The authors would like to thank Nathan Johnson for his assistance in preparing the figures. This research was supported by the Pan-omics Program that is funded by the U.S. Department of Energy&#39;s Office of Biological and Environmental Research (Genomic Science Program), the Microbiomes in Transition (MinT) Laboratory Directed Research Development Initiative at the Pacific Northwest National Laboratory, as well as the National Institutes of Health National Institute of Environmental Health Sciences (R01 ES022190) and NIH (P42 ES027704). KEBJ would like to thank R21 HD084788 for financial support to develop and validate novel multi-omic extraction techniques. This work was performed in the W. R. Wiley Environmental Molecular Sciences Laboratory (EMSL), a DOE national scientific user facility at the Pacific Northwest National Laboratory (PNNL). PNNL is a multi-program national laboratory operated by Battelle for the DOE under Contract DE-AC06-76RL01830.

NOTE: Very wet soils can be lyophilized prior to extraction without detriment to the effectiveness of the extraction. Wet soil can also be used but should be considered when adding reagents at specific ratios.
NOTE: It is recommended to use 20 g of dry soil weight per extraction, which must be split between two 50 mL tubes (maximum of 10 g soil per 50 mL tube). Extractions can be scaled up or down dependent upon available sample.
NOTE: Dry soil samples can be sieved through a 3 mm screen in order to homogenize and remove small roots and rocks. Do not sieve wet soil samples, as the sample will get stuck in the screen.
1. Soil Cell Lysis and Extraction of Metabolites, Proteins, and Lipids (Timing ~1 d)
<ol>
	<li>Per 20 g soil sample, weigh 10 g into two separate 50 mL tubes that are methanol/chloroform compatible. Be extremely careful with the tubes chosen, as chloroform will leach most plastics, contaminating the samples. Use glass whenever possible or plastic tubes made from polypropylene or polytetrafluoroethylene (PTFE). 
	NOTE: Keep the soil on ice and as cool as possible during the initial weighing and homogenization steps.</li>
	<li>Add 10 mL of chloroform-washed stainless steel and garnet beads to each tube. On ice, add 4 mL of cold ultrapure water (see Table of Materials for purification system) to each tube (one sample split between two tubes) and transfer the samples in an ice bucket into a fume hood.</li>
	<li>Using a 25 mL glass serological pipette, quickly add 20 mL of ice-cold 2:1 chloroform:methanol (v/v). 
	Caution: Chloroform:methanol has acute potential health effects: skin irritation, possible chemical burns, and irritation to the respiratory system. It may affect the kidneys, liver, and heart. Wear suitable protective glasses, clothing, and gloves, and always work in a fume hood.</li>
	<li>Tighten the lids and vortex into solution. 2:1 choloroform:methanol helps break down the cell wall of prokaryotes and also inactivates enzymatic activities.</li>
	<li>Attach the tubes to 50 mL tube vortex attachments and horizontally vortex for 10 min at 4 &#176;C inside a fridge if possible. Then, place the samples inside a -80 &#176;C freezer for ~15 min in order to cool them down completely.</li>
	<li>Using a probe sonicator inside a fume hood, sonicate each sample with a 6 mm (1/4&#8221;) probe at amplitude 60% for 30 s each on ice. 
	Caution: It is recommended to use a sound abating enclosure and ear protection while sonicating. High voltage is present in the power supply and high frequency cable. Avoid touching the bottom or sides of the sample vessel with an active probe; it may crack or melt the plastic.</li>
	<li>Place the samples in the -80 &#176;C freezer for ~15 min, as sonicating can generate a lot of heat.</li>
	<li>Repeat steps 1.5 - 1.7. 
	NOTE: Make sure the samples stay cold throughout the lysis procedure.</li>
	<li>Centrifuge the samples at 4,000 x g for 5 min, 4 &#176;C. At this point, the sample will be separated into the upper metabolite layer, the protein interlayer, and the lower lipid layer (and remaining debris pellet). See Figure 1.</li>
	<li>Place the samples on ice in an ice bucket and, inside a fume hood and using a 10 mL glass serological pipette, remove the upper metabolite layers from the two 50 mL tubes into one large glass vial. 
	NOTE: Avoid aspirating any of the protein layer into the pipette; leave a small layer of metabolite if necessary.</li>
	<li>Slightly tilt the 50 mL tube to release the protein interlayer so that it is free floating upon the lower lipid layer. Using a clean stainless steel flat-head lab spatula, carefully scoop both of the protein interlayers and place them together into one new 50 mL tube.</li>
	<li>Using a 25 mL glass serological pipette, remove the lower lipid layers into one large glass vial. 
	NOTE: Try to avoid aspiring soil particles; however, some soil debris and particles aspired along with the lipids will be removed at a later time.</li>
	<li>Place breathable membranes over the top of the metabolite and lipid glass vials and dry in a vacuum concentrator until dryness (lipids ~4 - 5 h, metabolites overnight).</li>
	<li>To the protein sample tube and the remaining debris pellet tubes, add 20 mL of ice-cold methanol to each and vortex. This is done to the debris pellets to rinse off chloroform before attempting to solubilize any possible remaining protein out of the debris before tossing it.</li>
	<li>Centrifuge the debris pellets and protein interlayer at 4,000 x g for 5 min, 4 &#176;C, then decant the methanol into a hazardous waste container inside a fume hood.</li>
	<li>Freeze the protein and debris pellets in liquid nitrogen and dry in a lyophilizer (with a collector temperature capable of -105 &#176;C due to methanol having a very low freezing point of -98 &#176;C) for ~2 hours. 
	NOTE: Do not over-dry the pellets, as they will be more difficult to solubilize.</li>
	<li>Add 10 mL of protein solubilization buffer (4% SDS, 100mM DTT (DL-dithiothreitol) in 50mM tris buffer, pH 8.0; see Supplementary Reagent Set-up) to the protein interlayer tube and 20 mL to each of the debris pellets. 
	Caution: SDS causes acute toxicity and is flammable. It is a skin, eye, and airway irritant. Wear gloves and safety glasses.</li>
	<li>In the fume hood, probe sonicate the samples at 20% amplitude for 30 s to bring them into solution. Vortex for 2 min.</li>
	<li>Place the protein interlayer sample into a lab tube rotator for 30 min at 300 rpm, 50 &#176;C, to solubilize the protein.</li>
	<li>Horizontally vortex the debris samples for an additional 10 min to lyse any remaining intact cells, then rotate with the protein interlayer samples for the remaining 20 min.</li>
	<li>Centrifuge all of the samples at 4,500 x g for 10 min, room temperature (RT), and collect the supernatant from each tube per sample into two 50 mL tubes.</li>
	<li>Add 10 mL of solubilization buffer to the protein interlayer pellet, then sonicate and vortex back into solution and centrifuge as before.</li>
	<li>Combine the supernatants into the two 50 mL tubes equally and centrifuge at 8,000 x g for 10 min, 4 &#176;C, in a fixed angle bucket rotor. 
	NOTE: A final centrifugation is necessary to remove excessive contaminating humic substances.</li>
	<li>Decant the supernatants into two 50 mL tubes so that there is 30 mL in each. Then, using a 10 mL glass serological pipette, add 7.5 mL of TCA (trichloroacetic acid) to each tube. Vortex into solution. This makes 20% TCA in each 30 mL sample (adjust accordingly), 
	Caution: TCA is caustic, toxic and may cause skin burns. Wear gloves and safety glasses.</li>
	<li>Place the samples in a -20 &#176;C freezer for 2 h to overnight. 
	NOTE: Proteins will typically precipitate within 1 h but can be left overnight (up to 18 h). 
	NOTE: Do not let the TCA extraction go longer than 18 h due to possible acid hydrolysis of the protein. If the sample is frozen, thaw it on ice; do not let the sample warm up past thaw.</li>
	<li>To pellet the precipitated protein, centrifuge the sample at 4,500 x g for 10 min, 4 &#176;C and decant the supernatant into waste.</li>
