マルチ弔土壌試料の分析のための MPLEx プロトコル

This method is used in the proteomics, metabolomics, and lipidomics fields by helping obtain comprehensive information about the molecular changes taking place in soil-associated microbial communities due to environmental perturbations. The main advantage of this technique is that obtaining multiple analytes from a single soil sample increases precision and accuracy while decreasing sample preparation time and effort. To begin, for each 20-gram soil sample, weigh 10 grams into two separate 50-milliliter methanol/chloroform-compatible tubes.

Use plastic tubes made from PTFE, as chloroform will leach most plastics, contaminating the samples. Add approximately 10 milliliters of chloroform-washed stainless steel and garnet beads to each tube. Then, while on ice, add four milliliters of cold ultrapure water to each tube, and transfer the samples in an ice bucket into a fume hood.

Using a 25-milliliter glass serological pipette, quickly add 20 milliliters of ice-cold two to one chloroform to methanol volume/volume to the tubes. Tighten the lids and vortex the soil mixture into solution. The chloroform-methanol helps break down the cell wall of prokaryotes and also inactivates enzymatic activities.

Next, attach the tubes to 50-milliliter tube vortex attachments, and horizontally vortex for 10 minutes at four degrees Celsius inside a fridge if possible. Then, place the samples inside a negative 80 degree Celsius freezer for approximately 15 minutes in order to cool them down completely. Then, using a probe sonicator inside a fume hood, sonicate each sample with a six millimeter, 1/4-inch probe at 60%amplitude for 30 seconds on ice.

Following sonication, place the samples in a negative 80 degree Celsius freezer, as sonicating can generate a lot of heat. After repeating the horizontal vortexing, sonication, and freezing steps, centrifuge the samples at 4, 000 times gravity and four degrees Celsius for five minutes. At this point, the sample will be separated into the upper metabolite layer, the protein interlayer, and the lower lipid layer.

Place the samples on ice. Then, inside a fume hood, use a 10-milliliter glass serological pipette to transfer the upper metabolite layers from the two 50-milliliter tubes into one large glass vial. Now, slightly tilt the 50-milliliter tube to release the protein interlayer so that it is free-floating upon the lower lipid layer.

Then, using a clean stainless steel flat-head lab spatula, carefully scoop both of the protein interlayers, and place them together into one new 50-milliliter tube. Using a 25-milliliter glass serological pipette, transfer the lower lipid layers into one large glass vial. Add 20 milliliters of ice-cold methanol to the two tubes with debris and to the one tube with the protein interlayer.

Briefly vortex the tubes, and centrifuge the debris pellets and protein interlayer at 4, 000 times gravity and four degrees Celsius for five minutes. Then, decant the methanol into a hazardous waste container inside a fume hood. Freeze the protein and debris pellets in liquid nitrogen, and dry them in a lyophilizer for about two hours.

Add 20 milliliters of protein solubilization buffer in 50-millimolar tris buffer, pH 8.0, to each of the debris pellets and 10 milliliters to the protein interlayer tube. In the fume hood, probe sonicate the samples at 20%amplitude for 30 seconds to bring them into solution. Vortex the samples for two minutes.

Then, place the protein interlayer sample into a lab tube rotator at 50 degrees Celsius for 30 minutes to solubilize the protein. Horizontally vortex the debris samples for an additional 10 minutes to lyse any remaining intact cells. Then, rotate with the protein interlayer samples for the remaining 20 minutes.

Centrifuge all of the samples at 4, 500 times gravity at room temperature for 10 minutes, and collect the supernatant from each tube per sample into two 50-milliliter tubes. Add 10 milliliters of solubilization buffer to the protein interlayer pellet. Then, sonicate and vortex the pellet back into solution.

Following centrifugation, equally combine the supernatants into the two 50-milliliter tubes with the debris pellet supernatants, and centrifuge the tubes in a fixed-angle bucket rotor at 8, 000 times gravity and four degrees Celsius for 10 minutes in order to pellet any remaining debris. Divide the supernatants into two 50-milliliter tubes. Then, using a 10-milliliter glass serological pipette, add 7.5 milliliters of TCA to each tube.

Vortex the samples into solution. Then, place the samples in a negative 20 degree Celsius freezer for two hours to overnight. To pellet the precipitated protein, centrifuge the sample at 4, 500 times gravity and four degrees Celsius for 10 minutes, and decant the supernatant into waste.

Add 10 milliliters of 100%ice-cold acetone to each protein pellet. Vortex the tubes, and combine like pellets into one tube. Centrifuge and then decant the acetone.

Using a smaller volume of acetone, wash the pellet twice, and transfer the suspension to a 1.5 or two-milliliter tube for the final spin. Decant the supernatant into waste, and allow the pellet to dry inverted on a paper wipe in a fume hood for approximately 20 minutes. Depending on the size of the pellet, add 100 to 200 microliters of the protein solubilization buffer.

Sonicate and vortex the pellet into solution. Note that the sample may be viscous due to humic substances precipitating along with the protein, which will be removed with a subsequent centrifugation. Finally, shake the sample in an incubator or shaker at 300 rpm and 40 degrees Celsius for 30 minutes, to solubilize the protein into solution before proceeding to protein digestion or snap freezing for future processing.

Using the MPLEx protocol demonstrated in this video, 3, 376 peptides, 105 lipids, and 102 polar metabolites were extracted from Kansas native prairie soil, and the protocol's extraction of proteins is compared to protein extraction kits and SDS extractions using reverse-phase LC-MS/MS. When initially comparing three replicates of the commercial protein extraction kit and SDS methods, only 12%of the peptides overlapped between the two techniques, showing the complexity of the microbial community and different extraction effects. Upon comparison of MPLEx with a commercial protein extraction kit and SDS extracts, about 38%of the peptides observed using the MPLEx method were also detected when using the SDS and/or commercial kit extractions.

Considering the number of species in the Kansas soil bacterial community and its metagenome, the overlap of peptide identifications is reasonable. As shown here, the broad applicability of the MPLEx approach has been previously evaluated using the archaeon S.acidocaldarius, a unicyanobacterial consortium, mouse brain cortex tissue, human urine, and leaves from A.thaliana. In all cases, the number of proteins observed from the MPLEx method was similar to that for the controls, indicating its utility for many different sample types.

After watching this video, you should have a good understanding of how to obtain proteins, metabolites, and lipids from a single soil sample. While attempting this procedure, it's important to be flexible, as all soil types are different. For example, the size of the protein interlayer will vary, depending on the soil biomass and humic substances present in the soil.

Don't forget that working with chloroform can be extremely hazardous, and precautions such as working in a fume hood should always be taken while attempting this procedure.