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High-resolution atmospheric-pressure MALDI mass spectrometry imaging workflow for lipidomic analysis of late fetal mouse lungs.

ABSTRACT: Mass spectrometry imaging (MSI) provides label-free, non-targeted molecular and spatial information of the biomolecules within tissue. Lipids play important roles in lung biology, e.g. as surfactant, preventing alveolar collapse during normal and forced respiration. Lipidomic characterization of late fetal mouse lungs at day 19 of gestation (E19) has not been performed yet. In this study we employed high-resolution atmospheric pressure scanning microprobe matrix-assisted laser desorption/ionization MSI for the lipidomic analysis of E19 mouse lungs. Molecular species of different lipid classes were imaged in E19 lung sections at high spatial and mass resolution in positive- and negative-ion mode. Lipid species were characterized based on accurate mass and on-tissue tandem mass spectrometry. In addition, a dedicated sample preparation protocol, homogenous deposition of matrices on tissue surfaces and data processing parameters were optimized for the comparison of signal intensities of lipids between different tissue sections of E19 lungs of wild type and Pex11?-knockout mice. Our study provides lipid information of E19 mouse lungs, optimized experimental and data processing strategies for the direct comparison of signal intensities of metabolites (lipids) among the tissue sections from MSI experiments. To best of our knowledge, this is the first MSI and lipidomic study of E19 mouse lungs.

SUBMITTER: Garikapati V

PROVIDER: S-EPMC6395778 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Project description:A liquid matrix-assisted laser desorption/ionization (liquid MALDI) method has been developed for high-throughput atmospheric pressure (AP) mass spectrometry (MS) analysis of the molecular content of crude bioliquids for disease diagnostics. The presented method is rapid and highly robust, enabling its application in environments where speed and low-cost high-throughput analyses are required. Importantly, because of the creation of multiply charged analyte ions, it provides additional functionalities that conventional solid MALDI MS profiling is lacking, including the use of high-performance mass analyzers with limited m/z range. The concomitant superior MS/MS performance that is achieved similar to ESI MS/MS adds greater analytical power and specificity to MALDI MS profiling while retaining the advantages of a fast laser-based analysis system and off-line large-scale sample preparation. The potential of this MALDI MS profiling method is demonstrated on the detection of dairy cow mastitis, which is a substantial economic burden on the dairy industry with losses of hundreds of dollars per diseased cow per year, equating to a total annual loss of billions of dollars, as well as leading to the use of large quantities of antibiotics, adding to the proliferation of antimicrobial resistance. Only small amounts of aliquots obtained from the daily farm milking process were prepared for liquid MALDI MS profiling using a simple one-pot/two-step analyte extraction. Automated analysis was performed using a custom-built AP-MALDI ion source, enabling the simultaneous detection of lipids, peptides, and proteins. Diagnostic, multiply charged, proteinaceous ions were easily sequenced and identified by MS/MS experiments. Samples were classified according to mastitis status using multivariate analysis, achieving 98.5% accuracy (100% specificity) determined by "leave 20% out" cross-validation. The methodology is generally applicable to AP-MALDI MS profiling on most commercial high-resolution mass spectrometers, with the potential for expansion into hospitals for rapid assessment of human and other biofluids.

Project description:BackgroundNeuropeptides are a diverse category of signaling molecules in the nervous system regulating a variety of processes including food intake, social behavior, circadian rhythms, learning, and memory. Both the identification and functional characterization of specific neuropeptides are ongoing fields of research. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of nervous tissues from a variety of organisms allows direct detection and identification of neuropeptides. Here, we demonstrate an analysis workflow that allows for the detection of differences in specific neuropeptides amongst a variety of neuropeptides being simultaneously measured. For sample preparation, we describe a straight-forward and rapid (minutes) method where individual adult Drosophila melanogaster brains are analyzed. Using a MATLAB-based data analysis workflow, also compatible with MALDI-TOF mass spectra obtained from other sample preparations and instrumentation, we demonstrate how changes in neuropeptides levels can be detected with this method.ResultsOver fifty isotopically resolved ion signals in the peptide mass range are reproducibly observed across experiments. MALDI-TOF MS profile spectra were used to statistically identify distinct relative differences in organ-wide endogenous levels of detected neuropeptides between biological conditions. In particular, three distinct levels of a particular neuropeptide, pigment dispersing factor, were detected by comparing groups of preprocessed spectra obtained from individual brains across three different D. melanogaster strains, each of which express different amounts of this neuropeptide. Using the same sample preparation, MALDI-TOF/TOF tandem mass spectrometry confirmed that at least 14 ion signals observed across experiments are indeed neuropeptides. Among the identified neuropeptides were three products of the neuropeptide-like precursor 1 gene previously not identified in the literature.ConclusionsUsing MALDI-TOF MS and preprocessing/statistical analysis, changes in relative levels of a particular neuropeptide in D. melanogaster tissue can be statistically detected amongst a variety of neuropeptides. While the data analysis methods should be compatible with other sample preparations, the presented sample preparation method was sufficient to identify previously unconfirmed D. melanogaster neuropeptides.

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High-resolution atmospheric-pressure MALDI mass spectrometry imaging workflow for lipidomic analysis of late fetal mouse lungs.

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