	<li>Add 10 mL of 100% ice-cold acetone to each protein pellet, vortex and combine like pellets into one tube.</li>
	<li>Centrifuge the tube containing the combined pellet (using a balance), and then decant the supernatant into waste. 
	Caution: Acetone may cause respiratory tract and skin and eye irritation, and is a flammable liquid and vapor. Wear safety glasses gloves and a lab coat, work in a fume hood.</li>
	<li>Wash the pellet twice&#160;using 1.5mL of acetone&#160;and finally transferring to a 2 mL tube for the final spin at 10,000 x g for 5 min.</li>
	<li>Decant the supernatant into waste and allow the pellet to dry inverted on a paper wipe in a fume hood for ~20 min or under a nitrogen stream until the pellet slightly begins to crack.</li>
	<li>Add 100 - 200 &#181;L of the protein solubilization buffer, depending on the size of the pellet. 
	NOTE: Keep the volume of solubilization buffer added to the sample as low as possible. Subsequent Filter Aided Sample Preparation (FASP) can only use up to 50 &#956;L of solubilized sample per column; any more must be split into multiple FASP columns.</li>
	<li>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.</li>
	<li>Shake the sample in an incubator/shaker for 30 min at 300 rpm, 40 &#176;C, to solubilize the protein into solution and proceed to protein digestion.</li>
	<li>Snap freeze the sample in liquid nitrogen and store in a -80 &#176;C freezer until ready for protein digestion.</li>
</ol>
2. Lipid Preparation (Timing ~20 min)
<ol>
	<li>Once the lipid samples are dry, add 200 &#956;L of 2:1 chloroform:methanol to the vial, vortex into solution and transfer into a 1.5 mL polypropylene tube, add an additional 200 &#956;L to the glass vial, vortex and pipette the remaining lipids and transfer to the tube.</li>
	<li>Centrifuge the debris out of the sample at 12,000 x g for 5 min at 4 &#176;C.</li>
	<li>Transfer the supernatant into a glass lipid vial and store at -20 &#176;C until LC-MS/MS analysis (additional details for LC-MS/MS in Supplementary Methods).</li>
	<li>If the samples cannot be analyzed immediately after preparation, the lipids need to be stored in solvent at -20 &#176;C to prevent oxidation and degradation.</li>
</ol>
3. Metabolite Preparation and Derivatization (Timing ~5 h)
<ol>
	<li>On the day following extraction, remove the metabolite samples from the Speed Vac and store dry at -20 &#176;C until ready for derivatization and analysis on the GC. If not running the metabolites on a GC, then prepare them using the appropriate protocol for the instrument.</li>
	<li>Immediately before derivatizing the metabolites for analysis on the GC, transfer them from the large glass vials by adding 200 &#956;L of methanol, vortexing and adding to a 1.5 mL polypropylene tube. Repeat once.</li>
	<li>Centrifuge the tube at 12,000 x g for 10 min, 4 &#176;C. Transfer the supernatant into smaller glass vials, add a breathable membrane to the top, and completely dry in a Speed Vac.</li>
	<li>Derivatize the metabolites by adding 20 &#181;L of methoxyamine solution to the sample vial and vortex for 30 s on a vortexer at medium speed. 
	Caution: Methoxyamine hydrochloride causes severe burns and serious damage to eyes, may cause sensitization by skin contact. Wear safety glasses, gloves and lab coat, and work in a fume hood.</li>
	<li>Use a bath sonicator to ensure the sample is completely dissolved.</li>
	<li>Incubate the sample in an incubator with a condensation prevention lid maintained at 37 &#176;C for 1 h 30 min with 1,000 rpm shaking.</li>
	<li>Invert the vial one time to mix the samples with condensed drops at the cap surface. Spin the sample down at 1,000 x g for 1 min, RT.</li>
	<li>Perform silylation by adding 80 &#181;L (using a syringe) of N-Methyl-N-(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane. Vortex for 10s. 
	Caution: MSTFA + 1% TMCS can cause skin corrosion, serious eye damage, and specific target organ toxicity, and is a flammable liquid and vapor. Wear safety glasses, gloves and lab coat, and work in a fume hood.</li>
	<li>Incubate the sample in an incubator with a condensation prevention lid maintained at 37 &#176;C for 30 min with 1,000 rpm shaking.</li>
	<li>Invert the vial one time to mix the samples with condensed drops at the cap surface.</li>
	<li>Spin the sample down for 5 min at 2,000 x g, RT.</li>
	<li>Transfer the reacted solution into vials appropriate for the GC-MS analysis.</li>
</ol>
4. Protein Digestion (Timing ~1 d)
<ol>
	<li>Centrifuge the sample at 15,000 x g for 5 min, RT, to pellet any debris.</li>
	<li>Perform Filter-Aided-Sample-Preparation (FASP) for the digestion using FASP kits (see Table of Materials) following modified manufacturer&#8217;s instructions31,32:
	<ol>
		<li>Add 400 &#956;L of the 8 M urea buffer into a FASP column (500 &#956;L 30K MWCO spin filter).</li>
		<li>Once the sample has finished centrifuging, pipette off the supernatant (discard the pellet) and add up to 50 &#956;L of the sample to the 400 &#956;L of 8 M urea in the column. 
		NOTE: Add only up to 50 &#956;L of sample per column. If there is more than 50 &#956;L of sample, use multiple columns and combine the peptides after digestion. It&#8217;s best to leave this volume as low as possible (30 &#181;L is ideal) in order to ensure the FASP column will remove all of the SDS which can dramatically interfere with mass spec analysis.</li>
		<li>Place the column into the included 1.5 mL tube and centrifuge the sample at 14,000 x g for 30 min, RT.</li>
		<li>Remove the flow-through into waste and add 400 &#956;L of 8 M urea to the column and centrifuge again.</li>
		<li>Repeat previous step one more time for a total of 3 urea rinses. 
		NOTE: Make sure the sample goes close to dryness on the column, if there is any more than ~ 30 &#181;L remaining on the filter after each spin, continue centrifuging.</li>
		<li>Add 400 &#956;L of 50 mM NH4HCO3 and centrifuge at 14,000 x g for 20 min, discard the waste, and repeat once.</li>
		<li>Transfer the columns to a clean 1.5 mL centrifuge tube.</li>
		<li>Add 75 &#956;L of trypsin digestion buffer to the columns and incubate at 37 &#176;C for 3 h in an incubator with a condensation prevention lid at 750 rpm.</li>
		<li>Add 40 &#181;L of 50 mM NH4HCO3 to the columns.</li>
		<li>Centrifuge the sample at 14,000 x g for 15 min to collect the peptides into the tube while keeping the high molecular weight contaminants on top of the column.</li>
		<li>Add 40 &#956;L of 50mM NH4HCO3 to the column and centrifuge again.</li>
	</ol>
	</li>
	<li>Discard the column, dry down the peptides in the tube in a Speed Vac to ~30 &#181;L, and BCA assay (Bicinchoninic acid protein assay kit; see Table of Materials) the peptides.</li>
	<li>Optionally, perform a clean-up step if SDS contamination is suspected (visible bubbles)33. A solid phase extraction must be done afterwards if choosing this option. Detailed information about this clean-up method is available in the Supplementary Methods.</li>
	<li>Dilute the sample for MS analysis or optionally proceed to HPLC fractionation (next two steps).</li>
	<li>To fractionate, dilute samples to a volume of 400 &#181;L with 10 mM ammonium formate buffer (pH 10.0).</li>
	<li>Resolve on an C18 column (see Table of Materials) by separating at 0.5 mL/min using an HPLC system with mobile phases (A) 10 mM ammonium formate, pH 10.0 and (B) 10 mM ammonium formate, pH 10.0/acetonitrile (10:90).
	<ol>
		<li>Adjust the gradient from at 100% A to 95% A over the first 10 min, 95% A to 65% A over min 10 to 70, 65% A to 30% A over min 70 to 85, maintain at 30% A over min 85 to 95.</li>
		<li>Re-equilibrate with 100% A over min 95 to 105 and hold at 100% A until min 120.</li>
		<li>Collect fractions every 1.25 min (96 fractions over the entire gradient) and finally combine every row for a total of 12 samples or every other row for 24 samples (each with n = 8 or n = 4 fractions pooled).</li>
	</ol>
	</li>
	<li>Dry all fractions under vacuum and add 15 &#181;L of ultrapure water to each for storage at -20 &#176;C until LC-MS/MS analysis.</li>
</ol>

