Experimental environmental metabolomics studies with 1H-NMR spectroscopy

Summary

Environmental metabolomics is a branch of metabolomics that focuses on the metabolic changes of organisms in response to environmental stress. Because this approach does not rely on knowledge of an organism's genome, it is ideal for studying multiple species in an ecosystem. The determination of an organism's metabolic components can, in principle, be used to identify new biomarkers and mechanisms of action of stimuli. This chapter describes an experimental protocol for metabolite extraction from biological samples, measurement of metabolites using1 H nuclear magnetic resonance (NMR) spectroscopy, and ultimately the analysis of metabolic data using a multiparametric statistical approach. The preparation of body fluids for NMR analysis and the methanol-chloroform protocol for metabolite extraction from tissue samples are first described. Then NM R methods are presented, including the standard one-dimensional (I-D)1 H-NMR method and the two-dimensional U-DVH-1 H J-resolution test. The advantages and disadvantages of each method are discussed. Finally, two methods for analyzing multiparametric metabolic NMR data are presented, including the traditional fingerprinting method consisting of a spectral preprocessing step followed by a multiparametric statistical analysis step. Although relatively reliable and effective, this method produces unidentified metabolites of limited value to the biologist. In a second, more recent, profiling method, NMR spectra are converted into a series of metabolites and their concentrations. Although more biologically meaningful, this method is more labor intensive. By Martin, this experiment is from "Environmental Genomics Lab Guide".

Operation method

Experimental environmental metabolomics research using 1H-NMR spectroscopy

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I. Materials 1. Organization of collection and preservation

(1) Liquid nitrogen and thermos flasks.

2. Collection and preservation of body fluids

(1) Liquid nitrogen and thermos flasks.

(2) Heparin-containing blood collection tubes (e.g., BD Vacutainer Heparin Tubes).

3. Combined extraction of polar and lipophilic metabolites from tissues using methanol-chloroform

(1) Straight-walled glass vials with aluminum-lined black urea threaded caps, including "large" (46 mm H X 12.5 mm diameter) and "small" (36 mm X 11 mm) vials (Fisher Scientific).

(2) Methanol (HPLC grade; Fisher Scientific): Place on ice throughout.

(3) Chloroform (pesticide analytical grade; Fisher Scientific): Keep on ice throughout.

4. NMR Preparation of Body Fluid and Polar Tissue Extracts

(1) NMR buffer: Sodium Mercate Buffer (composed of NaH2PO4 and Na2 HPO4; FisherScientific) pH 7.0, prepared with D2O (99. 9 % purity; Goss Scientific Instrument, GreatBaddon,UK) containing I mmol/L 3 - (trimethylsilyl)_propionate-2,2,3,3,3,-sodium (TMSP; 9 8 % purity; Goss Scientific Instrument, GreatBaddon,UK). Sodium 3-(trimethylsilyl)_propionate-2 , 2 , 3 , 3 , -san (TMSP; 98 % purity; Goss Scientific Instruments) was used as an internal reference for the chemical shift standard. Store in a desiccator at room temperature.

(2) Norell 5 mm NMR tube and cap.

5. Preparation of NMR lipophilic tissue extracts

(1) Deuterated NMR solvents: a 2 : 1 mixture of chloroform-d (CDCl3; 99. 8 % purity; Goss Scientific Instruments) and methanol-mountain (CD3OD; 99. 8 % purity; Goss Scientific Instruments) containing 0-5 mmol/l tetramethyl cinnamon (TMS; 99. 9 % purity; Goss Scientific Instruments) as internal reference for chemical shift standards. L tetramethylcinnamate (TMS; 99. 9 % pure; Goss Scientific Instruments) were used as internal references for the chemical shift standards. Store in a desiccator at room temperature.

(2) Norell 5 mm NMR tube and cap.

6. High resolution 1 H NMR

(1) 500 MHz or 600 MHz NMR spectrometers are available for metabolomics research.

(2) Conventional NMR probes, although the more sensitive NMR ultra-low temperature probes or cold probes are a definite advantage.

II. Methods 1. Sample collection and preparation

Preparation of metabolomic student samples involves three major steps. First, the samples are quickly collected and frozen to burst metabolism and maintain metabolite concentrations (see 1.1 and 1.2). Samples should then be stored at 80°C to prevent metabolic degradation (18). The second step is for tissue samples only and consists of mechanically breaking the tissue in the presence of a solvent and removing the protein (see 1.3). Although there are many extraction methods available, here we recommend the methanol-chloroform procedure that can separate polar and lipophilic metabolites into two fractions (19). The final step optimizes the solution for high-resolution NMR.For body fluid and polar tissue extracts, this step consists of adjusting the PH of the sample (to minimize chemical shift differences in the NMR resonance), addition of D2O (to provide a frequency lock for the NMR spectrometer), and addition of an NMR chemical shift standard (see 1.4). For lipophilic tissue extracts, this step requires the addition of deuterated solvent as a frequency lock and chemical shift standard (see 1.5).

