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Given the complexity of nervous tissues, understanding neurochemical pathophysiology puts high demands on bioanalytical techniques with respect to specificity and sensitivity. Mass spectrometry imaging (MSI) has evolved to become an important, biochemical imaging technology for spatial biology in biological and translational research. The technique facilitates comprehensive, sensitive elucidation of the spatial distribution patterns of drugs, lipids, peptides, and small proteins in situ. Matrix-assisted laser desorption ionization (MALDI)-based MSI is the dominating modality due to its broad applicability and fair compromise of selectivity, sensitivity price, throughput, and ease of use. This is particularly relevant for the analysis of spatial lipid patterns, where no other comparable spatial profiling tools are available. Understanding spatial lipid biology in nervous tissue is therefore a key and emerging application area of MSI research. The aim of this review is to give a concise guide through the MSI workflow for lipid imaging in central nervous system (CNS) tissues and essential parameters to consider while developing and optimizing MSI assays. Further, this review provides a broad overview of key developments and applications of MALDI MSI-based spatial neurolipidomics to map lipid dynamics in neuronal structures, ultimately contributing to a better understanding of neurodegenerative disease pathology.

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Mass spectrometry imaging (MSI) has emerged as an invaluable analytical technique for investigating the spatial distribution of molecules within biological systems. In the realm of plant science, MSI is increasingly employed to explore metabolic processes across a wide array of plant tissues, including those in leaves, fruits, stems, roots, and seeds, spanning various plant systems such as model species, staple and energy crops, and medicinal plants. By generating spatial maps of metabolites, MSI has elucidated the distribution patterns of diverse metabolites and phytochemicals, encompassing lipids, carbohydrates, amino acids, organic acids, phenolics, terpenes, alkaloids, vitamins, pigments, and others, thereby providing insights into their metabolic pathways and functional roles. In this review, we present recent MSI studies that demonstrate the advances made in visualizing the plant spatial metabolome. Moreover, we emphasize the technical progress that enhances the identification and interpretation of spatial metabolite maps. Within a mere decade since the inception of plant MSI studies, this robust technology is poised to continue as a vital tool for tackling complex challenges in plant metabolism.

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Detecting bovine tuberculosis (bTB) primarily relies on the tuberculin skin test, requiring two separate animal handling events with a period of incubation time (normally 3 days) between them. Here, we present the use of liquid atmospheric pressure (LAP)-MALDI for the identification of bTB infection, employing a three-class prediction model that was obtained by supervised linear discriminant analysis (LDA) and tested with bovine mastitis samples as disease-positive controls. Noninvasive collection of nasal swabs was used to collect samples, which were subsequently subjected to a short (<4 h) sample preparation method. Cross-validation of the three-class LDA model from the processed nasal swabs provided a sensitivity of 75.0% and specificity of 90.1%, with an overall classification accuracy of 85.7%. These values are comparable to those for the skin test, showing that LAP-MALDI MS has the potential to provide an alternative single-visit diagnostic platform that can detect bTB within the same day of sampling.

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Lipids and metabolites are small biological molecules that act major roles in cellular functions. Multicellular tumor spheroids (MCTS) are a highly beneficial three-dimensional cellular model for cancer research due to their ability to imitate numerous characteristics of tumor tissues. Increasing studies have performed spatial lipidomics and metabolomics in MCTS using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). However, these approaches often lack the sensitivity and specificity to offer a comprehensive characterization of lipids and metabolites within MCTS. In this study, we addressed this challenge by utilizing MALDI combined with laser-induced postionization (MALDI-2) and trapped ion mobility spectrometry (TIMS) imaging in H295R adrenocortical MCTS. Our results showed that MALDI-2 could detect more lipids and metabolites in MCTS than the traditional MALDI. TIMS data revealed a successful separation of many isomeric and isobaric ions of lipids and metabolites with different locations (e.g., proliferative region and necrotic region) within MCTS, suggesting an enhanced peak capacity for spatial lipidomics and metabolomics. To further identify these isomeric and isobaric ions, we performed MS/MS imaging experiments to compare the differences in signal intensities and spatial distributions of product ions. Our data highlight the strong potential of MALDI-2 and TIMS imaging for analyzing lipids and metabolites in MCTS, which may serve as valuable tools for numerous fields of biological and medical research.

