Name: Fluorescence-Guided Matrix-assisted Laser Desorption/Ionization with Laser-Induced Postionization Mass Spectrometry of Individual Rat Neural Cells (Video) | JoVE Journal
Uploaded: 2025-05-23T14:50:00.000+00:00
Duration: 8 min 48 s
Description: Discover single-cell MALDI-2 mass spectrometry for fluorescence-guided molecular profiling of rat neural cells. This computational method reveals lipid-based cellular heterogeneity across brain regions.

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5 minutes

S.W.C acknowledges support provided by the Peixin He and Xiaoming Chen PhD4 Fellowship and the University of Illinois Block Grant Fellowship. This work was also supported by the National Institute on Drug Abuse under award No. P30DA018310, the National Institute on Aging under Award No. R01AG078797, and by the Office of The Director, of the National Institutes of Health under Award Number S10OD032242.

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<li>Chan-Andersen, P. C., Romanova, E. V., Rubakhin, S. S., Sweedler, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Profiling+26%2C000+Aplysia+californica+neurons+by+single+cell+mass+spectrometry+reveals+neuronal+populations+with+distinct+neuropeptide+profiles%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Profiling 26,000 Aplysia californica neurons by single cell mass spectrometry reveals neuronal populations with distinct neuropeptide profiles.</a> J Biol Chem. 298 (8), 102254(2022).</li>
<li>Castro, D. C., Xie, Y. R., Rubakhin, S. S., Romanova, E. V., Sweedler, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Image-guided+MALDI+mass+spectrometry+for+high-throughput+single-organelle+characterization%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Image-guided MALDI mass spectrometry for high-throughput single-organelle characterization.</a> Nat Methods. 18 (10), 1233-1238 (2021).</li>
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<li>Ibáñez, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mass+spectrometry-based+metabolomics+of+single+yeast+cells%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci%22%5BJournal%5D)">Mass spectrometry-based metabolomics of single yeast cells.</a> Proc Natl Acad Sci. 110 (22), 8790-8794 (2013).</li>
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The authors have no competing interests to disclose.

High-throughput, image-guided single-cell MALDI MS is a valuable tool for understanding chemical heterogeneity on a single-cell scale. The addition of laser-induced post-ionization (MALDI-2) provides deeper analyte coverage, which is critical for mass- and volume-limited samples such as isolated mammalian cells.
While the overwhelming majority of published single-cell lipid and metabolite MS workflows use cultured cells, our approach is applied to relatively quickly isolated primary cells. Thi...

Lipids and metabolites are fundamental to cellular function and serve as essential components of membranes, energy sources, and signaling molecules1,2. However, their composition and abundance can vary significantly between individual cells, reflecting the differences in cell types and developmental and functional states3,4,5. Analyzing these differences is crucial for understanding biological variability and identifying distinct cell subpopulations. Single-cell measurement techniques, such as RNA sequencing, provide useful cell-specific transcript profiles6. However, these transcript-level measurements do not directly reflect the actual cellular amounts of lipids and metabolites, as gene expression does not always correlate with the actual abundance of these analytes. Specialized methods for direct measurements of lipids and metabolites are, therefore, required for comprehensive analysis of the chemical composition of single cells and their populations.
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is a tool of choice for label-free spatial mapping of endogenous biomolecules in situ7,8. Typically, with MALDI, a UV laser is used to ablate material from a thin sample layer co-crystallized with an organic matrix, forming a plume of ions and neutral molecules. The formed ions are then separated by an MS analyzer and detected within a mass spectrometer. Given the accurate positioning of modern mass spectrometer stages, the laser can be positioned to target specific sample regions or rastered across regions to generate molecular images by MSI. MSI at cellular or subcellular spatial resolution (&#60; 10 &#181;m), achieved with a focused laser beam and accurate stage movement, can be used to obtain chemical information about individual cells9,10. However, MSI measurements at this scale are inefficient, especially in the case of samples with low targeted cell densities, due to the amount of time spent imaging empty regions between cells. Further, the detectability of many analytes is limited due to the small volume sampled. To overcome these challenges, we have developed an image-guided MALDI MS approach for high-throughput single-cell analysis11,12. In this approach, the locations of dispersed cells are programmatically determined from whole-slide fluorescence images and used to guide the mass spectrometer stage to positions where individual cells with specific parameters (e.g., size and shape) are located and are then analyzed via irradiation with the MALDI laser. Previous work using this targeted approach has been used for characterizing lipids, peptides, and other biomolecules in heterogeneous cell populations5,13.
Given the mass-limited nature of single cells, the number of analytes detected in such samples is generally less than that observed directly from tissue. Therefore, to increase analyte coverage in single-cell MS analysis, increasing the analyte detection sensitivity is critical. One recently developed approach that aids in overcoming this detection challenge is MALDI with laser post-ionization (MALDI-2), which has been shown to enhance sensitivity for a broad range of analytes9,14,15,16. As a result, MALDI-2 generates more comprehensive single-cell datasets and provides deeper molecular coverage in mass-limited samples, such as isolated cells.
The goal of this method is to obtain lipid measurements from thousands of individual cells. To this end, we describe a workflow that enables high-throughput single-cell MALDI-2 mass spectrometry and is generally extendable to any probe-based mass spectrometry approach with precise stage control17. In this workflow, tissue from the brain region(s) of interest is dissected, and individual cells are obtained from the tissue after a papain dissociation procedure. The cells are then labeled with a nuclear stain and are dispersed onto conductive glass slides etched with fiducial markers, where they are allowed to adhere. Next, whole-slide images are taken using fluorescence microscopy. Matrix is deposited by sublimation, generating a repeatable homogenous crystal layer and high signal-to-noise for single-cell MS analysis. Using the open-source software microMS11, the relative coordinates of cell locations from the microscopy image are mapped and aligned with mass spectrometer stage coordinates by a point-set registration using fiducial markers etched onto the glass slides where the cells were deposited. Lastly, using this information, precise, targeted MS spectra are acquired from each individual cell, allowing for thousands of cells to be profiled in a single run (&#60;1 h)12,13.