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<li>Diamond, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Temporal+proteome+and+lipidome+profiles+reveal+hepatitis+C+virus-associated+reprogramming+of+hepatocellular+metabolism+and+bioenergetics%5BTitle%5D)+AND+%22PLoS+Pathog%22%5BJournal%5D)">Temporal proteome and lipidome profiles reveal hepatitis C virus-associated reprogramming of hepatocellular metabolism and bioenergetics.</a> PLoS Pathog. 6, e1000719(2010).</li>
<li>Alquier, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deletion+of+GPR40+impairs+glucose-induced+insulin+secretion+in+vivo+in+mice+without+affecting+intracellular+fuel+metabolism+in+islets%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Deletion of GPR40 impairs glucose-induced insulin secretion in vivo in mice without affecting intracellular fuel metabolism in islets.</a> Diabetes. 58, 2607-2615 (2009).</li>
<li>Ding, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Application+of+the+accurate+mass+and+time+tag+approach+in+studies+of+the+human+blood+lipidome%5BTitle%5D)+AND+%22J+Chromatogr+B+Analyt+Technol+Biomed+Life+Sci%22%5BJournal%5D)">Application of the accurate mass and time tag approach in studies of the human blood lipidome.</a> J Chromatogr B Analyt Technol Biomed Life Sci. 871, 243-252 (2008).</li>
<li>Rasmussen, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Systems+virology+identifies+a+mitochondrial+fatty+acid+oxidation+enzyme%2C+dodecenoyl+coenzyme+A+delta+isomerase%2C+required+for+hepatitis+C+virus+replication+and+likely+pathogenesis%5BTitle%5D)+AND+%22J+Virol%22%5BJournal%5D)">Systems virology identifies a mitochondrial fatty acid oxidation enzyme, dodecenoyl coenzyme A delta isomerase, required for hepatitis C virus replication and likely pathogenesis.</a> J Virol. 85, 11646-11654 (2011).</li>
<li>Manza, L. L., Stamer, S. L., Ham, A. J., Codreanu, S. G., Liebler, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sample+preparation+and+digestion+for+proteomic+analyses+using+spin+filters%5BTitle%5D)+AND+%22Proteomics%22%5BJournal%5D)">Sample preparation and digestion for proteomic analyses using spin filters.</a> Proteomics. 5, 1742-1745 (2005).</li>
<li>Wisniewski, J. R., Zougman, A., Nagaraj, N., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Universal+sample+preparation+method+for+proteome+analysis%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Universal sample preparation method for proteome analysis.</a> Nat Methods. 6, 359-362 (2009).</li>
<li>Zhou, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simple+sodium+dodecyl+sulfate-assisted+sample+preparation+method+for+LC-MS-based+proteomics+applications%5BTitle%5D)+AND+%22Anal+Chem%22%5BJournal%5D)">Simple sodium dodecyl sulfate-assisted sample preparation method for LC-MS-based proteomics applications.</a> Anal Chem. 84, 2862-2867 (2012).</li>
<li>Anderson, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Decreased+abundance+of+type+III+secretion+system-inducing+signals+in+Arabidopsis+mkp1+enhances+resistance+against+Pseudomonas+syringae%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Decreased abundance of type III secretion system-inducing signals in Arabidopsis mkp1 enhances resistance against Pseudomonas syringae.</a> Proc Natl Acad Sci U S A. 111, 6846-6851 (2014).</li>
<li>Chourey, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+cellular+lysis%2Fprotein+extraction+protocol+for+soil+metaproteomics%5BTitle%5D)+AND+%22J+Proteome+Res%22%5BJournal%5D)">Direct cellular lysis/protein extraction protocol for soil metaproteomics.</a> J Proteome Res. 9, 6615-6622 (2010).</li>
<li>Kim, S., Gupta, N., Pevzner, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spectral+probabilities+and+generating+functions+of+tandem+mass+spectra%3A+a+strike+against+decoy+databases%5BTitle%5D)+AND+%22J+Proteome+Res%22%5BJournal%5D)">Spectral probabilities and generating functions of tandem mass spectra: a strike against decoy databases.</a> J Proteome Res. 7, 3354-3363 (2008).</li>
<li>Kim, S., Pevzner, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MS-GF%2B+makes+progress+towards+a+universal+database+search+tool+for+proteomics%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">MS-GF+ makes progress towards a universal database search tool for proteomics.</a> Nat Commun. 5, 5277(2014).</li>
<li>Cole, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phototrophic+biofilm+assembly+in+microbial-mat-derived+unicyanobacterial+consortia%3A+Model+systems+for+the+study+of+autotroph-heterotroph+interactions%5BTitle%5D)+AND+%22Front+Microbiol%22%5BJournal%5D)">Phototrophic biofilm assembly in microbial-mat-derived unicyanobacterial consortia: Model systems for the study of autotroph-heterotroph interactions.</a> Front Microbiol. 5, (2014).</li>
<li>Isaacson, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sample+extraction+techniques+for+enhanced+proteomic+analysis+of+plant+tissues%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Sample extraction techniques for enhanced proteomic analysis of plant tissues.</a> Nat Protoc. 1, 769-774 (2006).</li>
</ol>

