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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 resulting from the presence of isobaric molecules or limited resolving power of the instrument, have encouraged the MSI scientific community to improve MALDI-MSI sample preparation workflows with innovations in chemistry. Recent developments of novel small organic MALDI matrices play a part in the improvement of image quality and the expansion of the application areas of MALDI-MSI. This includes rationally designed/synthesized as well as commercially available small organic molecules whose superior matrix properties in comparison with common matrices have only recently been discovered. Furthermore, on-tissue chemical derivatization (OTCD) processes get more focused attention, because of their advantages for localization of poorly ionizable metabolites and their‚ in several cases‚ more specific imaging of metabolites in tissue sections. This review will provide an overview about the latest developments of novel small organic matrices and on-tissue chemical derivatization reagents for MALDI-MSI.

Pathological microglia activation can promote neuroinflammation in many neurodegenerative diseases, and it has therefore emerged as a potential therapeutic target. Increasing evidence suggests alterations in lipid metabolism as modulators and indicators in microglia activation and its effector functions. Yet, how lipid dynamics in activated microglia is affected by inflammatory stimuli demands additional investigation to allow development of more effective therapies. Here, we report an extensive matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) whole cell fingerprinting workflow to investigate inflammation-associated lipid patterns in SIM-A9 microglial cells. By combining a platform of three synergistic MALDI MS technologies we could detect substantial differences in lipid profiles of lipopolysaccharide (LPS)- stimulated and unstimulated microglia-like cells leading to the identification of 21 potential inflammation-associated lipid markers. LPS-induced lipids in SIM-A9 microglial cells include phosphatidylcholines, lysophosphatidylcholines (LysoPC), sphingolipids, diacylglycerols and triacylglycerols. Moreover, MALDI MS-based cell lipid fingerprinting of LPS-stimulated SIM-A9 microglial cells pre-treated with the non-selective histone deacetylase inhibitor suberoylanilide hydroxamic acid revealed specific modulation of LPS-induced-glycerolipids and LysoPC(18:0) with a significant reduction of microglial inflammation response. Our study introduces MALDI MS as a complementary technology for fast and label-free investigation of stimulus-dependent changes in lipid patterns and their modulation by pharmaceutical agents.

Cell-based assays for compound screening and profiling are fundamentally important in life sciences, chemical biology and pharmaceutical research. Most cell assays measure the amount of a single reporter molecule or cellular endpoint, and require the use of fluorescence or other labeled materials. Consequently, there is high demand for label-free technologies that enable multiple biomolecules or endpoints to be measured simultaneously. Here, we describe how to develop, optimize and validate MALDI-TOF mass spectrometry (MS) cell assays that can be used to measure cellular uptake of transporter substrates, to monitor cellular drug target engagement or to discover cellular drug-response markers. In uptake assays, intracellular accumulation of a transporter substrate and its inhibition by test compounds is measured. In drug response assays, changes to multiple cellular metabolites or to abundant posttranslational protein modifications are monitored as reporters of drug activity. We detail a ten-part optimization protocol with every part taking 1–2 d that leads to a final 2 d optimized procedure, which includes cell treatment, transfer, MALDI MS-specific sample preparation, quantification using stable-isotope-labeled standards, MALDI-TOF MS data acquisition, data processing and analysis. Key considerations for validation and automation of MALDI-TOF MS cell assays are outlined. Overall, label-free MS cell-based assays offer speed, sensitivity, accuracy and versatility in drug research. MALDI-TOF mass spectrometry (MS) can detect multiple compounds simultaneously. This protocol describes how to develop and optimize high-throughput, cell-based assays that use MALDI-TOF MS to detect drug uptake or biochemical markers of drug activity.

Accurate and detailed tissue characterization is a central goal in medical diagnostics, often requiring the combination of multiple imaging modalities. This study presents a multimodal imaging system that integrates mid-infrared (MIR) scanning with fluorescence imaging to enhance the chemical specificity and spatial resolution in biological samples. A motorized mirror allows rapid switching between MIR and fluorescence modes, enabling efficient, co-registered data acquisition. The MIR modality captures label-free chemical maps based on molecular vibrations, while the fluorescence channel records endogenous autofluorescence for additional biochemical contrast. Applied to mouse brain tissue, the system enabled the clear differentiation of gray matter and white matter, supported by the clustering analysis of spectral features. The addition of autofluorescence imaging further improved anatomical segmentation and revealed fine structural details. In mouse skin, the approach allowed the precise mapping of the layered tissue architecture. These results demonstrate that combining MIR scanning and fluorescence imaging provides complementary, label-free insights into tissue morphology and chemistry. The findings support the utility of this approach as a powerful tool for biomedical research and diagnostic applications, offering a more comprehensive understanding of tissue composition without relying on staining or external markers.

