Nuclear magnetic resonance spectroscopy offers several distinct advantages for environmental metabolomics research, but is relatively insensitive in this procedure. A filtration method for the concentration and extraction of whole community metabolites from platonic systems suitable for NMR is reported. This is accomplished by first specially preparing filters for sample collection by removing extractables.
The second step of the procedure is to filter the sample, either natural or experimental onto the filters. The third step of the procedure is to lifealize the sample material and extract metabolites. The final step of the procedure is to analyze the samples using nuclear magnetic resonance spectroscopy and integrate the data as appropriate.
Ultimately, results can be obtained that show metabolic changes in natural samples across environmental gradients or in response to experimental manipulations through metabolomics and transams approaches. The main advantage of this technique over existing methods, such as centrifugation, is that the total community can be sampled and larger sample volumes are possible for low density samples. This method can help answer key question in the environmental metal mix field, such as how micro growth respond to environmental changes, Demonstrating the procedure will be stage and From my laboratory.
Begin this Protocol by placing filters in a clean 500 milliliter Pyrex speaker using tweezers. Cover the filter with distilled water to pre rinse swirl well to prevent the filters from sticking to each other. Pour off the water and repeat the rinse twice.
After the final rinse, add 300 milliliters of Milli Q water to the beaker and autoclave the filters. Once the filters have been autoclaved. Pour off the milli Q water and triple rinse the filters as before except use Milli Q Water.
Place the individual filters on a clean, dry surface, such as aluminum foil with tweezers dry at a reasonable temperature. For example, at 37 degrees or air dry, the filters are now ready to use for demonstrating this protocol. A millipore stainless steel three place filter manifold equipped with 25 millimeter microanalysis Filter columns with glass supports and a mechanical pump is employed using aseptic technique.
Place a single 25 millimeter filter on the filter column base. Apply the column and clamp the apparatus together. Load 15 milliliters of sample here obtained from a high nutrient microcosm to the column.
Open the stop valve on the filter manifold and turn on the pump filter under gentle pressure to minimize cell breakage. For lower density samples, successive additions of water may be necessary. Taking care not to let the filter go dry for an extended period of time between additions of water.
Two, reduce residual paramagnetic salts from marine samples and optional freshwater rinse may be performed. Once filtering is finished, turn off the pump and leave the valve open so there is still negative pressure under the filter. Remove the clamp and filter column with one hand.
Use clean tweezers to take hold of the filter. Fold the filter across itself but do not crease with the other hand. Use the lip of a sterile two milliliter micro centrifuge tube to hold the filter down.
Place the filter into the two milliliter tube and release it so that it opens With the sample side facing inwards. Freeze in Liquid nitrogen immediately to extract the aqueous Soluble metabolites. First lifealize the samples overnight or for at least 10 hours after lyophilization, add a Stainless steel crusher to each tube thin.
Add 750 microliters of standardized potassium phosphate buffer and deuterium oxide with DSS standard. Sonicate the samples For five minutes at four degrees in a water sonicate to remove the cell material from the filter. Remove the filters with clean tweezers.
Disrupt the cells using a mill crusher at 1600 RPM for five minutes. Next, incubate at 65 degrees Celsius. Was shaking on a benchtop shaker for 15 minutes.
Following incubation, remove the metal crusher with clean tweezers. Centrifuge the sample at 13, 000 G for five minutes. After centrifugation, draw off the SUP natin and transfer directly to an NMR tube for NM r spectroscopy.
Load The sample into the NMR spectrometer here. Samples are run on a Bruker DRX 500 spectrometer equipped with TXI probe with triple axis gradient operating at 298 Kelvin. Lock, tune and shim the magnet.
Obtain 1D proton NMR Spectra using the top spin interface and previously described methods or utilize other spectrometer interface software as needed. Here Spectra were acquired with suppression of residual water signals by the Watergate pulse sequence with a repetition time of 1.2 seconds. In the present example, 128 transients were collected for each sample.
Transfer the NMR data directories to a personal computer with NMR pipe software installed. Process the raw data, set the DSS signal as zero PPM reference, and then manually phase the spectra. Digitize the spectral data by bin here using the publicly available FT two B package from the eCommerce website.
Other software utilities such as RNMR or Atomics can also be used for bin.Bin. Data can be normalized to total or DSS signal to show proportional signal changes between samples. Output data can now be used for downstream statistical analysis, such as principle components analysis or PCA using free software packages like R shown.
Here is an example of proton NMR Spectra obtained using the demonstrated procedure. The spectra are from two time points of a microcosm experiment and show clear differences due to algal metabolic activities. The day four spectrum shows considerable abundance of peaks, particularly in the three to 4:00 PM range as compared to the day one sample.
These peaks can be attributed to sugars produced by blooming diatoms within the microcosm. In a similar experiment, A PCA score plot derived from bend in Mr.Spectra showing the first two principle components was used to compare the growth of natural plankton communities in artificial and natural seawater. This statistical approach reduces multidimensional data into simpler format here, two dimensional and simply reveal structure in the underlying data points close on.
Each or both axes are more similar here. Clear metabolic differences between the two treatments can be seen. The data from this experiment was used to perform multi omics analysis.
Combining NMR with genomic data community composition based on denaturing gradient gel electrophoresis of 18 s and 16 S-R-R-N-A genes also show distinct microbial community patterns between natural and artificial seawater microcosms. Such correspondence between metabolome and genome from natural systems demonstrates the usefulness of this approach. While attempting this procedure, it is important to remember to use a subject technique to minimize the contamination Following this procedure.
Other methods like creation analysis with omics levels can be performed in order to answer additional questions like how microbiome correlate with certain metabolites.