The authors declare that they have no competing financial interests.

It is important to note that not all laboratories will have the same available equipment so certain methods, for example the lysis step, can be adapted. Here we use vortexing and sonicating, however the use of a large 50 mL bead beater would work. If a lyophilizer with a collector temperature capable of -105 &#176;C is not available, then samples can be dried under a nitrogen stream. Also soil types vary greatly and can include sand, silt, clay, peat, and loam (etc.), and they can also vary based on pH, salinity, and organic matter richness. Each of these variances can have an impact on the protocol, so it is important to be flexible and to make adjustments when needed. It is critical, however, that the ratios and percentages remain the same.
The MPLEx protocol shows great promise for application in numerous biological and environmental systems and the ability to enable multi-omic analyses of soil microbial communities. Previous comparisons of MPLEx to other extraction protocols for diverse systems illustrated a high degree of peptide overlap10. However, one observed limitation was that MPLEx was not applicable to blood plasma proteins that need to be depleted. In these cases, the precipitated proteins were not well recognized by the antibodies in the immunodepletion columns needed for broad coverage of the plasma proteome. Therefore, more standard extraction approaches should be used under these circumstances. One additional limitation noted for the MPLex extraction approach is that it does not discern between intracellular and extracellular protein, however this is true for all other soil protein extraction protocols as well.
In this manuscript, MPLEx provided results for 3376&#160;peptides, 105 lipids, and 102 polar metabolites in Kansas native prairie soil using the extraction and analysis methods detailed in the protocol section. Better overlap was observed between the peptides extracted in the control and MPLEx approaches of individual systems compared to the soil microbial community (Figure 2). This is not a limitation, but one consequence of working with the extremely complicated soil microbial communities. These results are further noted as the well-known soil extraction techniques of SDS and MoBio did not overlap well either due to the great diversity and difficulties extracting soil proteins equally. Interestingly, the MPLEx protocol allowed the identification of more total peptides than either SDS or MoBio and also detected additional components not seen in either analysis.
These observations make MPLEx a very promising technique for working with small samples sizes, reducing overall multi-omic experimental variability, and reducing sample preparation time. The advantages possible with MPLEx look to enable the multi-omic capabilities and analyses required for large-scale microbial community studies.

Evaluating soil microbial communities has important implications for understanding carbon cycling and climate change. Recent studies have however highlighted difficulties, such as the lack of sequenced genomes for microbiota in various soil types and the unknown function of many of the proteins detected. These challenges result due to soil being the most complex microbial community known to date1,2,3. Multi-omic analyses, which combine results from metagenomic, metatranscriptomic, metaproteomic, metabolomic, and lipidomic studies, have recently been implemented in numerous soil studies to gain a greater understanding into the microbes present, while obtaining comprehensive information about the molecular changes taking place due to environmental perturbations1,4,5. One challenge with multi-omic studies is that the mass spectrometry (MS)-based metaproteomic, metabolomic, and lipidomic measurements typically require a specific extraction process for each omic to be MS compatible6,7,8,9. These precise procedures make their implementation extremely difficult or impossible when only a limited quantity of sample is available. These challenges have prompted us to investigate a simultaneous metabolite, protein, and lipid extraction (MPLEx) method capable of using smaller sample volumes or masses, improving accuracy, and providing faster sample preparations for all three analyses10. To date, there are no alternate soil extraction procedures that can achieve all of these goals.
To enable global multi-omic analyses of a single soil sample, an organic solvent extraction protocol based upon chloroform, methanol, and water separations was utilized10. This method was originally developed for total lipid extractions9,11 and more recently was amended for the simultaneous extraction of metabolites, proteins, and lipids from a single sample12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30, enabling less sample quantity and experimental variability10. In the MPLEx protocol, chloroform is not miscible with water, which provides the basis for the triphasic chemical separation of sample constituents into distinct fractions. The top aqueous phase therefore contains the hydrophilic metabolites, followed by a protein disk, and then a lipid layer in the bottom chloroform phase (Figure 1). When MPLEx is applied to most soils, particulate debris accumulates at the very bottom of the sampling tubes and can be discarded after all layers are collected. Each soil type can be different, however, and in highly organic soil such as peat, the soil debris stays in the middle layer and does not fall to the bottom of the sampling tube. MPLEx provides several advantages when isolating multiple molecule types from the same sample such as 1) smaller sample quantities can be used for multi-omic analyses, 2) multi-omic extractions from the same sample decrease overall experimental variability, and 3) greater numbers of samples can be prepared much faster for higher throughput studies10. Together these benefits are vital for providing better measurement capabilities for evaluating soil samples and their complex microbial communities.

