LM-GlycomeAtlas Ver. 1.0: A Novel Visualization Tool for Lectin Microarray-Based Glycomic Profiles of Mouse Tissue Sections.

For the effective discovery of the biological roles and disease-specific alterations concerning protein glycosylation in tissue samples, it is important to know beforehand the quantitative and qualitative variations of glycan structures expressed in various types of cells, sites, and tissues. To this end, we used laser microdissection-assisted lectin microarray (LMA) to establish a simple and reproducible method for high-throughput and in-depth glycomic profiling of formalin-fixed paraffin-embedded tissue sections. Using this "tissue glycome mapping" approach, we present 234 glycomic profiling data obtained from nine tissue sections (pancreas, heart, lung, thymus, gallbladder, stomach, small intestine, colon, and skin) of two 8-week-old male C57BL/6J mice. We provided this LMA-based dataset in the similar interface as that of GlycomeAtlas, a previously developed tool for mass spectrometry-based tissue glycomic profiling, allowing easy comparison of the two types of data. This online tool, called "LM-GlycomeAtlas", allows users to visualize the LMA-based tissue glycomic profiling data associated with the sample information as an atlas. Since the present dataset allows the comparison of glycomic profiles, it will facilitate the evaluation of site- and tissue-specific glycosylation patterns. Taking advantage of its extensibility, this tool will continue to be updated with the expansion of deposited data.

Molecular glycopathology by capillary electrophoresis: Analysis of the N-glycome of formalin-fixed paraffin-embedded mouse tissue samples.

Capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection was used to analyze endoglycosidase released and fluorophore-labeled N-glycans from formalin-fixed paraffin-embedded (FFPE) mouse tissue samples of lung, brain, heart, spleen, liver, kidney and intestine. The FFPE samples were first deparaffinized followed by solubilization and glycoprotein retrieval. PNGase F mediated release of the N-linked oligosaccharides was followed by labeling with aminopyrene trisulfonate. After CE-LIF glycoprofiling of the FFPE mouse tissues, the N-glycan pool of the lung specimen was subject to further investigation by exoglycosidase array based carbohydrate sequencing. Structural assignment of the oligosaccharides was accomplished by the help of the GUcal software and the associated database, based on the mobility shifts after treatments with the corresponding exoglycosidase reaction mixtures. Sixteen major N-linked carbohydrate structures were sequenced from the mouse lung FFPE tissue glycome and identified, as high mannose (3) neutral biantennary (3) sialylated monoantennary (1) and sialylated bianennary (9) oligosaccharides. Two of these latter ones also possessed alpha(1-3) linked galactose residues.

An application of RP-HPLC for determination of the activity of cystathionine ß-synthase and γ-cystathionase in tissue homogenates.

The RP-HPLC-based method of determination of the activity of cystathionine ß-synthase and γ-cystathionase was undertaken in mouse liver, kidney and brain. Products of the reactions, such as cystathionine, α-ketobutyrate, cysteine and glutathione, were measured using the RP-HPLC method. A difference in the cystathionine level between homogenates with totally CTH-inhibiting concentrations of DL-propargylglycine and without the inhibitor was employed to evaluate the activity of cystathionine ß-synthase. Gamma-cystathionase activity was measured using DL-homoserine as a substrate and a sensitive HPLC-based assay to measure α-ketobutyrate. The results confirmed high cystathionine ß-synthase activity and no γ-cystathionase activity in brain, and high γ-cystathionase activity in mouse liver. The method presented here allows for evaluating the relative contribution of CBS and CTH to generation of H2S in tissues. Additionally, it provides results, which reflect the redox status (GSH/GSSG) of a tissue.

Rapid qPCR-based quantitative immune cell phenotyping in mouse tissues.

The immune microenvironment plays an important role in the regulation of diseases. The characterization of the cellular composition of immune cell infiltrates in diseases and respective models is a major task in pathogenesis research and diagnostics. For the assessment of immune cell populations in tissues, fluorescence-activated cell sorting (FACS) or immunohistochemistry (IHC) are the two most common techniques presently applied, but they are cost intensive, laborious, and sometimes limited by the availability of suitable antibodies. Complementary rapid qPCR-based approaches exist for the human situation but are lacking for experimental mouse models. Accordingly, we developed a robust, rapid RT-qPCR-based approach to determine and quantify the abundance of prominent immune cell populations such as T cells, helper T (Th) cells, cytotoxic T cells, Th1 cells, B cells, and macrophages in mouse tissues. The results were independently validated by the gold standards IHC and FACS in corresponding tissues and showed high concordance.