1.1 Tissue Collection and Preservation

(1) Rapidly excise the tissue (ideally 100 m g wet weight, although the methods mentioned here have been successfully used for 2 0 m g of tissue) and immediately freeze into liquid nitrogen to fragment the metabolic reaction. Transfer the samples to labeled cryopreservation tubes and return to liquid nitrogen or place on dry ice (see Note 1).

(2) Samples should always be kept at 80°C (or lower) or in liquid nitrogen, dry ice, or refrigerator. Long-term storage in a refrigerator at 80°C is the most convenient method.

1.2 Collection and Preservation of Body Fluids
(1) Collect body fluids using an appropriate method (ideally 250-500 ML), e.g., with a syringe and needle.

(2) For blood, transfer each sample to a heparin-containing blood collection tube (see Note 3). Centrifuge to remove the cells, and then transfer the blood raffinose samples to a freezing tube and freeze in liquid nitrogen or dry ice.

(3) For urine, transfer each sample directly to a freezing tube and freeze in liquid nitrogen or dry ice.

(4) Samples should be stored at 80°C (or lower).

1.3 Combined Extraction of Polar and Lipophilic Metabolites from Tissues Using Methanol-Chloroform

(1) Label four glass vials (three small and one large) for each tissue sample.

(2) Remove the tissue samples from the refrigerator and place on dry ice. Weigh each sample quickly (ideally 100 mg), being careful not to let it melt.

(3) Add 4 mL/g (wet weight) of ice methanol and 0.85 mL/g ice deionized water to a large glass vial, add the first tissue sample, and homogenize with a homogenizer for 5 to 10 s. The first tissue sample will be removed from the refrigerator and placed on dry ice.

(4) Add 2 mL/g ice chloroform to the homogenized sample, vortex for 30 s, and place on ice. Repeat the process for all samples.

(5) Shake all samples on ice for l0 min using an orbital oscillator. All solutions must be single phase (see Note 4).

(6) Centrifuge the samples at 1800 g■ for 5 min at 4° C. Transfer each supernatant to a small glass vial.

(7) Add 2 mL/g of chloroform and 2 mL/g of deionized water to each sample (see Notes 4 and 5) and vortex for 30 s.

(8) Centrifuge the samples at 1800 g at 4°C for l 0 min. The solution will be dispersed into an upper methanol-water phase (polar metabolites) and a lower chloroform phase (lipophilic complex) with a thin layer of cellular debris in the center.

(9) Using two Hamilton syringes with metal needles, transfer the upper and lower layers of each sample to a small glass vial (see Note 6).

(10) Remove solvent from all samples using a rapid vacuum concentrator and store at -80°C until use.

1.4 Preparation of N M R Body Fluid and Polar Tissue Extracts

(1) For dried polar tissue extracts (from the methanol phase in 3.1.3), resuspend the sample in 550 uL of NM R buffer and vortex for 1Os (see Note 7). In this example, the NMR phosphate buffer concentration should be 100 mmol/L.

(2) For body fluids, mix each 300 fxL sample with 300 NMR Buffer and vortex mix for l0 s. Here, the starting NMR Phosphate Buffer concentration should be 200 mmol/L, and therefore the diluted concentration is 100 mmol/L.

(3) Centrifuge at 12 OOOg for 5 min at room temperature.

(4) Transfer 520uL to each labeled NMR tube.

1.5 Preparation of NMR lipophilic tissue extracts

(1) Resuspend the lipophilic tissue extract (from the dried chloroform phase in 3) in 550 uL of deuterated NMR solvent and vortex for 10 s. The extract should be prepared in the same way as the dried chloroform phase in 3, but with the same solvent as the dried chloroform phase.

(2) Centrifuge at IOOOg for 5 min at room temperature.

(3) Transfer 520 uL to each labeled NMR tube.

2. High Resolution1 H-NMR

NMR spectrometers are specialized analytical instruments that require considerable operator training prior to use. The methods described here assume that the operator has a basic working knowledge of NMR. A number of companies produce NMR systems, including Bmker BioSpin, JEOL, and Varian, and the methods described below work best with the Bruker system (Xif has the most experience with this system). Of course these methods can be used directly with other NMR systems. 2.1 and 2.2 describe
2.1 and 2.2 describe the detailed one-dimensional (I-D) and two-dimensional (2-D) NMR experiments used for environmental metabolomics, including the strengths and weaknesses of each method. General sample optimization strategies commonly used prior to data collection are described in 2.2 (see Note 8).

(1) Load a 5 mm NMR tube into the spectrometer.

(2) Set the sample temperature to 300 K and allow the solution to thermally equilibrate in the spectrometer for a few minutes (see Note 8).

