Stability-based approaches in chemoproteomics.

Target deconvolution can help understand how compounds exert therapeutic effects and can accelerate drug discovery by helping optimise safety and efficacy, revealing mechanisms of action, anticipate off-target effects and identifying opportunities for therapeutic expansion. Chemoproteomics, a combination of chemical biology with mass spectrometry has transformed target deconvolution. This review discusses modification-free chemoproteomic approaches that leverage the change in protein thermodynamics induced by small molecule ligand binding. Unlike modification-based methods relying on enriching specific protein targets, these approaches offer proteome-wide evaluations, driven by advancements in mass spectrometry sensitivity, increasing proteome coverage and quantitation methods. Advances in methods based on denaturation/precipitation by thermal or chemical denaturation, or by protease degradation are evaluated, emphasising the evolving landscape of chemoproteomics and its potential impact on future drug-development strategies.

Inhibition of macrophage infectivity potentiator in Burkholderia pseudomallei suppresses pro-inflammatory responses in murine macrophages.

Introduction: Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is a disease endemic in many tropical countries globally. Clinical presentation is highly variable, ranging from asymptomatic to fatal septicemia, and thus the outcome of infection can depend on the host immune responses. The aims of this study were to firstly, characterize the macrophage immune response to B. pseudomallei and secondly, to determine whether the immune response was modified in the presence of novel inhibitors targeting the virulence factor, the macrophage infectivity potentiator (Mip) protein. We hypothesized that inhibition of Mip in B. pseudomallei would disarm the bacteria and result in a host beneficial immune response. Methods: Murine macrophage J774A.1 cells were infected with B. pseudomallei K96243 in the presence of small-molecule inhibitors targeting the Mip protein. RNA-sequencing was performed on infected cells four hours post-infection. Secreted cytokines and lactose dehydrogenase were measured in cell culture supernatants 24 hours post-infection. Viable, intracellular B. pseudomallei in macrophages were also enumerated 24 hours post-infection. Results: Global transcriptional profiling of macrophages infected with B. pseudomallei by RNA-seq demonstrated upregulation of immune-associated genes, in particular a significant enrichment of genes in the TNF signaling pathway. Treatment of B. pseudomallei-infected macrophages with the Mip inhibitor, AN_CH_37 resulted in a 5.3-fold reduction of il1b when compared to cells treated with DMSO, which the inhibitors were solubilized in. A statistically significant reduction in IL-1ß levels in culture supernatants was seen 24 hours post-infection with AN_CH_37, as well as other pro-inflammatory cytokines, namely IL-6 and TNF-α. Treatment with AN_CH_37 also reduced the survival of B. pseudomallei in macrophages after 24 hours which was accompanied by a significant reduction in B. pseudomallei-induced cytotoxicity as determined by lactate dehydrogenase release. Discussion: These data highlight the potential to utilize Mip inhibitors in reducing potentially harmful pro-inflammatory responses resulting from B. pseudomallei infection in macrophages. This could be of significance since overstimulation of pro-inflammatory responses can result in immunopathology, tissue damage and septic shock.

Comparison of Quantitative Mass Spectrometric Methods for Drug Target Identification by Thermal Proteome Profiling.

Thermal proteome profiling (TPP) provides a powerful approach to studying proteome-wide interactions of small therapeutic molecules and their target and off-target proteins, complementing phenotypic-based drug screens. Detecting differences in thermal stability due to target engagement requires high quantitative accuracy and consistent detection. Isobaric tandem mass tags (TMTs) are used to multiplex samples and increase quantification precision in TPP analysis by data-dependent acquisition (DDA). However, advances in data-independent acquisition (DIA) can provide higher sensitivity and protein coverage with reduced costs and sample preparation steps. Herein, we explored the performance of different DIA-based label-free quantification approaches compared to TMT-DDA for thermal shift quantitation. Acute myeloid leukemia cells were treated with losmapimod, a known inhibitor of MAPK14 (p38α). Label-free DIA approaches, and particularly the library-free mode in DIA-NN, were comparable of TMT-DDA in their ability to detect target engagement of losmapimod with MAPK14 and one of its downstream targets, MAPKAPK3. Using DIA for thermal shift quantitation is a cost-effective alternative to labeled quantitation in the TPP pipeline.