Cryo-plasma FIB/SEM volume imaging of biological specimens.

Serial focussed ion beam scanning electron microscopy (FIB/SEM) enables imaging and assessment of subcellular structures on the mesoscale (10 nm to 10 µm). When applied to vitrified samples, serial FIB/SEM is also a means to target specific structures in cells and tissues while maintaining constituents' hydration shells for in situ structural biology downstream. However, the application of serial FIB/SEM imaging of non-stained cryogenic biological samples is limited due to low contrast, curtaining, and charging artefacts. We address these challenges using a cryogenic plasma FIB/SEM. We evaluated the choice of plasma ion source and imaging regimes to produce high-quality SEM images of a range of different biological samples. Using an automated workflow we produced three-dimensional volumes of bacteria, human cells, and tissue, and calculated estimates for their resolution, typically achieving 20-50 nm. Additionally, a tag-free localisation tool for regions of interest is needed to drive the application of in situ structural biology towards tissue. The combination of serial FIB/SEM with plasma-based ion sources promises a framework for targeting specific features in bulk-frozen samples (>100 µm) to produce lamellae for cryogenic electron tomography.

Evaluation of Lipid Extraction Protocols for Untargeted Analysis of Mouse Tissue Lipidome.

Lipidomics refers to the full characterization of lipids present within a cell, tissue, organism, or biological system. One of the bottlenecks affecting reliable lipidomic analysis is the extraction of lipids from biological samples. An ideal extraction method should have a maximum lipid recovery and the ability to extract a broad range of lipid classes with acceptable reproducibility. The most common lipid extraction relies on either protein precipitation (monophasic methods) or liquid-liquid partitioning (bi- or triphasic methods). In this study, three monophasic extraction systems, isopropanol (IPA), MeOH/MTBE/CHCl3 (MMC), and EtOAc/EtOH (EE), alongside three biphasic extraction methods, Folch, butanol/MeOH/heptane/EtOAc (BUME), and MeOH/MTBE (MTBE), were evaluated for their performance in characterization of the mouse lipidome of six different tissue types, including pancreas, spleen, liver, brain, small intestine, and plasma. Sixteen lipid classes were investigated in this study using reversed-phase liquid chromatography/mass spectrometry. Results showed that all extraction methods had comparable recoveries for all tested lipid classes except lysophosphatidylcholines, lysophosphatidylethanolamines, acyl carnitines, sphingomyelines, and sphingosines. The recoveries of these classes were significantly lower with the MTBE method, which could be compensated by the addition of stable isotope-labeled internal standards prior to lipid extraction. Moreover, IPA and EE methods showed poor reproducibility in extracting lipids from most tested tissues. In general, Folch is the optimum method in terms of efficacy and reproducibility for extracting mouse pancreas, spleen, brain, and plasma. However, MMC and BUME methods are more favored when extracting mouse liver or intestine.

Isolation of Mouse Neutrophils.

Neutrophils represent the first line of defense against bacterial and fungal pathogens. Indeed, patients with inherited or acquired qualitative and quantitative neutrophil defects are at high risk for developing bacterial and fungal infections and suffering adverse outcomes from these infections. Therefore, research aiming at defining the molecular factors that modulate neutrophil effector function under homeostatic conditions and during infection is essential for devising strategies to augment neutrophil function and improve the outcomes of infected individuals. This article describes reproducible density-gradient-centrifugation-based as well as positive and negative immunomagnetic selection protocols that can be applied in any laboratory to harvest large numbers of highly enriched and highly viable neutrophils from the bone marrow of mice. In another protocol, we also present a method that combines gentle enzymatic tissue digestion with a positive immunomagnetic selection technique or fluorescence-activated cell sorting (FACS) to harvest highly pure and highly viable preparations of neutrophils directly from mouse tissues such as the kidney, the liver, or the spleen. Mouse neutrophils isolated by these protocols can be used to examine several aspects of cellular function ex vivo, including pathogen binding, phagocytosis, and killing, neutrophil chemotaxis, oxidative burst, degranulation, and cytokine production, and for performing neutrophil adoptive transfer experiments. © 2023 Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. Basic Protocol 1: Isolation of Neutrophils from Mouse Bone Marrow Using Positive Immunomagnetic Separation Alternate Protocol 1: Purification of Neutrophils from Bone Marrow Using Negative Immunomagnetic Separation Alternate Protocol 2: Purification of Neutrophils from Bone Marrow Using Histopaque-Based Density Gradient Centrifugation Basic Protocol 2: Isolation of Neutrophils from Mouse Tissues Using Positive Immunomagnetic Separation Alternate Protocol 3: Isolation of Neutrophils from Mouse Tissues Using FACS.

