Nuclear Magnetic Resonance
NMR RESOURCES AT MONTANA STATE UNIVERSITY
The Department of Chemistry and Biochemistry currently houses three NMR spectrometers, including an older DRX 500 MHz NMR, and a recently upgraded 600 MHz (1H Larmor Frequency) NMR spectrometer, both located in a specifically designed instrumentation room in the basement of the recently built (2008) Chemistry and Biochemistry Building (CBB room 18 and 18A). Both 500 and 600 NMR instruments are equipped with four RF channels, deuterium decoupling capabilities, and 5 mm triple (1H, 15N, 13C) resonance room temperature probes with triple axis gradients.
The 600 MHz NMR was upgraded in 2011 from a DRX to an AVANCE III spectrometer, and is equipped with a helium-cooled 1H-optimized, inverse detection, (1H, 15N, 13C) TCI cryoprobe and a SampleJetTM automatic sample loading system. The 600 MHz NMR is dedicated to protein structure determination, characterization of macromolecular complexes (protein-protein or protein-nucleic acid interactions), protein dynamics studies (using 15N and 13C NMR relaxation approaches) and, more recently, is being used for broadly based untargeted NMR metabolomic analyses.
The TCI cryoprobe installed on MSU’s 600 MHz NMR provides a significant increase in sensitivity for the NMR experiments (> 4 fold, amounting to a > 16 time saving in data acquisition time requirements for mM concentrated samples). It also allows for detection of low abundance metabolite in the sub-micromolar concentrations. The cryoprobe is fully operational.
Acquisition of the cryoprobe has opened new research opportunities for identification of small molecules metabolites that may be present in low concentrations and together with the 600 MHz automatic sample loading system (SampleJetTM), opens new avenue for automated high-throughput profiling of metabolites (for example the SampleJet has five positions for 96 well-plate sized NMR tube racks. This allows the handling of batches with up to 480 sample tubes. It also accommodates and provides 96 positions for standard 3 mm and 5 mm NMR tubes). A multi-user license (> 5) to operate the ChenomxTM metabolite profiler software required to process and analyze NMR metabolomics data has been purchased.
In addition to the DRX 500 MHz NMR and the AVANCE III 600 MHz NMR, the NMR facility of the Department of Chemistry and Biochemistry houses an old two-channel Bruker DPX 300 MHz NMR spectrometer, which is used for the most routine organic and bioinorganic work, and occasionally for characterization of metal clusters.
Most recently, Dr. Copié (recently appointed Director of MSU’s NMR Center by MSU’s VPR) and colleagues applied to the NSF-MRI (awarded) and Murdock Charitable Trust Foundation (awarded) grant programs to acquire a ~ $1M state-of-the-art integrated LC-SPE-NMR-MS instrument, involving the acquisition of a new ProdigyTM cryoprobe-equipped (e.g. X-nucleus detection optimized) 500 MHz NMR system, which will be coupled to a mass spectrometer instrument for simultaneous MS and NMR analysis (i.e. MS and NMR analysis of the same LC analyte fractions) of metabolites. This instrument will be particularly useful for the identification and structural characterization of unknowns (ie. Metabolites that are not annotated in current NMR or MS databases of small molecules). The new system has been ordered from Bruker Inc. and should be delivered at MSU in June-July 2016.
During operation of the LC-SPE-NMR-MS, a tiny fraction of the liquid chromatography (LC) effluent (1-5%) will be directed to the MS system that detects and commands which fractions of the LC flow appear to contain interesting compounds; those will be captured on a fraction collection system for most of the LC sample stream, using a movable array of solid phase extraction (SPE) cartridges. The compounds trapped on the SPE cartridges will then be eluted following the LC run, and dried in NMR sample tubes. NMR solvents will be subsequently added and racks of tubes moved to the automatic sample loading system (autosampler) of the NMR instrument, where a variety of 1D and 2D NMR experiments will be used for small molecule identification and structure determination.
• Four-channel AVANCE III 600 MHz Bruker Solution NMR Spectrometer equipped with a Bruker SampleJetTM automatic sampler allows for rapid quantification of metabolites in complex biological samples.
• A sensitivity-enhanced cryogenically cooled TCI (1H, 15N, 13C) NMR Probe (cryoprobe)
• Chenomx Professional NMR Suite (~ 5 user licenses) is a high-throughput metabolomics data analysis software that allows for preprocessing of spectra, metabolite identification and quantification by comparison to a pH sensitive 600 MHz spectral library, which contains hundreds of compounds.
• XLSTAT is a multivariate-data-analysis software compatible with Microsoft Excel tables, which allows for the identification of patterns, outliers, and trends in metabolomics data sets using techniques such as principal component analysis.
• Integrated state-of-the-art, ProdigyTM cryoprobe-equipped, LC-SPE-500 MHz-NMR-MS system: The system has been ordered and is expected to be delivered and operational sometimes in summer of 2016.