Development and refinement of an in situ 'molecular microscope' utilizing ultrahigh resolution mass spectrometry
EMSL Project ID
50592
Abstract
The ability to elucidate complex metabolic behavior of living organisms in situ and with cellular resolution is a bioanalytical grand challenge that is yet to be realized. Mass spectrometry imaging (MSI) using laser ablation electrospray ionization mass spectrometry (LAESI) has recently been demonstrated, and holds promise for direct spatially resolved molecular profiling in a variety of living systems of DOE relevance (e.g., plant leaf, stem, and root tissue).The LAESI MSI approach has revealed cell-to-cell heterogeneity of metabolites within living plant tissue, in a multiplexed fashion and without the need for labeling. Cellular resolution by in situ, ambient pressure MSI methods are far from routine, however, and have inadequate spatial resolution for many biological questions of interest. We propose to develop next-generation LAESI approaches, using advanced fiber optics and transmission-based geometries, in efforts to attain dynamic bioanalysis of living systems at better than 20 µm lateral resolution. In parallel with LAESI innovations, attendant increases in sensitivity and dynamic range of the mass spectrometer system will also be necessary for detection of the limited amount of material being ionized. To respond to this need, a new, high magnetic field (21 Tesla) Fourier Transform Ion Cyclotron Resonance (FTICR) MS at the EMSL/PNNL will be coupled with the newly developed LAESI source. This mass spectrometer, currently being assembled, will be one of only two such systems in the world, and the only one in a DOE laboratory or user facility. Fundamentally, all key figures of merits of FTICR MS performance improve with increased magnetic field strength, providing the ability to achieve unparalleled molecular specificity (near unequivocal molecular constituent identification) at the highest possible mass resolving power and sensitivity. The work proposed here will greatly increase the utility of this FTICR MS instrument, providing unique capabilities to support BER-relevant science.
The research plan is comprised of three tasks, two of which are directed by the academic leads (University of Missouri and George Washington University), and one of which will be performed in conjunction with the BER user facility at EMSL/PNNL. In task 1, advancements of the LAESI technology will be achieved, with oversight and leadership of Professor Akos Vertes of George Washington University. In task 2, incorporation of this ion source with EMSL’s new 21T FTICR MS system will be undertaken, supplemented by global omics measurements and calibrated against more conventional MSI platforms also available at EMSL (Drs. Pasa-Tolic, Anderton, and Koppenaal team). In the final task, the resultant innovations and advancements will be tested and validated by addressing important BER relevant science questions on model systems of interest under the direction of Professor Gary Stacey of the University of Missouri. This research follows on key recommendations of the recent BER “Research in Sustainable Bioenergy” report that, for example, stressed that “The design of sustainable biofuel systems requires knowledge about key plant-microbe-environment interactions that provide a range of ecosystem services.” This task will focus on validation of the instrumentation developed through this proposal, advertising this capability to the community through demonstration and, finally, discovering new biology relevant to the BER mission. This final objective will make use of Setaria viridis, a model C4 grass species, as well as switchgrass and sorghum, two promising biofuel crops. Specifically, we will demonstrate applicability to plant-microbe rhizosphere interactions focusing on the biological nitrogen fixation by monitoring metabolic changes in real-time with nutrient, growth condition, and other perturbations.
Project Details
Start Date
2018-11-26
End Date
2021-09-30
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Agtuca B.J., S.A. Stopka, S. Evans, L.Z. Samarah, Y. Liu, D. Xu, and M. Stacey, et al. 2020. "Metabolomic Profiling of Wild-type and Mutant Soybean Root Nodules Using Laser-ablation Electrospray Ionization Mass Spectrometry Reveals Altered Metabolism." The Plant Journal. PNNL-SA-144662. doi:10.1111/tpj.14815
Agtuca B.J., S.A. Stopka, T. Tuleski, F.P. do Amaral, S. Evans, Y. Liu, and D. Xu, et al. 2020. "In situ Metabolomic Analysis of Setaria Viridis Roots Colonized by Beneficial Endophytic Bacteria." Molecular Plant-Microbe Interactions, MPMI 33, no. 2:272-283. PNNL-SA-145863. doi:10.1094/MPMI-06-19-0174-R
Samarah L.Z., R. Khattar, T.H. Tran, S.A. Stopka, C.A. Brantner, P. Parlanti, and D. Velickovic, et al. 2020. "Single-Cell Metabolic Profiling: Metabolite Formulas from Isotopic Fine Structures in Heterogeneous Plant Cell Populations." Analytical Chemistry 92, no. 10:7289-7298. PNNL-SA-152789. doi:10.1021/acs.analchem.0c00936