2D-DIGE Analysis of Liver Disease in Mice.

Hepatocellular carcinoma (HCC) is the major type of primary liver cancer. In this chapter, we describe our routine two-dimensional difference gel electrophoresis (2D-DIGE) workflow for analysis of mouse liver tissue in physiological conditions, as well as of mouse HCC. 2D-DIGE still constitutes a valuable comparative proteomics technique, not only providing information on global protein expression in a sample but also on potential posttranslational protein modifications, occurrence of protein degradation fragments, and the existence of protein isoforms. Thus, 2D-DIGE analysis provides highly complementary data to non-gel-based shotgun mass spectrometry (MS) methods (e.g., liquid chromatography (LC)-MS/MS)-allowing, for example, identification of novel protein biomarkers for HCC or increasing insights into the molecular mechanisms underlying hepatocarcinogenesis.

Isolation and Culture of Mouse Lymphatic Endothelial Cells from Lung Tissue.

There is increasing interest in the study of the mammalian lymphatic system, including the lymphatic endothelial cells (LECs) that make up lymphatic vessels. The ability to isolate primary LECs from tissue of normal and genetically modified mice permits detailed analysis of this unique cell type. Here, we describe a robust protocol for the isolation and in vitro expansion of LECs from mouse lung by antibody-based magnetic separation.

Mapping Microproteins and ncRNA-Encoded Polypeptides in Different Mouse Tissues.

Small open reading frame encoded peptides (SEPs), also called microproteins, play a vital role in biological processes. Plenty of their open reading frames are located within the non-coding RNA (ncRNA) range. Recent research has demonstrated that ncRNA-encoded polypeptides have essential functions and exist ubiquitously in various tissues. To better understand the role of microproteins, especially ncRNA-encoded proteins, expressed in different tissues, we profiled the proteomic characterization of five mouse tissues by mass spectrometry, including bottom-up, top-down, and de novo sequencing strategies. Bottom-up and top-down with database-dependent searches identified 811 microproteins in the OpenProt database. De novo sequencing identified 290 microproteins, including 12 ncRNA-encoded microproteins that were not found in current databases. In this study, we discovered 1,074 microproteins in total, including 270 ncRNA-encoded microproteins. From the annotation of these microproteins, we found that the brain contains the largest number of neuropeptides, while the spleen contains the most immunoassociated microproteins. This suggests that microproteins in different tissues have tissue-specific functions. These unannotated ncRNA-coded microproteins have predicted domains, such as the macrophage migration inhibitory factor domain and the Prefoldin domain. These results expand the mouse proteome and provide insight into the molecular biology of mouse tissues.

Development of a Quantitative FMRP Assay for Mouse Tissue Applications.

Fragile X syndrome results from the absence of the FMR1 gene product-Fragile X Mental Retardation Protein (FMRP). Fragile X animal research has lacked a reliable method to quantify FMRP. We report the development of an array of FMRP-specific monoclonal antibodies and their application for quantitative assessment of FMRP (qFMRPm) in mouse tissue. To characterize the assay, we determined the normal variability of FMRP expression in four brain structures of six different mouse strains at seven weeks of age. There was a hierarchy of FMRP expression: neocortex > hippocampus > cerebellum > brainstem. The expression of FMRP was highest and least variable in the neocortex, whereas it was most variable in the hippocampus. Male C57Bl/6J and FVB mice were selected to determine FMRP developmental differences in the brain at 3, 7, 10, and 14 weeks of age. We examined the four structures and found a developmental decline in FMRP expression with age, except for the brainstem where it remained stable. qFMRPm assay of blood had highest values in 3 week old animals and dropped by 2.5-fold with age. Sex differences were not significant. The results establish qFMRPm as a valuable tool due to its ease of methodology, cost effectiveness, and accuracy.

Bioanalytical assay for the new-generation ROS1/TRK/ALK inhibitor repotrectinib in mouse plasma and tissue homogenate using liquid chromatography-tandem mass spectrometry.