<table><tbody><tr><td>Chloroform</td><td>Sigma-Aldrich</td><td>650498</td><td>Stored at -20&amp;deg;C !Caution chloroform has acute potential health effects, skin irritation and possible chemical burns, irritation to the respiratory system, may affect the kidneys, liver, heart. Wear suitable protective glasses, clothing and gloves, work in a fume hood.</td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>34860</td><td>Stored at -20&amp;deg;C !Caution Methanol may cause respiratory tract, skin and eye irritation, may damage the nerves, kidneys and liver. Wear suitable protective glasses, clothing and gloves, work in a fume hood.</td></tr><tr><td>Purified water from Millipore</td><td>Milli-Q</td><td></td><td>Water purification system.</td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sigma-Aldrich</td><td>L6026</td><td>!Caution SDS causes acute toxicity and is flammable. It is a skin, eye and airway irritant. Wear gloves and safety glasses.</td></tr><tr><td>Soil protein extraction kit</td><td>MoBio, NoviPure Soil Protein Extraction Kit, Qiagen</td><td>30000-20</td><td></td></tr><tr><td>DL-dithiothreitol</td><td>Sigma-Aldrich</td><td>43815</td><td></td></tr><tr><td>1M Trizma HCL</td><td>Sigma-Aldrich</td><td>T2694</td><td></td></tr><tr><td>Trichloroacetic acid</td><td>Sigma-Aldrich</td><td>T0699</td><td>!Caution TCA is caustic, toxic and may cause skin burns. Wear gloves and safety glasses.</td></tr><tr><td>Acetone</td><td>Sigma-Aldrich</td><td>650501</td><td>Stored at -20&amp;deg;C !Caution Acetone may cause respiratory tract and skin and eye irritation. Flammable liquid and vapor. Wear safety glasses gloves and a lab coat, work in a fume hood.</td></tr><tr><td>Urea</td><td>Sigma-Aldrich</td><td>208884</td><td>!Caution Urea is an eye and skin irritant, use gloves and safety glasses</td></tr><tr><td>Ammonium bicarbonate</td><td>Fluka</td><td>09830</td><td></td></tr><tr><td>Trypsin</td><td>Promega</td><td>V528A</td><td>20&amp;micro;g vials</td></tr><tr><td>Bicinchoninic acid protein assay kit</td><td>Pierce</td><td>23227</td><td></td></tr><tr><td>Ammonium Formate</td><td>Sigma-Aldrich</td><td>09735</td><td></td></tr><tr><td>Acetonitrile</td><td>Sigma-Aldrich</td><td>34998</td><td>!Caution Acetonitrile is a skin and eye irritant. Highly flammable. Wear gloves and safety glasses. Work in a fume hood.</td></tr><tr><td>Trifluoroacetic acid</td><td>Sigma-Aldrich</td><td>T6508</td><td>!Caution TFA is extremely hazardous in case of skin contact, eye contact, ingestion and inhalation. May produce tissue damage particularly on mucous membranes of eyes, mouth and respiratory tract. Skin contact may produce burns. Wear gloves, lab coat, safety glasses and work in a fume hood.</td></tr><tr><td>Methoxyamine hydrochloride</td><td>Sigma-Aldrich</td><td>226904</td><td>!Caution Methoxyamine hydrochloride causes severe burns and serious damage to eyes, may cause sensitization by skin contact. Wear safety glasses, gloves and lab coat, work in a fume hood.</td></tr><tr><td>Pyridine</td><td>Sigma-Aldrich</td><td>270970</td><td>!Caution Pyridine can cause skin and eye irritation, central nervous system depression. Vapor may cause flash fire. Wear safety glasses, gloves and lab coat, work in a fume hood.</td></tr><tr><td>N-Methyl-N-(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane</td><td>Sigma-Aldrich</td><td>69478</td><td>!Caution MSTFA + 1% TMCS can cause skin corrosion, serious eye damage and specific target organ toxicity. Flammable liquid and vapor. Wear safety glasses, gloves and lab coat, work in a fume hood.