Background Glioblastoma is the most frequent and a particularly malignant primary brain tumor with no efficacy-proven standard therapy for recurrence. It has recently been discovered that excitatory synapses of the AMPA-receptor subtype form between non-malignant brain neurons and tumor cells. This neuron-tumor network connectivity contributed to glioma progression and could be efficiently targeted with the EMA/FDA approved antiepileptic AMPA receptor inhibitor perampanel in preclinical studies. The PerSurge trial was designed to test the clinical potential of perampanel to reduce tumor cell network connectivity and tumor growth with an extended window-of-opportunity concept. Methods PerSurge is a phase IIa clinical and translational treatment study around surgical resection of progressive or recurrent glioblastoma. In this multicenter, 2-arm parallel-group, double-blind superiority trial, patients are 1:1 randomized to either receive placebo or perampanel ( n  = 66 in total). It consists of a treatment and observation period of 60 days per patient, starting 30 days before a planned surgical resection, which itself is not part of the study interventions. Only patients with an expected safe waiting interval are included, and a safety MRI is performed. Tumor cell network connectivity from resected tumor tissue on single cell transcriptome level as well as AI-based assessment of tumor growth dynamics in T2/FLAIR MRI scans before resection will be analyzed as the co-primary endpoints. Secondary endpoints will include further imaging parameters such as pre- and postsurgical contrast enhanced MRI scans, postsurgical T2/FLAIR MRI scans, quality of life, cognitive testing, overall and progression-free survival as well as frequency of epileptic seizures. Further translational research will focus on additional biological aspects of neuron-tumor connectivity. Discussion This trial is set up to assess first indications of clinical efficacy and tolerability of perampanel in recurrent glioblastoma, a repurposed drug which inhibits neuron-glioma synapses and thereby glioblastoma growth in preclinical models. If perampanel proved to be successful in the clinical setting, it would provide the first evidence that interference with neuron-cancer interactions may indeed lead to a benefit for patients, which would lay the foundation for a larger confirmatory trial in the future. Trial registration EU-CT number: 2023-503938-52-00 30.11.2023.

In spatial’omics, highly confident molecular identifications are indispensable for the investigation of complex biology and for spatial biomarker discovery. However, current mass spectrometry imaging (MSI)-based spatial ‘omics must compromise between data acquisition speed and biochemical profiling depth. Here, we introduce fast, label-free quantum cascade laser mid-infrared imaging microscopy (QCL-MIR imaging) to guide MSI to high-interest tissue regions as small as kidney glomeruli, cultured multicellular spheroid cores or single motor neurons. Focusing on smaller tissue areas enables extensive spatial lipid identifications by on-tissue tandem-MS employing imaging parallel reaction monitoring-Parallel Accumulation-Serial Fragmentation (iprm-PASEF). QCL-MIR imaging-guided MSI allowed for unequivocal on-tissue elucidation of 157 sulfatides selectively accumulating in kidneys of arylsulfatase A-deficient mice used as ground truth concept and provided chemical rationales for improvements to ion mobility prediction algorithms. Using this workflow, we characterized sclerotic spinal cord lesions in mice with experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, and identified upregulation of inflammation-related ceramide-1-phosphate and ceramide phosphatidylethanolamine as markers of white matter lipid remodeling. Taken together, widely applicable and fast QCL-MIR imaging-based guidance of MSI ensures that more time is available for exploration and validation of new biology by default on-tissue tandem-MS analysis. Confident molecular identification is key for studying complex biochemistry. Here, the authors employ Quantum-Cascade Laser-based Mid-infrared imaging for rapid identification of ROIs, followed by MALDI imaging prm-PASEF for in-depth lipid identifications directly on complex tissues.

Bantscheff et al . use chemoproteomics to measure the affinity of small molecules for megadalton protein complexes in cell extracts. Differences in the selectivity of HDAC inhibitors observed when native HDAC complexes are compared with their purified catalytic subunits suggest the limitations of using isolated recombinant proteins in certain drug screens. The development of selective histone deacetylase (HDAC) inhibitors with anti-cancer and anti-inflammatory properties remains challenging in large part owing to the difficulty of probing the interaction of small molecules with megadalton protein complexes. A combination of affinity capture and quantitative mass spectrometry revealed the selectivity with which 16 HDAC inhibitors target multiple HDAC complexes scaffolded by ELM-SANT domain subunits, including a novel mitotic deacetylase complex (MiDAC). Inhibitors clustered according to their target profiles with stronger binding of aminobenzamides to the HDAC NCoR complex than to the HDAC Sin3 complex. We identified several non-HDAC targets for hydroxamate inhibitors. HDAC inhibitors with distinct profiles have correspondingly different effects on downstream targets. We also identified the anti-inflammatory drug bufexamac as a class IIb (HDAC6, HDAC10) HDAC inhibitor. Our approach enables the discovery of novel targets and inhibitors and suggests that the selectivity of HDAC inhibitors should be evaluated in the context of HDAC complexes and not purified catalytic subunits.

