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6 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Ambient Single-Cell Analysis and Native Tissue Imaging Using Laser-Ablation Electrospray Ionization Mass Spectrometry with Increased Spatial Resolution

Laser ablation electrospray ionization mass spectrometry (LAESI-MS) is an emerging method that has the potential to transform the field of metabolomics. This is in part due to LAESI-MS being an ambient ionization method that requires minimal sample preparation and uses (endogenous) water for in situ analysis. This application note details the employment of the ‘LAESI microscope’ source to perform spatially resolved MS analysis of cells and MS imaging (MSI) of tissues at high spatial resolution. This source configuration utilizes a long-working distance reflective objective that permits both visualization of the sample and a smaller LAESI laser beam profile than conventional LAESI setups. In this work, we analyzed 200 single cells of Allium cepa (red onion) and imaged Fittonia argyroneura (nerve plant) in high spatial resolution using this source coupled to a Fourier transform mass spectrometer for high mass resolution and high mass accuracy metabolomics.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Metabolomic Profiling of Wild-type and Mutant Soybean Root Nodules Using Laser-ablation Electrospray Ionization Mass Spectrometry Reveals Altered Metabolism

The establishment of the nitrogen-fixing symbiosis between soybean and Bradyrhizobium japonicum is complex. In order to document the changes in plant metabolism due to the symbiosis, we utilized laser ablation electrospray ionization mass spectrometry (LAESI-MS) for in situ metabolic profiling of wild-type nodules, nodules infected with a B. japonicum nifH mutant unable to fix nitrogen, nodules doubly infected by both strains, and nodules formed on plants mutated in the stearoyl-acyl carrier protein desaturase (sacpd-c) gene, which were previously shown to have altered nodule ultrastructure. Results showed that the relative abundance of fatty acids, purines, and lipids was significantly changed in response to the symbiosis. The nifH mutant nodules had elevated levels of jasmonic acid (JA), correlating with signs of nitrogen deprivation. Nodules resulting from the mixed inoculant displayed similar, overlapping metabolic distributions within the sectors of effective (fix+) and ineffective (nifH mutant, fix-) endosymbionts. These data are inconsistent with the notion that plant sanctioning is cell autonomous. Nodules lacking sacpd-c displayed an elevation of soyasaponins and organic acids in the central necrotic regions. This study demonstrates the utility of LAESI-MS for high-throughput screening of plant phenotypes. Overall, nodules disrupted in the symbiosis were elevated in metabolites related to plant defense.

Agtuca, Beverly J.↗
DOE OSTI2021

Optical Microscopy-Guided Laser Ablation Electrospray Ionization Ion Mobility Mass Spectrometry: Ambient Single Cell Metabolomics with Increased Confidence in Molecular Identification

Single cell analysis is a field of increasing interest as new tools are continually being developed to understand intercellular differences within large cell populations. Laser-ablation electrospray ionization mass spectrometry (LAESI-MS) is an emerging technique for single cell metabolomics. Over the years, it has been validated that this ionization technique is advantageous for probing the molecular content of individual cells in situ. Here, we report the integration of a microscope into the optical train of the LAESI source to allow for visually informed ambient in situ single cell analysis. Additionally, we have coupled this ‘LAESI microscope’ to a drift-tube ion mobility mass spectrometer to enable separation of isobaric species and allow for the determination of ion collision cross sections in conjunction with accurate mass measurements. This combined information helps provide higher confidence for structural assignment of molecules ablated from single cells. Here, we show that this system enables the analysis of the metabolite content of Allium cepa epidermal cells with high confidence structural identification together with their spatial locations within a tissue.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Single-Cell Metabolomics with Rapid Determination of Chemical Formulas from Isotopic Fine Structures

Metabolomic measurements can provide functional readouts of cellular states and phenotypes. Here, we present a protocol for single-cell metabolomics that permits direct untargeted detection of a broad number of metabolites under ambient conditions, without the need for sample processing, and with high confidence in the discovery and identification of the molecular formulas for detected metabolites. This protocol describes combining fiber-based laser ablation electrospray ionization (f-LAESI) with a 21 Tesla Fourier transform ion cyclotron resonance mass spectrometer (21T-FTICR-MS) to obtain high confidence molecular formula information about detected metabolites. The f-LAESI source utilizes mid-infrared laser ablation through a sharp optical fiber tip, affording direct ambient analysis of cells without the need for sample processing. Using the 21T-FTICR-MS as a mass analyzer enabled measurement of the isotopic fine structure (IFS) for numerous metabolites simultaneously from single cells, and the IFSs were in turn computationally processed to rapidly determine the corresponding elemental compositions. This metabolomics technique complements other single cell omics measurement methods, helping to resolve complex molecular interactions that take place within cells unattainable from single cell transcriptomic and proteomics methods.

mass spectrometry, FTICR, FTMS, ultrahigh mass res↗
DOE OSTI2020

Single-Cell Metabolic Profiling: Metabolite Formulas from Isotopic Fine Structures in Heterogeneous Plant Cell Populations

Characterization of the metabolic heterogeneity in cell populations requires the analysis of single cells. Most current methods in single-cell analysis rely on cell manipulation, potentially altering the abundance of metabolites in individual cells. Small sample volume and the chemical diversity of metabolites are additional challenges in single-cell metabolomics. Here, we describe the combination of fiber-based laser ablation electrospray ionization (f-LAESI) with 21 tesla Fourier transform ion cyclotron resonance mass spectrometry (21TFTICR-MS) for in situ single-cell metabolic profiling in plant tissue. Single plant cells infected by bacteria were selected and sampled directly from the tissue without cell manipulation through mid-infrared ablation with a fine optical fiber tip for ionization by f-LAESI. Finally, ultrahigh performance 21T-FTICR-MS enabled the simultaneous capture of isotopic fine structures (IFSs) for 47 known and 11 unknown compounds, thus elucidating their elemental compositions from single cells, and providing information on metabolic heterogeneity in the cell population.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Spatially Resolved Mass Spectrometry at the Single Cell: Recent Innovations in Proteomics and Metabolomics

Biological systems are composed of heterogeneous populations of cells that intercommunicate to form a functional living tissue. Biological function varies greatly across populations of cells, as each single cell has a unique transcriptome, proteome, and metabolome that translates to functional differences within single species and across kingdoms. Over the past decade, substantial advancements in our ability to characterize omic profiles on a single cell level have occurred, including in multiple spectroscopic and mass spectrometry (MS)-based techniques. Of these technologies, spatially resolved mass spectrometry approaches, including mass spectrometry imaging (MSI), have shown the most progress for single cell proteomics and metabolomics. For example, reporter-based methods using heavy metal tags have allowed for targeted MS investigation of the proteome at the subcellular level, and development of technologies such as laser ablation electrospray ionization mass spectrometry (LAESI-MS) now mean that dynamic metabolomics can be performed in situ. In this Perspective, we showcase advancements in single cell spatial metabolomics and proteomics over the past decade and highlight important aspects related to high-throughput screening, data analysis, and more which are vital to the success of achieving proteomic and metabolomic profiling at the single cell scale. Finally, using this broad literature summary, we provide a perspective on how the next decade may unfold in the area of single cell MS-based proteomics and metabolomics.

59 BASIC BIOLOGICAL SCIENCES↗