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Organoids have emerged as a promising advancement of the two-dimensional (2D) culture systems to improve studies in organogenesis, drug discovery, precision medicine, and regenerative medicine applications. Organoids can self-organize as three-dimensional (3D) tissues derived from stem cells and patient tissues to resemble organs. This chapter presents growth strategies, molecular screening methods, and emerging issues of the organoid platforms. Single-cell and spatial analysis resolve organoid heterogeneity to obtain information about the structural and molecular cellular states. Culture media diversity and varying lab-to-lab practices have resulted in organoid-to-organoid variability in morphology and cell compositions. An essential resource is an organoid atlas that can catalog protocols and standardize data analysis for different organoid types. Molecular profiling of individual cells in organoids and data organization of the organoid landscape will impact biomedical applications from basic science to translational use.

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Mass Spectrometry Imaging (MSI) has emerged as a powerful imaging technique for the analysis of biological samples, providing valuable insights into the spatial distribution and structural characterization of lipids. The advancements in high-resolution MSI have made it an indispensable tool for single-cell or subcellular lipidomics. By preserving both intracellular and intercellular information, MSI enables a comprehensive analysis of lipidomics in individual cells and organelles. This enables researchers to delve deeper into the diversity of lipids within cells and to understand the role of lipids in shaping cell behavior. In this review, we aim to provide a comprehensive overview of recent advancements and future prospects of MSI for cellular/subcellular lipidomics. By keeping abreast of the cutting-edge studies in this field, we will continue to push the boundaries of the understanding of lipid metabolism and the impact of lipids on cellular behavior.

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The blood-brain barrier (BBB) is likely to be intact during the early stages of brain metastatic melanoma development, and thereby inhibits sufficient drug delivery into the metastatic lesions. Our laboratory has been developing a system for boron drug delivery to brain cells via cerebrospinal fluid (CSF) as a viable pathway to circumvent the BBB in boron neutron capture therapy (BNCT). BNCT is a cell-selective cancer treatment based on the use of boron-containing drugs and neutron irradiation. Selective tumor targeting by boron with minimal normal tissue toxicity is required for effective BNCT. Boronophenylalanine (BPA) is widely used as a boron drug for BNCT. In our previous study, we demonstrated that application of the CSF administration method results in high BPA accumulation in the brain tumor even with a low dose of BPA. In this study, we evaluate BPA biodistribution in the brain following application of the CSF method in brain-tumor-model rats (melanoma) utilizing matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI). We observed increased BPA penetration to the tumor tissue, where the color contrast on mass images indicates the border of BPA accumulation between tumor and normal cells. Our approach could be useful as drug delivery to different types of brain tumor, including brain metastases of melanoma.

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Lipopolysaccharides (LPSs) are the main components of the external leaflet of the outer membrane of Gram-negative bacteria. They exert multiple functions, starting from conferring stability to the bacterial membrane to mediating the interaction of the microbe with the external environment. The composition and the structure of LPSs present tremendous diversity even within bacteria of the same species, and for this reason, the determination of the structure of these molecules is crucial because it can provide information on the motifs key for the virulence of a pathogen or that are associated to a bacterium of the commensal or beneficial microbiota. In addition, structural data disclose the effects triggered from a mutation or from the use of an antibiotic, or they can be used as tools to check the quality of adjuvants and/or medications, as vaccines, that make use of LPS.The structural study of LPSs is complex, and it can be achieved with the right combination of different techniques. In this frame, this chapter focuses on the two MS-based approaches, the gas chromatography-mass spectrometry (GC-MS) and the matrix-assisted laser desorption/ionization (MALDI).