The dynamics and phenotypes of intratumoral myeloid cells during tumor progression are poorly understood. Here we define myeloid cellular states in gliomas by longitudinal single-cell profiling and demonstrate their strict control by the tumor genotype: in isocitrate dehydrogenase (IDH)-mutant tumors, differentiation of infiltrating myeloid cells is blocked, resulting in an immature phenotype. In late-stage gliomas, monocyte-derived macrophages drive tolerogenic alignment of the microenvironment, thus preventing T cell response. We define the IDH-dependent tumor education of infiltrating macrophages to be causally related to a complex re-orchestration of tryptophan metabolism, resulting in activation of the aryl hydrocarbon receptor. We further show that the altered metabolism of IDH-mutant gliomas maintains this axis in bystander cells and that pharmacological inhibition of tryptophan metabolism can reverse immunosuppression. In conclusion, we provide evidence of a glioma genotype-dependent intratumoral network of resident and recruited myeloid cells and identify tryptophan metabolism as a target for immunotherapy of IDH-mutant tumors.

Metabolic reprogramming within the tumor microenvironment (TME) plays a central role in cancer progression and immune evasion, with L-arginine metabolism emerging as a key regulatory axis. Arginase overexpression depletes intratumoral L-arginine, thus suppressing T-cell proliferation while fuelling tumor growth through polyamine biosynthesis. OATD-02, a novel dual arginase (ARG1/ARG2) inhibitor, reprograms tumor metabolism by restoring L-arginine availability and reducing the levels of polyamines, thereby shifting the TME toward a more immunostimulatory state. Unlike ARG1-selective inhibitors with limited intracellular uptake, OATD-02 effectively inhibits both extracellular and intracellular arginases, thereby addressing a major limitation of first-generation arginase inhibitors. To visualize the pharmacodynamic effects of OATD-02 dosing in mice with spatial resolution, we employed MALDI mass spectrometry imaging (MALDI-MSI), thus enabling direct mapping of metabolic changes within tumor tissues. In preclinical models, OATD-02 treatment led to widespread accumulation of intratumoral L-arginine with concomitant depletion of polyamines and resulted in metabolic shifts that correlated with increased immune cell infiltration and an improved response to immune checkpoint blockade. These findings underscore the role of dual arginase inhibition in reshaping tumor metabolism and overcoming immune suppression by restoring the metabolic fitness of immune cells to fight cancer. The metabolic changes caused by OATD-02 treatment resulted in significantly enhanced antitumor immune responses, increased T-cell infiltration in tumors, expansion of CD8⁺ T cells in draining lymph nodes, and systemic upregulation of T-cell activation markers. These effects translated into a substantial survival benefit in the CT26 tumor model, particularly when combined with anti-PD-1 therapy, where OATD-02 improved checkpoint blockade efficacy by relieving metabolic constraints affecting tumor-infiltrating lymphocytes. By leveraging the unique capabilities of MALDI-MSI, this study provides high-resolution metabolic insights into the mechanism of action of OATD-02, reinforcing its potential as a next-generation metabolic-immunotherapeutic agent. The observed metabolic reprogramming, coupled with enhanced immune activation and prolonged survival, supports the clinical development of OATD-02 as a promising strategy for enhancing cancer immunotherapy efficacy. OATD-02 is currently undergoing clinical evaluation in a phase I/II trial (NCT05759923), which will further elucidate its safety and therapeutic impact. These findings highlight the potential of arginase-targeted therapies in cancer treatment and underscore the value of MALDI-MSI as a powerful tool for tracking metabolic responses to therapy.

Potency assessment of monoclonal antibodies or corresponding biosimilars in cell-based assays is an essential prerequisite in biopharmaceutical research and development. However, cellular bioassays are still subject to limitations in sample throughput, speed, and often need costly reagents or labels as they are based on an indirect readout by luminescence or fluorescence. In contrast, whole-cell Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry (MS) has emerged as a direct, fast and label-free technology for functional drug screening being able to unravel the molecular complexity of cellular response to pharmaceutical reagents. However, this approach has not yet been used for cellular testing of biologicals. In this study, we have conceived, developed and benchmarked a label-free MALDI-MS based cell bioassay workflow for the functional assessment of complement-dependent cytotoxicity (CDC) of Rituximab antibody. By computational evaluation of response profiles followed by subsequent m/z feature annotation via fragmentation analysis and trapped ion mobility MS, we identified adenosine triphosphate and glutathione as readily MS-assessable metabolite markers for CDC and demonstrate that robust concentration–response characteristics can be obtained by MALDI-TOF MS. Statistical assay performance indicators suggest that whole-cell MALDI-TOF MS could complement the toolbox for functional cellular testing of biopharmaceuticals.