Assessing the Phagosome Proteome by Quantitative Mass Spectrometry.

The process of phagocytosis involves a series of defined steps, including the formation of a new intracellular organelle, i.e., the phagosome, and the maturation of the phagosome by fusion with endosomes and lysosomes to produce an acidic and proteolytic environment in which the pathogens are degraded. Phagosome maturation is associated with significant changes in the proteome of phagosomes due to the acquisition of new proteins or enzymes, post-translational modifications of existing proteins, as well as other biochemical changes that ultimately lead to the degradation or processing of the phagocytosed particle. Phagosomes are highly dynamic organelles formed by the uptake of particles through phagocytic innate immune cells; thus characterization of the phagosomal proteome is essential to understand the mechanisms controlling innate immunity, as well as vesicle trafficking. In this chapter, we describe how novel quantitative proteomics methods, such as using tandem mass tag (TMT) labelling or acquiring label-free data using data-independent acquisition (DIA), can be applied for the characterization of protein composition of phagosomes in macrophages.

Biological insights from multi-omics analysis strategies: Complex pleotropic effects associated with autophagy.

Research strategies that combine molecular data from multiple levels of genome expression (i.e., multi-omics data), often referred to as a systems biology strategy, has been advocated as a route to discovering gene functions. In this study we conducted an evaluation of this strategy by combining lipidomics, metabolite mass-spectral imaging and transcriptomics data from leaves and roots in response to mutations in two AuTophaGy-related (ATG) genes of Arabidopsis. Autophagy is an essential cellular process that degrades and recycles macromolecules and organelles, and this process is blocked in the atg7 and atg9 mutants that were the focus of this study. Specifically, we quantified abundances of ~100 lipids and imaged the cellular locations of ~15 lipid molecular species and the relative abundance of ~26,000 transcripts from leaf and root tissues of WT, atg7 and atg9 mutant plants, grown either in normal (nitrogen-replete) and autophagy-inducing conditions (nitrogen-deficient). The multi-omics data enabled detailed molecular depiction of the effect of each mutation, and a comprehensive physiological model to explain the consequence of these genetic and environmental changes in autophagy is greatly facilitated by the a priori knowledge of the exact biochemical function of the ATG7 and ATG9 proteins.

Advances in high-throughput mass spectrometry in drug discovery.

High-throughput (HT) screening drug discovery, during which thousands or millions of compounds are screened, remains the key methodology for identifying active chemical matter in early drug discovery pipelines. Recent technological developments in mass spectrometry (MS) and automation have revolutionized the application of MS for use in HT screens. These methods allow the targeting of unlabelled biomolecules in HT assays, thereby expanding the breadth of targets for which HT assays can be developed compared to traditional approaches. Moreover, these label-free MS assays are often cheaper, faster, and more physiologically relevant than competing assay technologies. In this review, we will describe current MS techniques used in drug discovery and explain their advantages and disadvantages. We will highlight the power of mass spectrometry in label-free in vitro assays, and its application for setting up multiplexed cellular phenotypic assays, providing an exciting new tool for screening compounds in cell lines, and even primary cells. Finally, we will give an outlook on how technological advances will increase the future use and the capabilities of mass spectrometry in drug discovery.

A high-throughput MALDI-TOF MS biochemical screen for small molecule inhibitors of the antigen aminopeptidase ERAP1.

MALDI-TOF MS is a powerful analytical technique that provides a fast and label-free readout for in vitro assays in the high-throughput screening (HTS) environment. Here, we describe the development of a novel, HTS compatible, MALDI-TOF MS-based drug discovery assay for the endoplasmic reticulum aminopeptidase 1 (ERAP1), an important target in immuno-oncology and auto-immune diseases. A MALDI-TOF MS assay was developed beginning with an already established ERAP1 RapidFire MS (RF MS) assay, where the peptide YTAFTIPSI is trimmed into the product TAFTIPSI. We noted low ionisation efficiency of these peptides in MALDI-TOF MS and hence incorporated arginine residues into the peptide sequences to improve ionisation. The optimal assay conditions were established with these new basic assay peptides on the MALDI-TOF MS platform and validated with known ERAP1 inhibitors. Assay stability, reproducibility and robustness was demonstrated on the MALDI-TOF MS platform. From a set of 699 confirmed ERAP1 binders, identified in a prior affinity selection mass spectrometry (ASMS) screen, active compounds were determined at single concentration and in a dose-response format with the new MALDI-TOF MS setup. Furthermore, to allow for platform performance comparison, the same compound set was tested on the established RF MS setup, as the new basic peptides showed fragmentation in ESI-MS. The two platforms showed a comparable performance, but the MALDI-TOF MS platform had several advantages, such as shorter sample cycle times, reduced reagent consumption, and a lower tight-binding limit.