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Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is a powerful tool in the analysis and imaging of small molecules. However, MALDI MS analysis is easily subjected to poor signal reproducibility and selectivity, especially for complex samples. In this study, a matrix glycosylation strategy was proposed to synthesize glycosylated matrices with excellent performances by enhancing the interaction of the matrix with small molecules. A series of glycosylated matrices including 3-glycosylaminoquinoline (3-GAQ), 6-glycosylaminoquinoline (6-GAQ), and 1-amino-5-glycosylaminoquinoline (GDAN) were synthesized by connecting glucose with the existing amine matrices. Compared with their parent matrices and the existing matrix (1,5-diaminonaphathelene, 1,5-DAN), the glycosylated matrices exhibited remarkably-improved sensitivity, higher signal reproducibility (RSD < 9%) in detecting metabolites, demonstrating the effectiveness of the glycosylation strategy. Among them, 6-GAQ exhibited the best performance. Using 6-GAQ, the detection limit of citric acid reached the low fmol range, and the calibration curve of citric acid had ideal linearity (R2 > 0.99), proving that 6-GAQ was capable of accurate quantitative analysis of metabolites. Furthermore, 6-GAQ was used for the imaging of metabolites in the mouse kidney section, showing higher sensitivity and lower background noise than the commonly-used matrices, 9-aminoquinoline (9-AA), and 1,5-DAN. More importantly, 6-GAQ can selectively detect the hydrophilic metabolites, especially the hydrophilic lipids in the mouse kidney. Overall, 6-GAQ is an ideal matrix potentially applied in the imaging and quantitative analysis of hydrophilic small molecules in complex samples.

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Boron neutron capture therapy (BNCT) is a cell-selective particle therapy for cancer using boron containing drugs. Boron compounds are accumulated in high concentration of tens ppm level of boron in target tumors to cause lethal damage to tumor tissue. The examination of boron distribution in target tumor and normal tissue is important to evaluate the efficiency of therapy. The matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is a powerful tool to visualize the distribution of target analyte in biological samples. In this manuscript, we report a trial to visualize the distribution of a typical BNCT drug, L-4-phenylalanine boronic acid (BPA) in a brain tumor model rat using MALDI-MSI technique. We performed a MALDI-MSI with high mass resolution targeting to [BPA+H]+ at m/z 210 in a BPA-treated rat brain section using a spiral orbit-type time of flight (SpiralTOF) mass spectrometer. Several BPA ion species, [BPA+H]+, [BPA-H2O+Na]+, [BPA+DHB-2H2O+Na]+ and [BPA+DHB-2H2O+K]+ were detected separate from peaks originated from biomolecules or matrix reagent by achieving the mass resolving power of approximately 20,000 (full width at half maximum; FWHM) at m/z 210. The mass images with 60 µm spatial resolution obtained from these BPA ion species in a mass window of 0.02 Da revealed their localization in the tumor region. Additionally, the mass image obtained from [BPA+H]+ also likely showed the distribution of BPA inside the tumor. MALDI-MSI with high mass resolution targeting to [BPA+H]+ has a great potential to visualize the distribution of BPA in brain tissue with tumor.

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Butyl-terminated poly(2-vinylpyridine) (P2VP), C4H9(C7H7N)nH, is evaluated for use as an external and internal mass calibrant in positive-ion matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). P2VP oligomers covering the m/z 450-4500 range are employed to calibrate a time-of-flight (TOF) mass spectrometer in linear and reflector mode, an ion mobility-quadrupole-time-of-flight (IM-Q-TOF) mass spectrometer, and a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. The proton affinity of P2VPs introduced by the numerous pyridyl groups leads to the almost exclusive formation of [M + H]+ ions with common acidic matrices like α-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB) as well as with the non-acidic and aprotic matrices 1,8-dihydroxy-10H-anthracen-9-on (dithranol) and 2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2-enylidene]malonitrile (DCTB). This prevalence of [M + H]+ ions evenly spaced at Δ(m/z) = 105.0578 renders butyl-terminated P2VP oligomers as convenient mass calibrants. The mass accuracies achieved across various m/z ranges with different mass analyzers and modes of operation are evaluated by using established standard compounds. Results as obtained by internal or external calibration are presented. Further, the compilation of mass reference lists tailored to suit the respective analyzer modes is discussed and those reference files are provided.

