This protocol describes the method for comprehensive, unbiased molecular characterization of a single sample using both mass spectrometry, using metabolomics, proteomics and lipidomics, and nuclear magnetic resonance spectroscopy platforms. The significance of this approach is that we can characterize a biological system downstream of the genome, through identifying different types of molecules, allowing us to infer metabolic and decomposition pathways. Sequential Extraction Technique, allow extraction of bioavailable, polar compounds using water, followed by MPLEx to capture any additional polar compounds or metabolites as well as proteins and lipids from a single sample.
Visual demonstration of this method is critical because it guides the scientist through separating the three layers during the second extraction step, and splitting the extracts between different analysis instruments. Integrative multi-omic analyses empowers more effective investigations and complete understanding of complex biological systems. This robust method extracts a wide diversity of molecules and is applicable to diverse sample types.
Begin this procedure with collection and preparation of the samples as described in the text protocol. Using an ethanol washed, stainless steel utensil, aliquot 50 milligrams of each of the dried samples into individual two milliliter glass tubes. Add one milliliter of distilled, degassed water to each sample.
Then cap the vials and shake for two hours on a shaker table. Centrifuge the samples at 15 thousand times gravity for 30 minutes. And then allow the solutions to stand at room temperature for 20 minutes.
Decant and save the supernatant from each sample. Conduct an MPLEx Extraction on the now water extracted residues by repeating these extraction steps. Except to substitute a minus 20 degree celsius four to three chloroform to methanol mixture for the distilled, degassed water.
Carefully separate the two resulting solvent layers, which will be visually distinguishable through removing the top layer by careful pipetting. Dry down the lower non polar layer in the freeze dryer. As soon as it is dry, add five microliters of chloroform and 195 microliters of methanol if analyzing within the next couple of days.
To improve the electrospray ionization efficiency for fourier-transform ion cyclotron resonance mass spectrometry, abbreviated FTICR-MS by direct injection. Dilute the chloroform extract one to one in methanol, and the water extract two to one in methanol. Calibrate the FTICR spectrometer by directly injecting 100 microliters of a tuning solution, spanning a mass range of approximately 100 to 1, 300 daltons, into the FTICR-MS.
Direct inject 100 microliters of the Suwannee River Fulvic Acid standard to the electrospray ionization source. Coupled to the FTICR spectrometer through a syringe pump set to a Flow Rate of 3.0 microliters per minute. Set the needle voltage to positive 4.4 kilovolts.
Queue one to 100 mass to charge ratio and the glass capillary at 180 degrees celsius. Inspect the resulting spectra using the analysis software to confirm the quality of the data. Now, introduce 100 microliters of each extract via direct injection to the electrospray ionization source, coupled to the FTICR spectrometer through a syringe pump set to a Flow Rate of 3.0 microliters per minute.
Set the parameters as before. Adjust the ion accumulation time for each sample or group of samples to account for variation in carbon concentration. Collect 144 scans for each sample, average the scans and then conduct an internal calibration using a homologous CH2 series.
Dry the extracts using a concentrator and save the remainder of the extracts for subsequent gas chromatography mass spectrometry, liquid chromatography mass spectrometry and nuclear magnetic resonance spectroscopy analysis. To prepare for GC-MS, first prepare blank control samples as detailed in the text protocol. To protect carbinol groups add 20 microliters of 30 milligrams per milliliter methoxamine hydrochloride in Paradyne to each of the samples, including the methanol extracts, the water extracts, the blanks and the FAME calibration samples.
Seal the vials with caps. Vortex the extracts for 20 seconds, then sonicate the extracts for 60 seconds. Centrifuge the extracts at 37 degrees celsius for 90 minutes at 100 times gravity.
Add 80 microliters of MSTFA with 1%trimethylchlorosilane to each sample. After vortexing and sonicating the extracts as before, centrifuge the extracts again at 37 degrees celsius for 30 minutes at 100 times gravity. After cooling extracts to room temperature, transfer into GC-MS autosampler vials.
Proceed to GC-MS analysis and data processing as described in the text protocol. To prepare for liquid state NMR analysis, dilute the remainder of the water extracts by 10%with a five millimolar DSS internal standard. Transfer the mixture into a high quality, three millimeter outer diameter borosilicate glass NMR tube.
Proceed to NMR analysis and data processing as described in the text protocol. To perform the LC-MS lipidomics analysis, inject 10 microliters of each extract into an ultra performance liquid chromatography system, coupled to an Orbitrap mass spectrometer using a reversed phase charged surface hybrid column. Set a 34 minute gradient as listed in the text protocol at a Flow Rate of 250 microliters per minute.
Use both negative and positive ionization modes with higher energy collision dissociation and collision-induced dissociation. To perform proteomics analysis, first extract proteins according to the MPLEx protocol for the remainder of the methanol phase as outlined in the text protocol. Peat was compared with depth in the S1 bog at the Spruce and Peatlands Response Under Changing Environments or SPRUCE site in Minnesota, USA.
3, 312 enzymes were identified in the proteomics analysis. An analysis of the enzyme activities with depth reveals that the number of enzymes declines sharply between 15 centimeters and 45 centimeters in the SPRUCE Bog. To show how sites may vary in metabolite and enzyme activities, SPRUCE results are compared to those from a Permafrost Bog and Fen in northern Sweden.
Overall 67, 040 metabolites were identified in all of the SPRUCE Peat samples from the combination of FTICR-MS, NMR, GC-MS and LC-MS analyses. Shown here is the relative fraction of different chemical classes identified via various techniques in the different depths. While amino acids and sugars decline with depth, this is not observed for lipids.
By cross validating metabolites identified in all analyses against the KEGG Database it was determined that the identified compounds are involved in common metabolic pathways such as tricarboxylic acid cycle, glycolysis and sugar metabolism. Throughout this procedure it is critical to be aware of potential sources of contamination. Beginning at sample collection through analysis, samples should not come in contact with pegged plastics which can negatively affect ionization.
Many of the solvents used during the extraction procedure are hazardous or flammable. Always wear appropriate PPE to avoid skin and eye contact, as well as to avoid contaminating samples.