Correction: Mugume et al. Complex Changes in Membrane Lipids Associated with the Modification of Autophagy in Arabidopsis. Metabolites 2022, 12, 190.

In the original article [...].

A Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Assay Identifies Nilotinib as an Inhibitor of Inflammation in Acute Myeloid Leukemia.

Inflammatory responses are important in cancer, particularly in the context of monocyte-rich aggressive myeloid neoplasm. We developed a label-free cellular phenotypic drug discovery assay to identify anti-inflammatory drugs in human monocytes derived from acute myeloid leukemia (AML), by tracking several features ionizing from only 2500 cells using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. A proof-of-concept screen showed that the BCR-ABL inhibitor nilotinib, but not the structurally similar imatinib, blocks inflammatory responses. In order to identify the cellular (off-)targets of nilotinib, we performed thermal proteome profiling (TPP). Unlike imatinib, nilotinib and other later-generation BCR-ABL inhibitors bind to p38α and inhibit the p38α-MK2/3 signaling axis, which suppressed pro-inflammatory cytokine expression, cell adhesion, and innate immunity markers in activated monocytes derived from AML. Thus, our study provides a tool for the discovery of new anti-inflammatory drugs, which could contribute to the treatment of inflammation in myeloid neoplasms and other diseases.

Complex Changes in Membrane Lipids Associated with the Modification of Autophagy in Arabidopsis.

Autophagy is a conserved mechanism among eukaryotes that degrades and recycles cytoplasmic components. Autophagy is known to influence the plant metabolome, including lipid content; however, its impact on the plant lipidome is not fully understood, and most studies have analyzed a single or few mutants defective in autophagy. To gain more insight into the effect of autophagy on lipid concentrations and composition, we quantitatively profiled glycerolipids from multiple Arabidopsis thaliana mutants altered in autophagy and compared them with wild-type seedlings under nitrogen replete (+N; normal growth) and nitrogen starvation (-N; autophagy inducing) conditions. Mutants include those in genes of the core autophagy pathway, together with other genes that have been reported to affect autophagy. Using Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry (MALDI-MS), we imaged the cellular distribution of specific lipids in situ and demonstrated that autophagy and nitrogen treatment did not affect their spatial distribution within Arabidopsis seedling leaves. We observed changes, both increases and decreases, in the relative amounts of different lipid species in the mutants compared to WT both in +N and -N conditions, although more changes were seen in -N conditions. The relative amounts of polyunsaturated and very long chain lipids were significantly reduced in autophagy-disrupted mutants compared to WT plants. Collectively, our results provide additional evidence that autophagy affects plant lipid content and that autophagy likely affects lipid properties such as chain length and unsaturation.

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.

Delayed induction of type I and III interferons mediates nasal epithelial cell permissiveness to SARS-CoV-2.

The nasal epithelium is a plausible entry point for SARS-CoV-2, a site of pathogenesis and transmission, and may initiate the host response to SARS-CoV-2. Antiviral interferon (IFN) responses are critical to outcome of SARS-CoV-2. Yet little is known about the interaction between SARS-CoV-2 and innate immunity in this tissue. Here we apply single-cell RNA sequencing and proteomics to a primary cell model of human nasal epithelium differentiated at air-liquid interface. SARS-CoV-2 demonstrates widespread tropism for nasal epithelial cell types. The host response is dominated by type I and III IFNs and interferon-stimulated gene products. This response is notably delayed in onset relative to viral gene expression and compared to other respiratory viruses. Nevertheless, once established, the paracrine IFN response begins to impact on SARS-CoV-2 replication. When provided prior to infection, recombinant IFNß or IFNλ1 induces an efficient antiviral state that potently restricts SARS-CoV-2 viral replication, preserving epithelial barrier integrity. These data imply that the IFN-I/III response to SARS-CoV-2 initiates in the nasal airway and suggest nasal delivery of recombinant IFNs to be a potential chemoprophylactic strategy.