Repotrectinib, a next-generation ROS1/TRK/ALK tyrosine kinase inhibitor, overcomes resistance due to acquired solvent-front mutations involving ROS1, NTRK1-3, and ALK. A bioanalytical assay for quantification of repotrectinib in mouse plasma and seven tissue-related matrices (brain, liver, spleen, kidney, small intestinal tissue, small intestinal content, and testis homogenates) was developed and validated using liquid chromatography with tandem mass spectrometric detection in a high-throughput 96-well format. Protein precipitation was performed by adding acetonitrile, also containing the internal standard axitinib, to 10-µl samples for all matrices. Chromatographic separation of analytes was done on an ACQUITY UPLC® BEH C18 column by gradient elution using ammonium hydroxide in water and methanol. Compounds were monitored with positive electrospray ionization using a triple quadruple mass spectrometer in selected reaction monitoring mode. The method was successfully validated in the 1-1000 ng/ml calibration range. Precisions (intra- and interday) were in the range of 1.3-8.7% and accuracies were in between 90.5% and 107.3% for all levels in all matrices. The developed method was successfully applied to investigate the plasma pharmacokinetics and tissue accumulation of repotrectinib in wild-type mice.

Phospholipid Acyl Chain Diversity Controls the Tissue-Specific Assembly of Mitochondrial Cardiolipins.

Cardiolipin (CL) is a phospholipid specific for mitochondrial membranes and crucial for many core tasks of this organelle. Its acyl chain configurations are tissue specific, functionally important, and generated via post-biosynthetic remodeling. However, this process lacks the necessary specificity to explain CL diversity, which is especially evident for highly specific CL compositions in mammalian tissues. To investigate the so far elusive regulatory origin of CL homeostasis in mice, we combine lipidomics, integrative transcriptomics, and data-driven machine learning. We demonstrate that not transcriptional regulation, but cellular phospholipid compositions are closely linked to the tissue specificity of CL patterns allowing artificial neural networks to precisely predict cross-tissue CL compositions in a consistent mechanistic specificity rationale. This is especially relevant for the interpretation of disease-related perturbations of CL homeostasis, by allowing differentiation between specific aberrations in CL metabolism and changes caused by global alterations in cellular (phospho-)lipid metabolism.

Bioanalytical assay for the novel TRK inhibitor selitrectinib in mouse plasma and tissue homogenates using liquid chromatography-tandem mass spectrometry.

Selitrectinib is a next generation tropomyosin receptor kinase (TRK) inhibitor developed to overcome acquired resistance to first generation TRK inhibitors. The drug is a cyclic analogue of larotrectinib. An existing bioanalytical assay for larotrectinib was therefore redesigned for selitrectinib. The assay used liquid chromatography-electrospray tandem mass spectrometry in positive selected reaction monitoring mode. Mouse plasma and tissue homogenates of brain, heart, kidney, liver, lung, small intestine, spleen, and testis were pretreated using acetonitrile protein precipitation with larotrectinib added as internal standard. Successful validation using current guidelines was obtained in the range 0.5-1000 ng/ml. Precision was within 5-12% and accuracy within 91-108% for all matrices investigated. The drug was stable in all matrices under the relevant storage conditions. Pharmacokinetics and tissue distribution of selitrectinib were monitored in a pilot study in mice demonstrating the applicability of the presented assay.

High-Resolution and Super-Resolution Immunofluorescent Microscopy Ex Vivo to Study Pneumococcal Interactions with the Host.

In vivo imaging, meaning imaging tissues in living animals, is still a developing technique. However, microscopy imaging ex vivo remains a very important tool that allows for visualization of biological and pathological processes occurring in vivo. As described in Chap. 5, imaging of animal and human tissue postmortem can be performed at high resolution. Recently, imaging of human tissues infected with pneumococci using an even higher resolution, the so-called super-resolution with STED, has been reported.

Quantification of FGFR4 inhibitor BLU-554 in mouse plasma and tissue homogenates using liquid chromatography-tandem mass spectrometry.

BLU-554 is a potent, highly selective oral FGFR4 inhibitor. A bioanalytical assay for quantification of BLU-554 in mouse plasma and six tissue homogenates (brain, kidney, liver, lung, small intestine, and spleen) was developed and validated using liquid chromatography with tandem mass spectrometric detection and with erlotinib as internal standard. After protein precipitation with acetonitrile in a 96-well format and separation on an XBridge® Peptide BEH C18 column by gradient elution using 0.2% (v/v) ammonium hydroxide (in water) and methanol, analytes were ionized by positive electrospray and monitored in the selected reaction monitoring mode by triple quadrupole mass spectrometry. The assay was validated in a 1-1000 ng/ml concentration range using calibration in mouse plasma. Precisions (intra-day and inter-day) were in the range 2.8-10.1% and accuracies were in between 88.5 and 96.6% for all levels in all matrices. The assay was successfully applied for a pilot pharmacokinetic and tissue distribution study in wild-type mice.

Immunophenotyping of Tissue Samples Using Multicolor Flow Cytometry.

