NMR SPECTROSCOPY of MICROSCOPIC SPECIMENS – HRmicroMAS

The project divides into five tasks:

Task 1: construct the HR-uMAS probe at JEOL in Japan
Task 2: establish sample preparation protocols for tissue, whole organism and cell
Task 3: implement, validate, optimize the HR-uMAS NMR experiments
Task 4: carry out HR-uMAS study on whole organism c. elegans
Task 5: carry out HR-uMAS study on brain tissue

Tasks 1 (2017, delivered): Despite a long delay on the shipment (international custom), the prototype HR-uMAS probe had been delivered from Japan to LSDRM (France) on 06/2017.

Task 2 (2017-2019, delivered): together with a postdoc, and after over 500 samplings in a span of 2-years, a general guideline of sample preparation has established for tissue, organism and cell. (Metabolites 2020;10:54) Note that the sample preparation is not trivial (for manipulating ug-scale specimens with a 1-mm sample rotor) considering the procedures must be adequate for biological studies.

Task 3 (2017-2018, delivered but still on-going): Project ran into a few technical issues, with sample-preparation and NMR optimization (field shimming), and had further delay the progress. But, we managed to overcome these issues with unconventional approach (shimming: App Magn Reson 2019;50:1305) and successfully carried out the first demonstrative study with a fruit tissue. (Anal Chem 2018;90:13736). The latter has led to a 'new' concept of NMR metabolomics 'homogeneous sampling in heterogeneous specimens' and has inspired us to develop NEW NMR methods with the classical HR-MAS (Anal Bioanal Chem 2019;411:1591. Analyst 2020;145:2520)

Task 4 (2018-2019, not successful): Unfortunately, due to the inefficient sensitivity for profiling just 20-30 individual worms, no significant results were acquired.

Task 5 (2018-2020, delivered): We have successfully mapped the metabolic profile across the resented glioma-bearing rat brain slice. The high-spectral and -spatial resolution permits delineate and characterize the tumoral region. Moreover, the extensive model with n=178 sampling demonstrates, for the first time, the reliable of the HR-uMAS measurements. (submitted to NMR Biomed) This study represents the first HR-uMAS NMR on the ug-tissue study. It opens a new NMR perspective to biopsy research, which essentially has led to a new project: HR-uMAS NMR on microscopy.

Project Summary
-HR-uMAS NMR has successfully been optimized and validated with the current state of the HR-uMAS probe for metabolomics.
-Of the three main types of specimens - tissue, organism and cell - it is found that HR-uMAS application is most suitable for tissue biopsy. This is due to the adequacy of sample preparation.
-The project has opened two new explorations: (1) HR-uMAS NMR microbiopsy; and (2) localized HR-MAS NMR spectroscopy. Both are in-line with the same conceptual theme 'homogeneous sampling from heterogeneous specimens'

Immediate Future prospectives
-continue the development towards HR-uMAS NMR microbiopsy. For example, the idea of minimal invasiveness with ug-scale excised from a living animal could open the door to the concept of a longitudinal study with living animals. These results could represent a stepping-stone to clinical NMR spectroscopy!
-development of localized HR-MAS spectroscopy could target on intact (or in vivo) NMR study of a single whole specimen such as mm-scale insect.

Journal articles:
HR-uMAS
[1] 'Metabolic 1H NMR mapping with HR-uMAS: delineation and characterization of glioma in rat model' submitted to NMR Biomed 2020.
[2] 'General guidelines for sample preparation strategies in HR-uMAS NMR based metabolomics of microscopic specimens' Metabolites 2020, 10, 54.[3] ‘Current developments in uMAS NMR analysis for metabolomics’ Metabolites 2019, 9, 29.
[4] ‘Supplemental shimming for HR-MAS NMR spectroscopy’ App. Magn. Reson. 2019, 50, 1305-1313.
[5] 'Localized metabolic profiling: a model study of HR-µMAS NMR-based metabolomics on ug garlic tissue' Anal. Chem. 2018, 90, 13736-13743
[6] 'Capillary-inserted rotor design for HR-uMAS NMR-based metabolomics on mass-limited neurosphere' Molecules 2017, 22, 1289.

Localized HR-MAS (same conceptual theme as HR-uMAS)
[1] C. Lucas-Torres, A. Wong 'Intact NMR spectroscopy: slow high-resolution magic angle spinning chemical shift imaging' Analyst 2020, 145, 2520-2524.
[2] C. Lucas-Torres, A. Wong. ‘Simultaneous metabolic mapping of different anatomies by 1H HR-MAS chemical shift imaging' Anal. Bioanal. Chem. 2019, 411, 1591.
70.