</td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich</td><td>P9541</td><td></td></tr><tr><td>Milli-Q water purification system</td><td>Millipore</td><td></td><td>model MPGP04001</td></tr><tr><td>Vortex</td><td>Scientific Industries</td><td>SI-0236</td><td>Vortex Genie 2</td></tr><tr><td>Probe sonicator</td><td>FisherBrand</td><td></td><td>model FB505</td></tr><tr><td>Refrigerated centrifuge</td><td>Eppendorf</td><td></td><td>model 5810R</td></tr><tr><td>50mL tube swinging bucket rotor</td><td>Eppendorf</td><td>A-4-44</td><td></td></tr><tr><td>50mL fixed angle rotor</td><td>Eppendorf</td><td>FA-45-6-30</td><td></td></tr><tr><td>Balance</td><td>OHAUS</td><td></td><td>model V22PWE150IT</td></tr><tr><td>Serological pipette controller</td><td>Eppendorf</td><td>12-654-100</td><td></td></tr><tr><td>10mL, 25mL glass serological pipettes</td><td>FisherBrand</td><td>13-678-27F, 13-678-36D</td><td></td></tr><tr><td>Thermomixer with Thermotop</td><td>Eppendorf</td><td>5382000015, 5308000003</td><td></td></tr><tr><td>0.9 &amp;ndash; 2.0 mm blend stainless steel beads</td><td>NextAdvance</td><td>SSB14B</td><td></td></tr><tr><td>0.15 mm garnet beads</td><td>MoBio</td><td>13122-500</td><td></td></tr><tr><td>Magnetic stir plate</td><td>FisherBrand</td><td>11-100-16SH</td><td></td></tr><tr><td>Magnetic stir bar</td><td>FisherBrand</td><td>14512130</td><td></td></tr><tr><td>pH paper strips, pH range 0&amp;ndash;14</td><td>FisherBrand</td><td>M95903</td><td></td></tr><tr><td>15mL, 50mL conical polypropylene centrifuge tube</td><td>Genesee Scientific</td><td>21-103 21-108</td><td>chloroform compatible</td></tr><tr><td>50mL vortex attachment</td><td>MoBio</td><td>13000-V1-50</td><td></td></tr><tr><td>Ice bucket</td><td>FisherBrand</td><td>02-591-44</td><td></td></tr><tr><td>27.25x70mm glass vials</td><td>FisherBrand</td><td>03-339-22K</td><td></td></tr><tr><td>Breathe Easier plate membranes</td><td>Midwest Scientific</td><td>BERM-2000</td><td></td></tr><tr><td>Alcohol wipes</td><td>Diversified Biotech</td><td>BPWP-1000</td><td></td></tr><tr><td>Heater shaker incubator</td><td>Benchmark, Incu-Shaker Mini</td><td></td><td></td></tr><tr><td>Analog rotisserie tube rotator</td><td>SoCal BioMed, LLC</td><td>82422001</td><td></td></tr><tr><td>Filter-Aided-Sample-Prep kit</td><td>FASP; Expedeon</td><td>44250</td><td></td></tr><tr><td>Microplate reader</td><td>Biotek, EPOCH</td><td></td><td></td></tr><tr><td>-20 Degree Celsius Freezer</td><td>Fisher</td><td>13986149</td><td></td></tr><tr><td>-80 Degree Celsius Freezer</td><td>Stirling Ultracold</td><td>SU78OUE</td><td></td></tr><tr><td>Q-Exactive ion trap mass spectrometer</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Agilent 7890A gas chromatograph coupled with a single quadrupole 5975C mass spectrometer</td><td>Agilent Technologies, Inc.</td><td></td><td></td></tr><tr><td>LTQ-Orbitrap Velo</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Waters NanoEquityTM UPLC system</td><td>Millford, MA</td><td></td><td></td></tr><tr><td>250mL media bottle</td><td>FisherBrand</td><td>1395-250</td><td></td></tr><tr><td>Waters vial</td><td>Waters</td><td>186002805</td><td></td></tr><tr><td>Glass MS sample vial and inserts</td><td>MicroSolv</td><td>9502S-WCV, 9502S-02ND</td><td></td></tr><tr><td>Glass HPLC vial and snap caps</td><td>MicroSolv</td><td>9512C-0DCV, 9502C-10C-B</td><td></td></tr><tr><td>HPLC 96-well plate</td><td>Agilent</td><td>5042-6454</td><td></td></tr><tr><td>Large glass vial 27.25x70mm</td><td>FisherBrand</td><td>03-339-22K</td><td></td></tr><tr><td>Lyophilizer</td><td>Labconco</td><td>7934021</td><td></td></tr><tr><td>Polished stainless steel flat head spatula</td><td>Spoonula; FisherBrand</td><td>14-375-10</td><td></td></tr><tr><td>Kim wipes</td><td>Kimberly-Clark</td><td>34721</td><td></td></tr><tr><td>XBridge C18, 250x4.6 mm, 5 &amp;mu;M with 4.6x20 mm guard column</td><td>Waters</td><td>186003117, 186003064</td><td></td></tr><tr><td>Agilent 1100 series HPLC system</td><td>Agilent Technologies</td><td>G1380-90000</td><td></td></tr><tr><td>1.7mL centrifuge tube</td><td>Sorenson</td><td>11700</td><td></td></tr><tr><td>Hamilton Glass Syringes, 5mL, 50&amp;micro;L and 250&amp;micro;L</td><td>Hamilton</td><td>81517, 80975, 81175</td><td></td></tr><tr><td>Pasteur Pipettes</td><td>FisherBrand</td><td>13-678-20A</td><td></td></tr><tr><td>Pasteur Pipette Bulbs</td><td>Sigma-Aldrich</td><td>Z111597</td><td></td></tr><tr><td>Bath Sonicator</td><td>Branson 1800 Ultrasonic Cleaner</td><td></td><td></td></tr><tr><td>Vacuum Centrifuge</td><td>Labconco Centrivap Acid-Resistant Concentrator System</td><td></td><td></td></tr><tr><td>MicroSpin Columns, C18 Silica</td><td>The Nest Group</td><td>SEM SS18V</td><td></td></tr></tbody></table>

