome sensitivity and other mass analyzer limitations. [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. ...
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.
... [1] MALDI MS imaging (MSI) has recently gained increasing attention in clinical [2] and pharmaceutical [3] research. In parallel, the investigation of the ionization mechanism [4] and the discovery of novel MALDI matrix chemicals [5] are active research topics. Besides theoretical arguments regarding requirements for a good MALDI matrix, [4] practical considerations for good laboratory practice, such as vacuum stability, solubility in common solvents, and chemical stability are key. ...
... [1] MALDI MS imaging (MSI) has recently gained increasing attention in clinical [2] and pharmaceutical [3] research. In parallel, the investigation of the ionization mechanism [4] and the discovery of novel MALDI matrix chemicals [5] are active research topics. Besides theoretical arguments regarding requirements for a good MALDI matrix, [4] practical considerations for good laboratory practice, such as vacuum stability, solubility in common solvents, and chemical stability are key. ...
Insufficient vacuum stability of matrix chemicals is a major limitation in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) of large tissue sample cohorts. Here, we designed and synthesized the photo-cleavable caged molecule 4,5-dimethoxy-2-nitrobenzyl-2,5-dihydroxyacetophenone (DMNB-2,5-DHAP) and employed it for lipid MALDI-MSI of mouse brain tissue sections. DMNB-2,5-DHAP is vacuum-stable in a high vacuum MALDI ion source for at least 72 h. Investigation of the uncaging process suggested that the built-in laser (355 nm) in the MALDI ion source promoted the in situ generation of 2,5-DHAP. A caging group is used for the first time in designing a MALDI matrix that is vacuum-stable, uncaged upon laser irradiation during the measurement process, and that boosts lipid ion intensity with MALDI-2 laser-induced postionization.
... Recently, on-tissue chemical derivatization (OTCD) coupled with MALDI-MSI has emerged as a powerful approach to overcome sensitivity and other mass analyzer limitations. [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. ...
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.
... For instance, the vacuum stability of the matrix is even more important since MALDI MSI experiments take much more time than the acquisition of a conventional MALDI spectrum. For further details, please check the recently published reviews (for instance, [4,12,17]) or some textbooks with a focus on MSI (e.g., [18]). ...
... A review dealing with useful matrices for MSI purposes has been recently published by the Hopf laboratory [12]. The data therein can be transferred to the acquisition of MALDI mass spectra of lipid solutions. ...
Matrix-assisted laser desorption and ionization (MALDI) is a widely used soft-ionization technique of modern mass spectrometry (MS). MALDI enables the analysis of nearly all chemical compounds—including polar and apolar (phospho)lipids—with a minimum extent of fragmentation. MALDI has some particular advantages (such as the possibility to acquire spatially-resolved spectra) and is competitive with the simultaneously developed ESI (electrospray ionization) MS. Although there are still some methodological aspects that need to be elucidated in more detail, it is obvious that the careful selection of an appropriate matrix plays the most important role in (lipid) analysis. Some lipid classes can be detected exclusively if the optimum matrix is used, and the matrix determines the sensitivity by which a particular lipid is detected within a mixture. Since the matrix is, thus, crucial for optimum results, we provide here an update on the progress in the field since our original review in this journal in 2018. Thus, only the development during the last five years is considered, and lipids are sorted according to increasing complexity, starting with free fatty acids and ending with cardiolipins and phosphoinositides.
... More details on the principles and workflows of MALDI MS methodology can also be found elsewhere [202][203][204]. Matrices are pivotal to successful MALDI MS experiments, and the advances in various novel MALDI matrices have been comprehensively reviewed in the literature [205,206]. MALDI MS is especially useful to analyze a mixture of analytes such as protein digests and polymers because singly charged ions are major forms produced during the process, which reduces the mass spectral complexity [207,208]. It also covers a wide range of sample types, including reaction mixtures [209], tissues [210,211] and biofluids [212][213][214][215]. ...
Technological developments and improvements in single-cell isolation and analytical platforms allow for advanced molecular profiling at the single-cell level, which reveals cell-to-cell variation within the admixture cells in complex biological or clinical systems. This helps to understand the cellular heterogeneity of normal or diseased tissues and organs. However, most studies focused on the analysis of nucleic acids (e.g., DNA and RNA) and mass spectrometry (MS)-based analysis for proteins and metabolites of a single cell lagged until recently. Undoubtedly, MS-based single-cell analysis will provide a deeper insight into cellular mechanisms related to health and disease. This review summarizes recent advances in MS-based single-cell analysis methods and their applications in biology and medicine.