Single-Cell Metabolomics by Mass Spectrometry Imaging.

Multicellular organisms achieve their complex living activities through the highly organized metabolic interplay of individual cells and tissues. This complexity has driven the need to spatially resolve metabolomics down to the cellular and subcellular level. Recent technological advances have enabled mass spectrometry imaging (MSI), especially matrix-assisted laser desorption/ionization (MALDI), to become a powerful tool for the visualization of molecular species down to subcellular spatial resolution. In the present chapter, we summarize recent advances in the field of MALDI-MSI, with respect to single-cell level resolution metabolomics directly on tissue. In more detail, we focus on advancements in instrumentation for MSI at single-cell resolution, and the applications towards metabolomic scale imaging. Finally, we discuss new computational tools to aid in metabolite identification, future perspective, and the overall direction that the field of single-cell metabolomics directly on tissue may take in the years to come.

On-tissue boronic acid derivatization for the analysis of vicinal diol metabolites in maize with MALDI-MS imaging.

Derivatization reactions are commonly used in mass spectrometry to improve analyte signals, specifically by enhancing the ionization efficiency of those compounds. Vicinal diols are one group of biologically important compounds that have been commonly derivatized using boronic acid. In this study, a boronic acid with a tertiary amine was adapted for the derivatization of vicinal diol metabolites in B73 maize tissue cross-sections for mass spectrometry imaging analysis. Using this method, dozens of vicinal diol metabolites were derivatized, effectively improving the signal of those metabolites. Many of these metabolites were tentatively assigned using high-resolution accurate mass measurements. In addition, reaction interference and cross-reactivity with various other functional groups were systematically studied to verify data interpretation.

Corrigendum: Towards Mass Spectrometry Imaging in the Metabolomics Scale: Increasing Metabolic Coverage Through Multiple On-Tissue Chemical Modifications.

[This corrects the article DOI: 10.3389/fpls.2019.00860.].

Toward Mass Spectrometry Imaging in the Metabolomics Scale: Increasing Metabolic Coverage Through Multiple On-Tissue Chemical Modifications.

Exploring the metabolic differences directly on tissues is essential for the comprehensive understanding of how multicellular organisms function. Mass spectrometry imaging (MSI) is an attractive technique toward this goal; however, MSI in metabolomics scale has been hindered by multiple limitations. This is most notable for single cell level high-spatial resolution imaging because of the limited number of molecules in small sampling size and the low ionization yields of many metabolites. Several on-tissue chemical derivatization approaches have been reported to increase MSI signals of targeted compounds, especially in matrix-assisted laser desorption/ionization (MALDI)-MSI. Herein, we adopt a combination of chemical derivatization reactions, to selectively enhance metabolite signals of a specific functional group for each consecutive tissue section. Three well-known on-tissue derivatization methods were used as a proof of concept experiment: coniferyl aldehyde for primary amines, Girard's reagent T for carbonyl groups, and 2-picolylamine for carboxylic acids. This strategy was applied to the cross-sections of leaves and roots from two different maize genotypes (B73 and Mo17), and enabled the detection of over six hundred new unique metabolite features compared to without modification. Statistical analysis indicated quantitative variation between metabolites in the tissue sections, while MS images revealed differences in localization of these metabolites. Combined, this untargeted approach facilitated the visualization of various classes of compounds, demonstrating the potential for untargeted MSI in the metabolomics scale.

Sputter-Coated Metal Screening for Small Molecule Analysis and High-Spatial Resolution Imaging in Laser Desorption Ionization Mass Spectrometry.