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The study of biological specimens by mass spectrometry imaging (MSI) has had a profound influence in the various forms of spatial-omics over the past two decades including applications for the identification of clinical biomarker analysis; the metabolic fingerprinting of disease states; treatment with therapeutics; and the profiling of lipids, peptides and proteins. No singular approach is able to globally map all biomolecular classes simultaneously. This led to the development of many complementary multimodal imaging approaches to solve analytical problems: fusing multiple ionization techniques, imaging microscopy or spectroscopy, or local extractions into robust multimodal imaging methods. However, each fusion typically requires the melding of analytical information from multiple commercial platforms, and the tandem utilization of multiple commercial or third-party software platforms-even in some cases requiring computer coding. Herein, we report the use of matrix-assisted laser desorption/ionization (MALDI) in tandem with desorption electrospray ionization (DESI) imaging in the positive ion mode on a singular commercial orthogonal dual-source Fourier transform ion cyclotron resonance (FT-ICR) instrument for the complementary detection of multiple analyte classes by MSI from tissue. The DESI source was 3D printed and the commercial Bruker Daltonics software suite was used to generate mass spectrometry images in tandem with the commercial MALDI source. This approach allows for the generation of multiple modes of mass spectrometry images without the need for third-party software and a customizable platform for ambient ionization imaging. Highlighted is the streamlined workflow needed to obtain phospholipid profiles, as well as increased depth of coverage of both annotated phospholipid, cardiolipin, and ganglioside species from rat brain with both high spatial and mass resolution.

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Compared with conventional optical microscopy techniques, mass spectrometry imaging (MSI) or imaging mass spectrometry (IMS) is a powerful, label-free analytical technique, which can sensitively and simultaneously detect, quantify, and map hundreds of biomolecules, such as peptides, proteins, lipid, and other organic compounds in cells and tissues. So far, although several soft ionization techniques, such as desorption electrospray ionization (DESI) and secondary ion mass spectrometry (SIMS) have been used for imaging biomolecules, matrix-assisted laser desorption/ionization (MALDI) is still the most widespread MSI scanning method. Here, we aim to provide a comprehensive review of MALDI-MSI with an emphasis on its advances of the instrumentation, methods, application, and future directions in single cell and biological tissues.

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The ubiquitin-proteasome system (UPS) is one of the major protein degradation pathways in eukaryotic cells. Abnormal functioning of this system has been observed in cancer and neurological diseases. The 20S proteasomes, essential components of the UPS, are present not only within the cells but also in the extracellular space, and their concentration in blood plasma has been found to be elevated and dependent upon the disease state, being of prognostic significance in patients suffering from cancer, liver diseases, and autoimmune diseases. However, functions of extracellular proteasomes and mechanisms of their release by cells remain largely unknown. The main mechanism of proteasome activity regulation is provided by modulation of their composition and post-translational modifications (PTMs). Moreover, diverse PTMs of proteins are known to participate in the loading of specific elements into extracellular vesicles. Since previous studies have revealed that the transport of extracellular proteasomes may occur via extracellular vesicles, we have set out to explore the PTMs of extracellular proteasomes in comparison to cellular counterparts. In this work, cellular and extracellular proteasomes were affinity purified and separated by SDS-PAGE for subsequent trypsinization and matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) analysis. In total, we could identify 64 and 55 PTM sites in extracellular and cellular proteasomes, respectively, including phosphorylation, ubiquitination, acetylation, and succinylation. We observed novel sites of acetylation at K238 and K192 of the proteasome subunits ß2 and ß3, respectively, that are specific for extracellular proteasomes. Moreover, cellular proteasomes show specific acetylation at K227 of α2 and ubiquitination at K201 of ß3. Interestingly, succinylation of ß6 at the residue K228 seems not to be present exclusively in extracellular proteasomes, whereas both extracellular and cellular proteasomes may also be acetylated at this site. The same situation takes place at K201 of the ß3 subunit where ubiquitination is seemingly specific for cellular proteasomes. Moreover, crosstalk between acetylation, ubiquitination, and succinylation has been observed in the subunit α3 of both proteasome populations. These data will serve as a basis for further studies, aimed at dissection of the roles of extracellular proteasome-specific PTMs in terms of the function of these proteasomes and mechanism of their transport into extracellular space.