Invited book-chapter
[1] 'High-resolution magic-angle spinning (HR-MAS) NMR spectroscopy' in NMR-based metabolomics (NMR developments). Edited by Hector C. Keun: RSC publisher UK, 2019, ch-5, p133-147.
International conference presentation
Oral
[1] ‘High-resolution µ-scaled MAS NMR mapping of diseased rat brains’ Metabolomics, The Hague, Netherlands, June 23-27, 2019.
[2] ‘HR-µMAS NMR localised metabolic profiling of µg specimens’ VIII IBero-American NMR meeting, Lisbon, Portugal, June 26-29, 2018.
[3] ‘The development of HR-MAS NMR towards µg biospecimens’ SMMAP 2017, Paris, October 2-5, 2017.

Poster
[1] ‘1H HR-MAS chemical shift imaging metabolic profiling’ VIII IBero- American NMR meeting, Lisbon, Portugal, June 26-29, 2018.
[2] Introducing HR-µMAS NMR probe for µg-scale samples’ Metabomeeting 2017, Birmingham, United Kingdom, December 11-13, 2017.

Nuclear Magnetic Resonance (NMR) has already proven to be a tremendous tool in –omics studies of Systems Biology, including the study of metabolome in biological systems known as metabolomics. Its major weakness is the low detection sensitivity that renders the analysis of microscopic quantities (submilligram) impractical, time consuming and often impossible. A cost-effective method is the use of micro(µ)-coils in NMR detections; however, implementing a NMR µcoil for heterogeneous samples such as tissues, cells and organisms is a challenging task. This is because of the necessity of spinning the sample at a 54.74° to the NMR magnetic field. This technique is known as Magic-Angle Spinning (MAS). The use of a spinning µcoil called High Resolution Magic Angle Coil Spinning (HRMACS) – developed by Alan Wong and his team at LSDRM marks the first successful µMAS NMR analyses of microscopic biospecimens, together with different biological expert-teams (INMG, ISA and CRMSB). Unfortunately HRMACS is greatly hindered by the impractical operation and the instability of µcoil. In 2015 without any financial supports, LSDRM has teamed up with a NMR-industry JEOL and developed an alternative technology, a standalone HRµMAS probe, specifically targeted to metabolomic applications. Unlike HRMACS, HRµMAS offers good metabolic analytical stability. However, further technological development and experimental validations are absolutely necessary in order for HRµMAS to have an impact in metabolomics. The aim of HRmicroMAS project is to implement and establish a truly convenient and accessible HRµMAS NMR-based application to the field of metabolomics. The project includes designing and constructing a stable HRµMAS probe, benchmarking and validating the HRµMAS NMR-based studies, and demonstrating its utilities with real applications. The success of the project will have a significant impact to the current NMR-platform for metabolomics, because it will be the first cornerstone of µMAS NMR-based metabolomics for microscopic specimens.

The first half of the project will be dedicated to designing and building a reliable HRµMAS probe, and to benchmarking and validating the HRµMAS NMR experiments from sample-preparations to data acquisitions. The goal here is to develop a reliable HRµMAS NMR application that is convenient to everyone. The second half will carry out two independent pilot metabolomic studies with different teams whose are experts in metabolomic studies: (1) with the teams INMG and ISA, an metabolic investigation will be carried out on the heterogeneity of phenotype associated to aging within isogenic populations of Caenorhabditis elegans nematodes; and (2) with CRMSB, the metabolic profile of the brain energy metabolism in rat will be explored. These biological studies are designed to exploit and evaluate the different bioapplications of HRµMAS with different biological specimens, and will be an important milestone for HRmicroMAS, because it will demonstrate the wide utility of this innovative technology for NMR applications to in the field of metabolomics.

HRmicroMAS fits well within the societal challenge #4 of “Life, Health and Well-Being” because it will be a vehicle that will lead to potential new discoveries and innovations in biological and medical science. And the strong collaborative nature in the multidisciplinary this project suits under the PRC financial instrument. It gathers µMAS experts (from LSDRM and JEOL), NMR spectroscopists (LSDRM and ISA) and biologists (INMG and CRMSB). This project also capitalizes a strong collaboration with a NMR industry, JEOL, whom will offer their involvements (probe design and construction) at zero-cost to the project. For this reason, HRmicroMAS can be considered a low-cost project (a requested fund of 304 k€) for a development of an innovative NMR application that could advance the analytical platform in metabolomics and open many new exciting venues.