Nanoparticles are efficient matrices in laser desorption/ionization (LDI) mass spectrometry (MS), especially for the profiling or imaging of small molecules. Recently, solvent-free physical vapor desorption (PVD), or sputter coating, was adopted as a homogenous method to rapidly apply metal nanoparticles (NPs) in situ to samples prior to LDI MS or MS imaging analysis. However, there has been no systematic study comparing different metal targets for the analysis of a variety of small molecule metabolites. Here, we present a screening and optimization of various sputter-coated metals, including Ag, Au, Cu, Pt, Ni, and Ti, for LDI analysis of small molecules in both positive and negative ion modes. Optimized sputter coating is then applied to high-spatial resolution LDI mass spectrometry imaging (MSI) of maize root and seed cross-sections. Noble metals, Ag, Au, and Pt, are found to be much more efficient than transition metals and organic matrices for most small metabolites. Sputter-coated metals are efficient for neutral lipids, such as triacylglycerols and diacylglycerols, but are very inefficient for most phospholipids. Graphical Abstract ᅟ.

Nanoparticle microarray for high-throughput microbiome metabolomics using matrix-assisted laser desorption ionization mass spectrometry.

A high-throughput matrix-assisted laser desorption/ionization mass spectrometry (MALDI)-MS-based metabolomics platform was developed using a pre-fabricated microarray of nanoparticles and organic matrices. Selected organic matrices, inorganic nanoparticle (NP) suspensions, and sputter coated metal NPs, as well as various additives, were tested for metabolomics analysis of the turkey gut microbiome. Four NPs and one organic matrix were selected as the optimal matrix set: α-cyano-4-hydroycinnamic acid, Fe3O4 and Au NPs in positive ion mode with 10 mM sodium acetate, and Cu and Ag NPs in negative ion mode with no additive. Using this set of five matrices, over two thousand unique metabolite features were reproducibly detected across intestinal samples from turkeys fed a diet amended with therapeutic or sub-therapeutic antibiotics (200 g/ton or 50 g/ton bacitracin methylene disalicylate (BMD), respectively), or non-amended feed. Among the thousands of unique features, 56 of them were chemically identified using MALDI-MS/MS, with the help of in-parallel liquid chromatography (LC)-MS/MS analysis. Lastly, as a proof of concept application, this protocol was applied to 52 turkey cecal samples at three different time points from the antibiotic feed trial. Statistical analysis indicated variations in the metabolome of turkeys with different ages or treatments. Graphical abstract ᅟ.

Characterizing virus-induced gene silencing at the cellular level with in situ multimodal imaging.

BACKGROUND: Reverse genetic strategies, such as virus-induced gene silencing, are powerful techniques to study gene function. Currently, there are few tools to study the spatial dependence of the consequences of gene silencing at the cellular level. RESULTS: We report the use of multimodal Raman and mass spectrometry imaging to study the cellular-level biochemical changes that occur from silencing the phytoene desaturase (pds) gene using a Foxtail mosaic virus (FoMV) vector in maize leaves. The multimodal imaging method allows the localized carotenoid distribution to be measured and reveals differences lost in the spatial average when analyzing a carotenoid extraction of the whole leaf. The nature of the Raman and mass spectrometry signals are complementary: silencing pds reduces the downstream carotenoid Raman signal and increases the phytoene mass spectrometry signal. CONCLUSIONS: Both Raman and mass spectrometry imaging show that the biochemical changes from FoMV-pds silencing occur with a mosaic spatial pattern at the cellular level, and the Raman images show carotenoid expression was reduced at discrete locations but not eliminated. The data indicate the multimodal imaging method has great utility to study the biochemical changes that result from gene silencing at the cellular spatial level of expression in many plant tissues including the stem and leaf. Our demonstrated method is the first to spatially characterize the biochemical changes as a result of VIGS at the cellular level using commonly available instrumentation.

Cellular and Subcellular Level Localization of Maize Lipids and Metabolites Using High-Spatial Resolution MALDI Mass Spectrometry Imaging.

Recent technological advances have pushed the achievable spatial resolution for mass spectrometry imaging (MSI) to cellular and subcellular levels. Direct visualization of maize tissues by this tool has provided key insights into the localization of metabolites and lipids. This chapter outlines methodology for sample preparation, data acquisition, and data analysis of maize tissue sections using high-spatial resolution matrix-assisted laser desorption ionization (MALDI)-MSI, as well as the incorporation of a multi-resolution optical system, which allows for simple inter-conversion between different resolution setups (5, 10, and 50 µm imaging).