A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample

The overall goal of this method is to recover and analyze all the major molecular entities, including polar and semi-polar metabolites, lipids, and proteins from a single sample using a simple fractionated methyl-tributyl ether extraction. Generally, scientists find it difficult to analyze multiple compound classes from a single sample. Using the MTBE extraction that we introduce here, you can extract and analyze multiple compound classes, like lipids, metabolites, and proteins from a single sample.

The main advantage of this technique is that it can robustly extract all molecular compound classes from a small amount of single sample aliquot. This method helps answer fundamental questions in systems biology, since it provides the experimental basis for multiomics analysis, providing fractions which can be used for analysis by proteomics, lipidomics, and metabolomics. The following protocol is demonstrated using Arabidopsis leaf tissue.

Arabidopsis are small flowering plants in the Brassicaceae family, and are related to cabbage. Begin this procedure by pre-cooling the tissue homogenizer tube holders in liquid nitrogen for at least 10 minutes. Remove the samples from liquid nitrogen and place them in the tube holders.

And then remove the tube holders from liquid nitrogen. Quickly put the tube holders in the tissue homogenizer, and set the homogenizer to grind the biological material into a fine and homogenous powder. For leaves, use 20 hertz for one minute.

The homogenization time and speed may be varied depending on the tissue. Be sure you homogenize until the resulting sample is a very fine powder. And the sample should be kept frozen at every step of homogenization.

Homogenize the samples, then remove the biological samples from the tube holders. And if they won't be used right away, place them into a minus 80 degrees Celsius freezer until further extraction. Label four two-milliliter round-bottom safe-lock microcentrifuge tubes with the sample number.

Pre-cool the tubes and some spatulas by submerging them in liquid nitrogen. Once the tubes and spatulas are cool, place one of the tubes on an analytical balance, and use a spatula to aliquot 25 milligrams of tissue powder into the microcentrifuge tube. Record the exact weight for each sample, then immediately place the aliquotic samples in liquid nitrogen.

Perform this step quickly to avoid defrosting the plant material. Store the aliquoted samples at minus 80 degrees Celsius until further extraction. To prepare for the extraction, pre-cool the methyl tert-butyl ether, or MTBE methanol extraction mixture, prepared as described in the accompanying document, in a minus 20 degrees Celsius freezer.

Take out the aliquoted samples, and add one milliliter of the pre-cooled extraction mixture to each sample tube. Perform this step quickly. MTBE has a low viscosity and can drip out of the pipette tip.

Mix each sample immediately on a vortex mixer until the tissue is well homogenized within the extraction mixture. Keep the tubes in a rack on the bench until all the samples have been extracted. This step is critical.

Here we precipitate proteins and inactivate their enzymatic activities. Incubate all the samples on an orbital shaker at 100 RPM for 45 minutes at four degrees Celsius. Then sonicate the samples for 15 minutes in an ice-cold sonication bath.

Next, to fractionate by phase separation, add 650 microliters of a three-to-one solution of water and methanol to each sample tube. Then mix by vortexing for one minute. Centrifuge the samples at a speed of 20, 000 times g for five minutes at four degrees Celsius.

After this step, handle the tubes with care to avoid mixing of the two liquid phases and avoid disrupting the precipitated pellet. At this stage, there are two admissible liquid phases with a solid pellet in the bottom of the tube. The non-polar upper phase contains lipids.

The lower aqueous phase contains polar and semi-polar metabolites. The pellet contains proteins, starches, and the cell wall. Transfer 500 microliters of the solvent from the upper lipid-containing phase into a labeled 1.5 milliliter microcentrifuge tube.

Then, using a 200 microliter pipette, carefully remove the remaining lipid phase and discard it. Next, transfer 400 microliters of the solvent from the lower phase into a labeled 1.5 milliliter microcentrifuge tube. Transfer an additional aliquot of 200 microliters to a micro-fuge tube to perform further analysis, such as gas chromatography-based metabolite analysis.

Remove and discard the remainder of the aqueous phase by pipetting off the excess volume. Then, to wash the obtained protein-starch-cell wall pellet, add 500 microliters of methanol and then vortex it for one minute. Centrifuge the samples at a speed of 10, 000 times g for five minutes at four degrees Celsius.

Evaporate the solvent from the lipid samples using a nitrogen flow evaporator to avoid oxidative modifications of the lipids. The resulting dried samples should be analyzed immediately. Evaporate the solvent from the aqueous samples overnight in a vacuum concentrator without heating.

The dried aqueous samples can be stored for several weeks at minus 80 degrees Celsius before analysis. Re-suspend the dried lipid fractions in 400 microliters of a solution of seven-to-three acetonitrile to 2-propanol. Transfer sufficient liquid to glass vials and cap tightly.

Then, put the glass vials in a cooled auto-sampler at four degrees Celsius. Inject two microliters per sample and separate the lipids on a reversed phase C8 column held at 60 degrees Celsius using a UPLC system running at a flow-rate of 400 microliters per minute. Use the mobile phases described in Table 1 of the accompanying document for the chromatographic separation.

Acquire the mass spectra in positive and negative ionization mode using a suitable MS instrument covering the mass range between 150 and 1500 charge-to-mass ratio. Re-suspend the polar phase in 200 microliters of a solution of one-to-one UPLC-grade methanol to water. Transfer sufficient liquid to glass vials and cap tightly.

Then, put the glass vials in a cooled auto-sampler, four degrees Celsius. Inject two microliters from each sample and separate the metabolites on an RP C-18 column held at 40 degrees Celsius using a UPLC system running at a flow-rate of 400 microliters per minute. Use the mobile phases for chromatographic separation with parameters given in Table 2 of the accompanying document.

Acquire full scan mass spectra in positive and negative ionization mode using a suitable mass spectrometer covering a mass range between 50 and 1500 mass-to-charge ratio. Finally, perform protein extraction, digestion, and analysis, as described in the accompanying document. 25 milligrams of Arabidopsis leaf tissue were harvested, ground, and extracted before subjecting it to three analytical UPLC-MS platforms.

The polar and semi-polar primary and secondary metabolites were analyzed from the polar phase by reversed-phase C-18 UPLC-MS. Base peak chromatograms of lipids, shown in the upper panel, and semi-polar metabolites, shown in the lower panel were analyzed in positive ionization mode. The pie chart in the upper right corner of each chromatogram shows the number of identified lipids and metabolites assigned to different chemical classes.

For example, 58 different lipids were assigned to the triacylglyceride group, indicated in the upper chart as TAG. More hydrophilic metabolites from this fraction, such as sugars and polar amino acids, which do not show good retention on the reversed-phase material, can be analyzed by other analytical methods, such as GCMS, or hydrophilic interaction liquid chromatography. The proteins that were retrieved from the extraction were in solution digested, and analyzed using shotgun LCMS.

The pie chart shown in the upper right corner indicates the number of identified proteins assigned to different biological processes. For example, 268 proteins were assigned to the localization category. In summary, more than 200 lipid species, 50 annotated semi-polar metabolites, and several thousand proteins can be routinely identified from samples like the one used in this example.

Additionally, the method showed broad applicability using different tissues, organs, and cell-culture material. After having watched this video, you should have a good understanding of how to extract and analyze most essential compounds from a single sample. While attempting this procedure, it is important to keep all samples frozen, grind the material properly, and use analytical-grade solvents and chemicals.

Following this procedure, all analytical methods can be employed to determine the molecular composition of the extracted samples.