(3) Rotate and match the NMR probe.

(4) Lock the spectrometer frequency to the deuterium resonance frequency generated by the NMR solvent (to D2O for analysis of body fluids or polar tissue extracts, or to CDCl3 for non-polar tissue extracts).

(5) Pad the sample by the automated method (see Note 9).

(6) Determine the pulse duration of a 360° cone angle peak in the NMR spectrum, from which the 60° and 90° cone angles can be easily calculated.

(7) Determine the frequency of the water resonance and center the spectrum at this frequency.

2.1 Criteria 1 Dimension ¹ H-NM R test

series, which is also dependent on the type of sample (see Remark 11).

(2) Process parameters: zero the M K data point; use an exponential line broadening of 0.5 Hz; use the Fourier transform to manually phase the spectrum (zero and first order corrections); manually calibrate the baseline with a polynomial function; calibrate the spectrum by setting the TM SF or TM S peak to 0.0 ppm.

(3) Record and process the data using the above parameters.

(4) Calculate the I-D horizon projection (p-JRES) of the 2-D spectrum.

3. Analysis of NMR metabolomics data

There are two related strategies for analyzing NMR metabolomic data. These two strategies can be categorized into traditional fingerprinting techniques (3.1) and new pattern methods (3.2).

3.2 Analysis of NM R Metabolomics Data Based on the Model Approach

(1) The modal approach is a more computationally challenging way to analyze NMR data and is based on the conversion of each NMR peak into a series of metabolites and their concentrations. Because the chemical shifts of the peaks in an NMR spectrum fluctuate depending on sample pH, temperature, and other "matrix effects," it is currently impossible to accurately identify and quantify metabolites in an I-D NMR spectrum using a fully automated method. Some human intervention is still required to guide the de-transformation procedure. A leading software program based on pattern-based analysis of I-D NMR metabolic data is the Chenomx NMR Suite (Chenomx, Edmonton, Canada), which now allows the identification and quantification of metabolites in body fluids using a library of 240 metabolic spectra.

(2) Once the spectra have been transformed, these data can be directly analyzed using the same multi-parameter statistical analysis methods as in 3.1 (e.g., PC A and PLS). The advantage of this modal approach is that the results of the multiparametric analysis are relatively more meaningful to the biologist because they consist of metabolite identification results rather than unidentified peaks. This information is clearly more useful and can be used for biomarker discovery, identification of the role of specific metabolic pathways, and non-instrument-dependent data archiving.

Caveat

(1) When cutting many samples, the fastest way is to put the cut samples directly into a freezing tube and then immediately into liquid nitrogen.(2) Samples can be stored for months. For example, plasma samples that were quickly frozen and maintained at 80°C were stored for 9 months without significant metabolic changes (18).(3) Do not use EDTA as an anticoagulant; it produces interfering peaks in the NMR spectrum.(4) B lig h and Dyer ( 2 3 ) determined that the volumes of solutions should follow the following ratios: monophasic solution for metabolite extraction: 2 : 1 : 0.8 methanol-chloroform-zK; biphasic solution for isolation of components: 2 : 1 : 1.8 methanol-chloroform-water. The total volume of water also includes the volume of water in the tissue plus the added volume of deionized water.(5) For better distribution of polar lipids (phospholipids) into the chloroform layer, this can be achieved by using 0 . 8 % K C l aqueous solution instead of water to increase the polarity of the methanol phase.(6) All sample handling and preservation must be carried out in glass containers because chloroform can dissolve compounds in plastic tubes and pipette tips, thereby contaminating the N M R spectrum .(7) If the extract is from a methanol-chloroform extraction only, vortex mix and transfer the sample from the glass vial to an Eppendorf tube.(8) Ideally, a consistent extraction method, N M R buffer, and volume of solution in the N M R tube should be used for all samples in a given metabolomics study. In this case, many of the optimization procedures for each sample in the NMR spectrometer can be reduced. In detail, only steps 1, 2, 4, and 5 are required, and the optimized mass can be calibrated with the standard in Note 9.(9) The quality of the padding, as measured by the full width of the half peak of T M S P or T M S, should be below 2. 0 H z (before the toe-cut method results in any line broadening).(10) For samples containing dosage of ~100% deuterated solvent (e.g., from tissue extraction), basic water presaturation can be used to suppress residual water resonance. However, for samples of body fluids containing high percentages of H 2O, more robust water suppression techniques must be used.(11) If the standard I-D 1 H -N M R spectrum exhibits broad resonance frequencies (from high molecular mass macromolecules and dynamically inhibited compounds), then a corrected pulse sequence can be used to more favor the observation of free, low molecular mass metabolites. This is often necessary in the analysis of plasma samples. The required pulse sequence is called the 1 H Carr-Purcell-Meiboom-Gill (C P M G ) spin-echo N M R sequence.

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