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Peptides have become a fast-growing segment of the pharmaceutical industry over the past few decades. It is essential to develop cutting edge analytical techniques to support the discovery and development of peptide therapeutics, especially to examine their absorption, distribution, metabolism and excretion (ADME) properties. Herein, we utilized two label-free mass spectrometry (MS) based techniques to investigate representative challenges in developing therapeutic peptides, such as tissue distribution, metabolic stability and clearance. A tool proof-of-concept cyclic peptide, melanotan II, was used in this study. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), which is a well-developed label-free imaging technique, was used to map the detailed molecular distribution of melanotan II and its metabolites. Droplet-based liquid microjunction surface sampling liquid chromatography-high resolution mass spectrometry (LMJ-SSP-LC-HRMS) was used in combination with MALDI-MSI to rapidly profile molecular information and provide structural insights on drug and metabolites. Using both techniques in parallel allowed a more comprehensive and complementary data set than using either technique independently. We envision MALDI-MSI and droplet-based LMJ-SSP-LC-HRMS, which can be used in combination or as standalone techniques, to become valuable tools for assessing the in vivo fate of peptide therapeutics in support of drug discovery and development.

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Flavan-3-ols, procyanidins and their monomers are major flavonoids present in peanuts that show a wide range of biological properties and health benefits, based on their potent antioxidant activity. Procyanidin oligomers, especially A-type, are reportedly abundant in peanut skin; however, their localization in the raw peanut testa remains poorly understood. Therefore, we performed matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) to investigate the localization of flavan-3-ols in peanut testa. 1,5-Diaminonaphthalene was coated onto the peanut section by matrix vapor deposition/recrystallization, and MALDI-MSI measurements were performed in the negative-ion mode. Peaks matching the m/z values of flavan-3-ol [M - H]- ions were observed in the mass spectrum extracted from the outer epidermis of the peanut testa, using the region of interest function. Catechin and/or epicatechin, five A-type, and one B-type procyanidins were assigned by the fragment ions generated by retro-Diels-Alder, heterocyclic ring fission, and quinone methide reactions detected in MALDI-tandem MS spectra. These flavan-3-ols were localized in the outer epidermis of the peanut testa. This information will contribute to improving the extraction and purification efficiencies of flavan-3-ols from peanut testa. As flavan-3-ols display anti-microbial activity, it is speculated that flavan-3-ols present in the outer epidermis of peanut testa act to prevent pathogen infection.

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Phosphatidylcholine (PC) is the major phospholipid in meat and influences meat qualities, such as healthiness. PC is classified into three groups based on the bond at the sn-1 position: Diacyl, alkylacyl, and alkenylacyl. To investigate their composition and distribution in pork tissues, including longissimus thoracis et lumborum (loin) spinalis muscles, intermuscular fat, and transparent tissues, we performed matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI). Eleven diacyl-, seven alkylacyl-, and six alkenylacyl-PCs were identified using liquid chromatography (LC)-tandem MS (MS/MS) analysis. Despite many alkylacyl- and alkenylacyl-PC species sharing identical m/z values, we were able to visualize these PC species using MALDI-MSI. Diacyl- and alkylacyl- and/or alkenylacyl-PC species showed unique distribution patterns in the tissues, suggesting that their distribution patterns were dependent on their fatty acid compositions. PCs are a major dietary source of choline in meat, and the amount was significantly higher in the muscle tissues. Consumption of choline mitigates age-related memory decline and neurodegenerative diseases; therefore, the consumption of pork muscle tissues could help to mitigate these diseases. These results support the use of MALDI-MSI analysis for assessing the association between PC species and the quality parameters of meat.