<table><tbody><tr><td>2&#039;,5&#039;-dihydroxyacetophenone</td><td>Sigma Aldrich</td><td>D107603</td><td>DHAP, 97% purity</td></tr><tr><td>Ammonium acetate</td><td>Sigma Aldrich</td><td>238074</td><td>ACS reagent, &amp;ge;97%</td></tr><tr><td>Axio M2 Imager</td><td>Zeiss</td><td>N/A</td><td>N/A</td></tr><tr><td>Biopsy punch, 2 mm</td><td>Fisher Scientific</td><td>12-460-399</td><td>integra miltex standard biopsy punch, 2mm</td></tr><tr><td>Calcium chloride</td><td>Sigma Aldrich</td><td>C4901</td><td>anhydrous, powder &amp;ge;97%</td></tr><tr><td>Eppendorf Centrifuge</td><td>Sigma Aldrich</td><td>EP5405000441</td><td>centrifuge 5425 with rotor FA-24x2</td></tr><tr><td>Gentamicin</td><td>Sigma Aldrich</td><td>G1272</td><td>liquid, BioReagent</td></tr><tr><td>Glass etching pen</td><td>Sigma Aldrich</td><td>Z225568</td><td>carbide time, pkg of 1</td></tr><tr><td>Glycerol</td><td>Sigma Aldrich</td><td>G7893</td><td>ACS reagent, &amp;ge;99.5%</td></tr><tr><td>HEPES buffer</td><td>Sigma Aldrich</td><td>H3375</td><td>&amp;ge;99.5% (titration)</td></tr><tr><td>Hoechst 33258 Solution</td><td>Sigma Aldrich</td><td>94403</td><td>1 mg/mL in H2O, &amp;ge;98.0% (HPLC)</td></tr><tr><td>In line HEPA Filter</td><td>Sigma Aldrich</td><td>WHA67225001</td><td>VACU-GUARD 60 mm disc, 0.45 PFTE housing</td></tr><tr><td>ITO-Coated Microscopy Slides</td><td>Delta Technologies</td><td>CG-90IN-S115</td><td>70-100&amp;Omega; resistance</td></tr><tr><td>Magnesium chloride</td><td>Sigma Aldrich</td><td>M8266</td><td>anhydrous, &amp;ge;98%</td></tr><tr><td>Magnesium sulfate</td><td>Sigma Aldrich</td><td>208094</td><td>anhydrous, &amp;ge;97%</td></tr><tr><td>Microcentrifuge tubes</td><td>Sigma Aldrich</td><td>HS4323K</td><td>tube capacity 1.5 mL, pack of 500</td></tr><tr><td>Papain dissociation system</td><td>Worthington Biochemical&amp;nbsp;</td><td>LK003150</td><td>one box, 5 single use vials</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma Aldrich</td><td>P4458</td><td>liquid, BioReagent</td></tr><tr><td>Potassium chloride</td><td>Sigma Aldrich</td><td>529552</td><td>Molecular biology grade</td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma Aldrich</td><td>P5379</td><td>Reagent Plus</td></tr><tr><td>Sodium biocarbonate</td><td>Sigma Aldrich</td><td>S6014</td><td>ACS reagent, &amp;ge;99.7%</td></tr><tr><td>Sodium chloride</td><td>Sigma Aldrich</td><td>S9888</td><td>ACS reagent, &amp;ge;99%</td></tr><tr><td>Sodium hydroxide</td><td>Sigma Aldrich</td><td>221465</td><td>ACS reagent, &amp;ge;97%, pellets</td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma Aldrich</td><td>S9763</td><td>ACS reagent, &amp;ge;99%</td></tr><tr><td>Sublimate</td><td>HTX</td><td>N/A</td><td>N/A</td></tr><tr><td>timsTOF FleX MALDI-2</td><td>Bruker</td><td>N/A</td><td>microGRID enabled</td></tr><tr><td>Vacuum tubing&amp;nbsp;</td><td>Thermo Scientific</td><td>8701-0080</td><td>Nalgene Non-phthalate PVC Tubing&amp;nbsp;</td></tr></tbody></table>

fluorescence-guided matrix-assisted laser desorption/ionization with laser-induced postionization mass spectrometry of individual rat neural cells