the mplex protocol for multi-omic analyses of soil samples

Mass spectrometry (MS)-based integrated metaproteomic, metabolomic, and lipidomic (multi-omic) studies are transforming our ability to understand and characterize microbial communities in environmental and biological systems. These measurements are even enabling enhanced analyses of complex soil microbial communities, which are the most complex microbial systems known to date. Multi-omic analyses, however, do have sample preparation challenges, since separate extractions are typically needed for each omic study, thereby greatly amplifying the preparation time and amount of sample required. To address this limitation, a 3-in-1 method for the simultaneous extraction of metabolites, proteins, and lipids (MPLEx) from the same soil sample was created by adapting a solvent-based approach. This MPLEx protocol has proven to be both simple and robust for many sample types, even when utilized for limited quantities of complex soil samples. The MPLEx method also greatly enabled the rapid multi-omic measurements needed to gain a better understanding of the members of each microbial community, while evaluating the changes taking place upon biological and environmental perturbations.

When the MPLEx protocol was used to extract molecules from Kansas native prairie soil (a Mollisol soil), the triplicate analyses provided results for 3376&#160;peptides, 105 lipids, and 102 polar metabolites (all unique identifications). While the MPLEx protocol has been well-established for general extraction of lipids and metabolites12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,34, its comparison to common soil protein extraction methods for microbial analyses, such as soil protein extraction kits (see Table of Materials) and SDS (sodium dodecyl sulfate) extractions35, is further evaluated here. To assess these techniques, Kansas native prairie soil proteins were extracted with each approach and analyzed directly with reversed-phase LC-MS/MS using a UPLC system coupled with a hybrid quadrupole/Orbitrap mass spectrometer. Detailed information about the method parameters are available in the Supplementary Methods. The resulting experimental peptide MS/MS spectra from each extraction approach were compared with predicted peptide sequences from the representative Kansas native prairie metagenome2 using a stringent MS-GF+ spectral probability cutoff of 1x10-10 36,37 and a mass error cutoff of less than 5 ppm. It should be noted that the analytes extracted from soil using the MPLEx protocol are suitable for MS-based studies and other analytical techniques. When initially comparing three replicates of the known methods of MoBio and SDS, only 12% of the peptides overlapped between the two techniques showing the complexity of the microbial community and different extraction effects (Figure 2). Upon comparison of MPLEx with MoBio and SDS extracts, ~38% of the peptides observed using the MPLEx method were also detected when using the SDS and/or MoBio extractions (Figure 2). Considering the number of species in the Kansas soil bacterial community and its metagenome2, the overlap of peptide identifications is reasonable and due to its complexity, the combination of the approaches extracts more peptides than each alone35. Also, since these samples were unfractionated and analyzed by only 1-dimensional LC-MS/MS, the extremely high sample complexity was possibly causing some bias in detection due to different peptide concentrations extracted by each approach. The MPLEx protocol has also been applied to other soil types such as permafrost and wetlands, and in all cases the preliminary proteomic results are of similar quality to that of the Kansas native prairie soil data presented here.
The broad applicability of the MPLEx approach, has been previously evaluated using a diverse set of samples, including the archaeon Sulfolobus acidocaldarius, a unicyanobacterial consortium38, mouse brain cortex tissue, human urine, and leaves from Arabidopsis thaliana (Figure 2)10. For all samples except A.thaliana, urea extractions are the most common way to extract proteins, so they were used as the control approach for evaluation. The A. thaliana MPLEx results were compared to an extraction performed with trichloroacetic acid (TCA) because plant leaves are rich in phenolic compounds that need to be removed prior to MS analyses39. In all cases, the number of proteins observed from the MPLEx method was similar to that for the controls10, indicating its utility for many different sample types. Also, there were no trends associated with the specific protein classes extracted in all the sample types (including soil). Therefore, the broad applicability of MPLEx to numerous biological and environmental systems makes it a promising approach.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57343/57343fig1.jpg" /> 
Figure 1. A schematic showing the MPLEx process, in which metabolites, proteins and lipids are simultaneously extracted from the same soil sample for MS analyses. <a href="/files/ftp_upload/57343/57343fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57343/57343fig2.jpg" /> 
Figure 2. Venn diagrams illustrating the peptide overlap for a diverse set of samples extracted using MPLEx and a standard urea-based protein extraction method normally performed. Data from MPLEx extraction of the archaeon S.acidocaldarius, the unicyanobacterial consortium, human urine, mouse brain cortex, and A.thaliana plant leaves adapted from Nakayasu, E. S. et al. 201610. <a href="/files/ftp_upload/57343/57343fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The MPLEx Protocol for Multi-omic Analyses of Soil Samples

A protocol is presented for simultaneously extracting metabolites, proteins, and lipids from a single soil sample, allowing reduced sample preparation times and enabling multi-omic mass spectrometry analyses of samples with limited quantities.

Mass spectrometry (MS)-based integrated metaproteomic, metabolomic, and lipidomic (multi-omic) studies are transforming our ability to understand and ...

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10.9K Views.  Pacific Northwest National Laboratory. A protocol is presented for simultaneously extracting metabolites, proteins, and lipids from a single soil sample, allowing reduced sample preparation times and enabling multi-omic mass spectrometry analyses of samples with limited quantities.

Video: The MPLEx Protocol for Multi-omic Analyses of Soil Samples

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about the overall structure of terrestrial microbial communities. PLFA profiling has been extensively used in a range of ecosystems as a biological index of overall soil quality, and as a quantitative indicator of soil response to land management and other environmental stressors.
The standard method presented here outlines four key steps: 1. lipid extraction from soil samples with a single-phase chloroform mixture, 2. fractionation using solid phase extraction columns to isolate phospholipids from other extracted lipids, 3. methanolysis of phospholipids to produce fatty acid methyl esters (FAMEs), and 4. FAME analysis by capillary gas chromatography using a flame ionization detector (GC-FID). Two standards are used, including 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (PC(19:0/19:0)) to assess the overall recovery of the extraction method, and methyl decanoate (MeC10:0) as an internal standard (ISTD) for the GC analysis.

Phospholipid fatty acids provide information about the structure of soil microbial communities. We present methods for extraction from soil samples with a single-phase chloroform mixture, fractionation of extracted lipids using solid phase extraction columns, and methanolysis to produce fatty acid methyl esters, which are analyzed by capillary gas chromatography.

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about ...

Environment

A Method to Define the Effects of Environmental Enrichment on Colon Microbiome Biodiversity in a Mouse Colon Tumor Model

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental enrichment (EE) reduces tumorigenesis by activating the mouse immune system, or affects tumor bearing animal survival by stimulating the wound repair response, including improved microbiome diversity, in the tumor microenvironment. Provided here is a detailed procedure to assess the effects of environmental enrichment on the biodiversity of the microbiome in a mouse colon tumor model. Precautions regarding animal breeding and considerations for animal genotype and mouse colony integration are described, all of which ultimately affect microbial biodiversity. Heeding these precautions may allow more uniform microbiome transmission, and consequently will alleviate non-treatment dependent effects that can confound study findings. Further, in this procedure, microbiota changes are characterized using 16S rDNA sequencing of DNA isolated from stool collected from the distal colon following long-term environmental enrichment. Gut microbiota imbalance is associated with the pathogenesis of inflammatory bowel disease and colon cancer, but also of obesity and diabetes among others. Importantly, this protocol for EE and microbiome analysis can be utilized to study the role of microbiome pathogenesis across a variety of diseases where robust mouse models exist that can recapitulate human disease.

Environmental Enrichment (EE) is an animal housing environment that is used to reveal mechanisms that underlie the connections between lifestyle, stress, and disease. This protocol describes a procedure that uses a mouse model of colon tumorigenesis and EE to specifically define alterations in microbiota biodiversity that may impact animal mortality.

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental ...

Cancer Research

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial communities, a relatively precise but effort-intensive technique to assay microbial community composition is phospholipid fatty acid (PLFA) analysis. PLFA was developed to analyze phospholipid biomarkers, which can be used as indicators of microbial biomass and the composition of broad functional groups of fungi and bacteria. It has commonly been used to compare soils under alternative plant communities, ecology, and management regimes. The PLFA method has been shown to be sensitive to detecting shifts in microbial community composition.
An alternative method, fatty acid methyl ester extraction and analysis (MIDI-FA) was developed for rapid extraction of total lipids, without separation of the phospholipid fraction, from pure cultures as a microbial identification technique. This method is rapid but is less suited for soil samples because it lacks an initial step separating soil particles and begins instead with a saponification reaction that likely produces artifacts from the background organic matter in the soil. 
This article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples. The method combines the accuracy achieved through PLFA profiling by extracting and concentrating soil lipids as a first step, and a reduction in effort by saponifying the organic material extracted and processing with the MIDI-FA method as a second step.

