Characterizing RNA Modifications in Single Neurons Using Mass Spectrometry

Post-transcriptional modifications (PTMs) of RNA represent an understudied mechanism involved in the regulation of translation in the central nervous ...

University of Illinois Urbana-Champaign

Mass spectrometry imaging (MSI), a rapidly growing subfield of chemical imaging, employs mass spectrometry (MS) technologies to create single- and multi-dimensional localization maps for a variety of atoms and molecules. Complimentary to other imaging approaches, MSI provides high chemical specificity and broad analyte coverage. This powerful analytical toolset is capable of measuring the distribution of many classes of inorganics, metabolites, proteins, and pharmaceuticals in chemically and structurally complex biological specimens in vivo, in vitro, and in situ. The MSI approaches highlighted in this Methods in Molecular Biology volume provide flexibility of detection, characterization, and identification of multiple known and unknown analytes. The goal of this chapter is to introduce investigators who may be unfamiliar with MS to the basic principles of the mass spectrometric approaches as used in MSI. In addition to guidelines for choosing the most suitable MSI method for specific investigations, cross-references are provided to the chapters in this volume that describe the appropriate experimental protocols.

A mass spectrometry primer for mass spectrometry imaging.

Pinpointing a specific cell from within a relatively uniform cell population to determine its chemical content presents a challenging bioanalytical task. Immunocytochemistry is the classical method used to localize specific molecules and, hence, selected cells. Mass spectrometry also probes endogenous molecules such as neuropeptides within a cell. Here, these two approaches are hyphenated to allow microchemical analysis of immunocytochemical-selected peptidergic neurons. This two-step strategy utilizes antibody-based localization of cells containing selected biomarkers to isolate the cell(s) of interest, followed by peptidomic analysis via mass spectrometry. Applicable to a broad range of analyte and cell types, the strategy was used to successfully profile neuropeptides from individual immunostained insect neurons stored for up to 2 weeks as well as from tissues preserved for 42 weeks.

Targeted single-cell microchemical analysis: MS-based peptidomics of individual paraformaldehyde-fixed and immunolabeled neurons.

The metabolome refers to the entire set of small molecules, or metabolites, within a biological sample. These molecules are involved in many fundamental intracellular functions and reflect the cell's physiological condition. The ability to detect and identify metabolites and determine and monitor their amounts at the single cell level enables an exciting range of studies of biological variation and functional heterogeneity between cells, even within a presumably homogenous cell population. Significant progress has been made in the development and application of bioanalytical tools for single cell metabolomics based on mass spectrometry, microfluidics, and capillary separations. Remarkable improvements in the sensitivity, specificity, and throughput of these approaches enable investigation of multiple metabolites simultaneously in a range of individual cell samples.

Progress toward single cell metabolomics.

Understanding and controlling the interactions occurring between cells and engineered materials are central challenges toward progress in the development of biomedical devices. In this work, we describe materials for direct ink writing (DIW), an extrusion-based type of 3D printing, that embed a custom synthetic protein (RGD-PDL) within the microfilaments of 3D-hydrogel scaffolds to modify these interactions and differentially direct tissue-level organization of complex cell populations in vitro. The RGD-PDL is synthesized by modifying poly-d-lysine (PDL) to varying extents with peptides containing the integrin-binding motif Arg-Gly-Asp (RGD). Compositional gradients of the RGD-PDL presented by both patterned and thin-film poly(2-hydroxyethyl) methacrylate (pHEMA) substrates allow the patterning of cell-growth compliance in a grayscale form. The surface chemistry-dependent guidance of cell growth on the RGD-PDL-modified pHEMA materials is demonstrated using a model NIH-3T3 fibroblast cell line. The formation of a more complex cellular system-organotypic primary murine dorsal root ganglion (DRG)-in culture is also achieved on these scaffolds, where distinctive forms of cell growth and migration guidance are seen depending on their RGD-PDL content and topography. This experimental platform for the study of physicochemical factors on the formation and the reorganization of organotypic cultures offers useful capabilities for studies in tissue engineering, regenerative medicine, and diagnostics.

3D-Printed pHEMA Materials for Topographical and Biochemical Modulation of Dorsal Root Ganglion Cell Response.

Microfluidic and mass spectrometry (MS) methods are widely used to sample and probe the chemical composition of biological systems to elucidate chemical correlates of their healthy and disease states. Though matrix-assisted laser desorption/ionization-mass spectrometry (MALDI)-MS has been hyphenated to droplet microfluidics for offline analyses, the effects of parameters related to droplet generation, such as the type of oil phase used, have been understudied. To characterize these effects, five different oil phases were tested in droplet microfluidics for producing samples for MALDI-MS analysis. Picoliter to nanoliter aqueous droplets containing 0.1 to 100 mM γ-aminobutyric acid (GABA) and inorganic salts were generated inside a polydimethylsiloxane microfluidic chip and deposited onto a conductive glass slide. Optical microscopy, Raman spectroscopy, and MALDI-mass spectrometry imaging (MSI) of the droplet samples and surrounding areas revealed patterns of solvent and oil evaporation and analyte deposition. Optical microscopy detected the presence of salt crystals in 50-100 μm diameter dried droplets, and Raman and MSI were used to correlate GABA signals to the visible droplet footprints. MALDI-MS analyses revealed that droplets prepared in the presence of octanol oil led to the poorest detectability of GABA, whereas the oil phases containing FC-40 provided the best detectability; GABA signal was localized to the footprint of 65 pL droplets with a limit of detection of 23 amol. The effect of the surfactant perfluorooctanol on analyte detection was also investigated.

Stanislav S. Rubakhin

Beckman Institute for Advanced Science and Technology,
Department of Chemistry

University of Illinois Urbana-Champaign

Characterizing RNA Modifications in Single Neurons Using Mass Spectrometry

Stanislav S. Rubakhin

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University of Illinois Urbana-Champaign

Characterizing RNA Modifications in Single Neurons Using Mass Spectrometry

Beckman Institute for Advanced Science and Technology,
Department of Chemistry