Flow cytometry enables the measurement of single cells in a flowing system. Heterogeneous mixtures of cells or particles can be analyzed with respect to their morphology, surface and intracellular protein expression, DNA content, and cellular physiology at high speed and purity. A series of key technical developments and improvements in flow cytometry hardware, software, and dye chemistry made it possible to measure more than 20 parameters simultaneously. Here, we provide a stepwise protocol for the preparation of single cell suspension samples from different murine lymphoid or tumor tissues and a detailed description of a 17-color polychromatic flow cytometry analysis of tumor-infiltrating leukocytes.

Quantitative bioanalytical assay for the tropomyosin receptor kinase inhibitor larotrectinib in mouse plasma and tissue homogenates using liquid chromatography-tandem mass spectrometry.

Larotrectinib is a promising tyrosine kinase inhibitor for solid tumors harboring tropomyosin receptor kinase gene fusions. A bioanalytical assay was developed for this drug in small volume samples using a 96-well format to efficiently support multiple mouse studies. The assay was completely validated for mouse plasma and partially for homogenates of eight different tissues: brain, heart, kidneys, liver, lungs, small intestine, spleen, and testes. Proteins in 10-µl samples were precipitated using acetonitrile containing momelotinib as internal standard. Chromatographic separation of analyte and internal standard from endogenous interferences was performed on an ethylene bridged octadecyl silica column using 0.1% (v/v) formic acid (in water) and methanol for gradient elution. Electrospray ionization and selected reaction monitoring on a triple quadrupole mass spectrometer were used for detection. In the range 1-2000 ng/ml the drug could be quantified in all 9 matrices with precisions (within-day and between-day) in the range 2.7-11.1% and accuracies in the range 87.4-101.4%. Compounds were sufficiently stable under all investigated conditions except for kidney homogenate. A pilot pharmacokinetic and tissue distribution study in mice demonstrated the applicability of the new presented assay for larotrectinib.

Simultaneous determination of tryptophan and its 31 catabolites in mouse tissues by polarity switching UHPLC-SRM-MS.

Tryptophan (TRP) and its catabolites have attracted a lot of attention because of their clinical significance to human health. Recently, microbiome-gut-brain axis was found to have links to many diseases based on the imbalance of TRP catabolism. By using ultra-high performance liquid chromatography coupled to electrospray ionization triple quadrupole mass spectrometry, we present a rapid, robust and comprehensive method to determine 31 TRP catabolites covering three major pathways - kynurenic, serotonergic and bacterial degradation - within 5 min. Polarity switching was employed to analyze catabolites in both ionization modes simultaneously for greatly improved analytical throughput. The intra-day and inter-day precision were 0.5-15.8% and 1.5-16.7%, respectively. Accuracy was between 75.8 and 126.9%. The developed method was applied to study the tissue level of TRP catabolites in the liver, ileum, ileal contents, brain and plasma samples from 8 mice, and clear differences in the distribution of TRP catabolites were observed in different tissues. Ratios of key catabolites to TRP were used to evaluate the activities of specific enzyme and pathway in respective tissues. This method has potential in high throughput analysis of TRP catabolites in biological matrices, which can facilitate understanding the influence of TRP catabolites on microbiome-gut-brain axis and on human health.

Bioanalytical liquid chromatography-tandem mass spectrometric assay for the quantification of the ALK inhibitors alectinib, brigatinib and lorlatinib in plasma and mouse tissue homogenates.

Several second and third generation ALK inhibitors have been introduced in recent years. A bioanalytical assay for simultaneous quantification of alectinib, brigatinib, and lorlatinib was developed and validated for human plasma. The method was also partially validated for diluted mouse plasma and tissue homogenates of brain, liver, kidney, and spleen. Samples (40 µl) were pretreated in a 96-well plate by protein precipitation with acetonitrile containing the internal standard [2H8]-alectinib. After chromatographic separation on an ethylene bridged octadecyl silica column by gradient elution at 600 µl/min using 1% (v/v) formic acid (in water) and acetonitrile, compounds were ionized by a turbo electrospray and monitored by selected reaction monitoring on a triple quadrupole mass spectrometer. Validation was performed in a 2-2000 ng/ml concentration range for alectinib and lorlatinib and a 4-4000 ng/ml range for brigatinib. Precisions (within-day and between-day) were in the range 2.2-15.0% and accuracies were in between 87.2 and 110.2% for all matrices and levels. Compounds were sufficiently stable under most investigated conditions. Results of a pilot pharmacokinetic and tissue distribution study for brigatinib in mice are reported. Finally, successful incurred samples reanalysis of tissue homogenate samples containing brigatinib and lorlatinib is presented. Lorlatinib homogenate samples were also successfully reanalyzed using a second independent assay (cross-validation).