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RATIONALE: Two Early Classic period (ca 250-600 CE) Maya carved, greenstone jade pendants, Specimens A and B, were recovered from the Pacbitun site in Belize in 1987. Mass scans of resolution ~600,000 were obtained for the jade pendants and two British Columbia jade ornaments. METHODS: Polyatomic ions from jade grit have been observed using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), specifically the Bruker Daltonics, SolariX XR, combined with matrix-assisted laser desorption/ionization (MALDI); the matrix material was α-cyano-4-hydroxycinnamicacid. RESULTS: Presented here is an interpretation of the mass spectrometric observations from multiplicities of silicate materials. Observation of novel polyatomic ion species of m/z 800-1400 indicate the ejection of charged ensembles composed of 60-180 atoms from jade grit. The charged ensembles represent minute regions of jade grit that have retained many of the spatial relationships of atoms within the jade; the required ejection energy is provided by laser photons accumulated in the matrix. Acid solutions of grit from Specimens A and ornament KMM were analyzed by inductively-coupled plasma mass spectrometry. The novelty of this application of mass spectrometry to jade was reported in the Journal of Archaeological Science: Reports. CONCLUSIONS: Isotopic fine structure analysis can yield elemental compositions for ions composed of base isotopes together with stable isotope intensities arising from ions with one and two additional neutrons. The analysis is challenging as jade is a multiplicity of similar solid solutions; thus, a multiple isotopic fine structure approach has been explored. Attempts to compose ensembles of atoms, of those elements found normally in coloured silicates, to match the observed ion mass/charge ratios were unsuccessful without the addition of multiple hydrogen atoms. The production of polyatomic ions of siliceous material may permit impinging fine glass coatings onto construction materials for protection against fire and weather.

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Background In recent years, mass spectrometry (MS) has been applied to clinical microbial biotyping, exploiting the speed of matrix-assisted laser desorption/ionization (MALDI) in recording microbe-specific MS profiles. More recently, liquid atmospheric pressure (AP) MALDI has been shown to produce extremely stable ion flux from homogenous samples and 'electrospray ionization (ESI)-like' multiply charged ions for larger biomolecules, whilst maintaining the benefits of traditional MALDI including high tolerance to contaminants, low analyte consumption and rapid analysis. These and other advantages of liquid AP-MALDI MS have been explored in this study to investigate its potential in microbial biotyping. Methods Genetically diverse bacterial strains were analyzed using liquid AP-MALDI MS, including clinically relevant species such as Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae. Bacterial cultures were subjected to a simple and fast extraction protocol using ethanol and formic acid. Extracts were spotted with a liquid support matrix (LSM) and analyzed using a Synapt G2-Si mass spectrometer with an in-house built AP-MALDI source. Results Each species produces a unique lipid profile in the m/z range of 400-1100, allowing species discrimination. Traditional (solid) MALDI MS produced spectra containing a high abundance of matrix-related clusters and an absence of lipid peaks. The MS profiles of the bacterial species tested form distinct clusters using principle component analysis (PCA) with a classification accuracy of 98.63% using a PCA-based prediction model. Conclusions Liquid AP-MALDI MS profiles can be sufficient to distinguish clinically relevant bacterial pathogens and other bacteria, based on their unique lipid profiles. The analysis of the lipid MS profiles is typically excluded from commercial instruments approved for clinical diagnostics.

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Cigarette smoke is a well-established exogenous risk factor containing toxic reactive molecules able to induce oxidative stress, which in turn contributes to smoking-related diseases, including cardiovascular, pulmonary, and oral cavity diseases. We investigated the effects of cigarette smoke extract on human bronchial epithelial cells. Cells were exposed to various concentrations (2.5-5-10-20%) of cigarette smoke extract for 1, 3, and 24 h. Carbonylation was assessed by 2,4-dinitrophenylhydrazine using both immunocytochemical and Western immunoblotting assays. Cigarette smoke induced increasing protein carbonylation in a concentration-dependent manner. The main carbonylated proteins were identified by means of two-dimensional electrophoresis coupled to MALDI-TOF mass spectrometry analysis and database search (redox proteomics). We demonstrated that exposure of bronchial cells to cigarette smoke extract induces carbonylation of a large number of proteins distributed throughout the cell. Proteins undergoing carbonylation are involved in primary metabolic processes, such as protein and lipid metabolism and metabolite and energy production as well as in fundamental cellular processes, such as cell cycle and chromosome segregation, thus confirming that reactive carbonyl species contained in cigarette smoke markedly alter cell homeostasis and functions.

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