Fluorescence-Guided Matrix-assisted Laser Desorption/Ionization with Laser-Induced Postionization Mass Spectrometry of Individual Rat Neural Cells

Single-cell measurements are critical to understanding the rich spatiochemical heterogeneity of the brain. Matrix-assisted laser/desorption ionization (MALDI) mass spectrometry (MS) is capable of label-free, high-throughput characterization of endogenous molecules in individual cells. The recent advances in the development of MALDI mass spectrometers with laser-induced post-ionization (MALDI-2) provide greatly enhanced sensitivity of detection for a variety of lipids and other small molecules. However, MS imaging of large samples with MALDI-2 at cellular resolution is prohibitively slow for most applications. In this protocol, primary cells are isolated and dispersed onto conductive slides. Relative cell locations are determined by whole-slide fluorescence microscopy, followed by accurate coregistration of the microscopy coordinates to the stage coordinates of the MALDI-2 mass spectrometer. Targeted MS analysis of only cell locations provides high-throughput, single-cell measurements with high analyte coverage and reduced data size as compared to MS imaging of the entire sample. We describe the critical steps necessary for single-cell preparation, whole-slide fluorescence imaging, matrix application, and MALDI-2 mass spectrometry.

An overview of the workflow for fluorescence-guided single-cell MALDI-2 MS is shown in Figure 1. First, the tissue dissected from targeted brain regions (Figure 1A) is dissociated into single cells and deposited onto conductive ITO-coated microscopy slides (Figure 1B). The locations of cells are determined by whole-slide fluorescence imaging (Figure 1C), followed by MALDI matrix application (<strong cla...

This protocol outlines the use of microMS for fluorescence-guided, single-cell MALDI-2 mass spectrometry, enabling enhanced molecular profiling of primary rat neuronal cells.

Single-cell measurements are critical to understanding the rich spatiochemical heterogeneity of the brain. Matrix-assisted laser/desorption ionization ...

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Research

JoVE Journal

Neuroscience

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Source: Hare, D. J., et.al. Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS). J. Vis. Exp. (2017)This video demonstrates the use of laser ablation–inductively coupled plasma–mass spectrometry (LA-ICP-MS) to visualize and quantify metal distributions in mouse brain tissue. The protocol involves laser ablation to generate an aerosol of sample particles, ionization of the particles in high-temperature plasma, and mass spectrometry to...

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We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

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Human neuroprogenitor cells (NPCs) were expanded under proliferating conditions. NPCs were differentiated into neuron-rich cultures in the presence of a combination of neurotrophins. Neuronal markers were detected by immunofluorescence staining. To isolate a pure population of neurons, NPCs were differentiated on PEN membrane slides and laser capture microdissection was performed.

Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

This protocol describes the design and surgical implantation of a head-restraining mechanism to monitor neuronal activity in sub-cortical brain structures in alert rats.  It delineates procedures to isolate single neurons in the juxtacellular configuration and to efficiently identify their anatomical locations.

Juxtacellular Monitoring and Localization of Single Neurons within Sub-cortical Brain Structures of Alert, Head-restrained Rats

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Gene expression analysis of a subset of cells in a specific tissue represents a major challenge. This article describes how to isolate high-quality total RNA from a specific cell population by combining a rapid immunolabeling method with laser capture microdissection.

RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

We describe a technique to label neurons and their processes via anterograde or retrograde tracer injections into brain nuclei using an in vitro preparation. We modified an existing method of in vitro tracer electroporation by taking advantage of fluorescently labeled mouse mutants and basic optical equipment in order to increase labeling accuracy.

Laser-guided Neuronal Tracing In Brain Explants

A protocol for the sublimation of DAN matrix onto rat brain tissue for the detection of gangliosides using MALDI Imaging Mass Spectrometry is presented.

Sublimation of DAN Matrix for the Detection and Visualization of Gangliosides in Rat Brain Tissue for MALDI Imaging Mass Spectrometry

This protocol explains how to collect single neurons, microglia, and astrocytes from the central nucleus of the amygdala with high accuracy and anatomic specificity using laser capture microdissection. Additionally, we explain our use of microfluidic RT-qPCR to measure a subset of the transcriptome of these cells.

Combining Laser Capture Microdissection and Microfluidic qPCR to Analyze Transcriptional Profiles of Single Cells: A Systems Biology Approach to Opioid Dependence

Discover single-cell MALDI-2 mass spectrometry for fluorescence-guided molecular profiling of rat neural cells. This computational method reveals lipid-based cellular heterogeneity across brain regions.

Fluorescence-Guided Matrix-assisted Laser Desorption/Ionization with Laser-Induced Postionization Mass Spectrometry of Individual Rat Neural Cells (Video) | JoVE Journal

Fluorescence-Guided Matrix-assisted Laser Desorption/Ionization with Laser-Induced Postionization Mass Spectrometry of Individual Rat Neural Cells

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