The article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples.

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial ...

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

Genetics

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

Immunology and Infection

Nanopore DNA Sequencing for Metagenomic Soil Analysis

This article describes the steps for construction of a DNA library from soil, preparation and use of the nanopore flow cell, and analysis of the DNA sequences identified using computer software. Nanopore DNA sequencing is a flexible technique that allows for rapid microbial genome sequencing to identify bacterial and viral species, to characterize bacterial strains, and to detect genetic mutations that confer resistance to antibiotics. The advantages of nanopore sequencing (NS) for life sciences include its low complexity, reduced cost, and rapid real-time sequencing of purified genomic DNA, PCR amplicons, cDNA samples, or RNA. NS is an example of "strand sequencing" which involves sequencing DNA by guiding a single stranded DNA molecule through a nanopore that is inserted into a synthetic polymer membrane. The membrane has an electrical current applied across it, so as the individual bases pass through the nanopore the electrical current is disrupted to varying degrees by the four nucleotide bases. The identification of each nucleotide occurs by detecting the characteristic modulation of the electrical current by the different bases as they pass through the nanopore. The NS system consists of a handheld, USB powered portable device and a disposable flow cell that contains a nanopore array. The portable device plugs into a standard laptop computer that reads and records the DNA sequence using computer software.

Nanopore technology for sequencing biomolecules has wide applications in the life sciences, including identification of pathogens, food safety monitoring, genomic analysis, metagenomic environmental monitoring, and characterization of bacterial antibiotic resistance. In this article, the procedure for metagenomic soil DNA sequencing for species identification using the nanopore sequencing technology is demonstrated.

This article describes the steps for construction of a DNA library from soil, preparation and use of the nanopore flow cell, and analysis of the DNA ...

Isolation and Analysis of Microbial Communities in Soil, Rhizosphere, and Roots in Perennial Grass Experiments

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The purpose of this manuscript is to provide detail on how to rapidly sample soil, rhizosphere, and endosphere of replicated field trials and analyze changes that may occur in the microbial communities due to sample type, treatment, and plant genotype. The experiment used to demonstrate these methods consists of replicated field plots containing two, pure, warm-season grasses (Panicum virgatum and Andropogon gerardii) and a low-diversity grass mixture (A. gerardii, Sorghastrum nutans, and Bouteloua curtipendula). Briefly, plants are excavated, a variety of roots are cut and placed in phosphate buffer, and then shaken to collect the rhizosphere. Roots are brought to the laboratory on ice and surface sterilized with bleach and ethanol (EtOH). The rhizosphere is filtered and concentrated by centrifugation. Excavated soil from around the root ball is placed into plastic bags and brought to the lab where a small amount of soil is taken for DNA extractions. DNA is extracted from roots, soil, and rhizosphere and then amplified with primers for the V4 region of the 16S rRNA gene. Amplicons are sequenced, then analyzed with open access bioinformatics tools. These methods allow researchers to test how the microbial community diversity and composition varies due to sample type, treatment, and plant genotype. Using these methods along with statistical models, the representative results demonstrate there are significant differences in microbial communities of roots, rhizosphere, and soil. Methods presented here provide a complete set of steps for how to collect field samples, isolate, extract, quantify, amplify, and sequence DNA, and analyze microbial community diversity and composition in replicated field trials.

Excavation of plant roots from the field as well as processing of samples into endosphere, rhizosphere, and soil are described in detail, including DNA extraction and data analysis methods. This paper is designed to enable other laboratories to use these techniques for the study of soil, endosphere, and rhizosphere microbiomes.

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The ...

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to characterize. However, to understand the thermodynamic and kinetic controls on greenhouse gas (carbon dioxide [CO2] and methane [CH4]) production resulting from the decomposition of NOM, a molecular-level characterization coupled with microbial proteome analyses is necessary. Further, climate and environmental changes are expected to perturb natural ecosystems, potentially upsetting complex interactions that influence both the supply of organic matter substrates and the microorganisms performing the transformations. A detailed molecular characterization of the organic matter, microbial proteomics, and the pathways and transformations by which organic matter is decomposed will be necessary to predict the direction and magnitude of the effects of environmental changes. This article describes a methodological throughput for comprehensive metabolite characterization in a single sample by direct injection Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), gas chromatography mass spectrometry (GC-MS), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography mass spectrometry (LC-MS), and proteomics analysis. This approach results in a fully-paired dataset which improves statistical confidence for inferring pathways of organic matter decomposition, the resulting CO2 and CH4 production rates, and their responses to environmental perturbation. Herein we present results of applying this method to NOM samples collected from peatlands; however, the protocol is applicable to any NOM sample (e.g., peat, forested soils, marine sediments, etc.).

This protocol describes a single throughput for complementary analytical and omics techniques culminating in a fully-paired characterization of natural organic matter and microbial proteomics in different ecosystems. This approach permits robust comparisons for identifying metabolic pathways and transformations important for describing greenhouse gas production and predicting responses to environmental change.

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to ...

A Multi-Omics Extraction Method for the In-Depth Analysis of Synchronized Cultures of the Green Alga Chlamydomonas reinhardtii

Microalgae have been the focus of research for their applications in the production of high value compounds, food and fuel. Moreover, they are valuable photosynthetic models facilitating the understanding of the basic cellular processes. System wide studies enable comprehensive and in-depth understanding of molecular functions of the organisms. However, multiple independent samples and protocols are required for proteomics, lipidomics and metabolomics studies introducing higher error and variability. A robust high throughput extraction method for the simultaneous extraction of chlorophyll, lipids, metabolites, proteins and starch from a single sample of the green alga Chlamydomonas reinhardtii is presented here. The illustrated experimental setup is for Chlamydomonas cultures synchronized using 12 h/12 h light/dark conditions. Samples were collected over a 24 h cell cycle to demonstrate that the metabolites, lipids and starch data obtained using various analytical platforms are well conformed. Furthermore, protein samples collected using the same extraction protocol were used to conduct detailed proteomics analysis to evaluate their quality and reproducibility. Based on the data, it can be inferred that the illustrated method provides a robust and reproducible approach to advance understanding of various biochemical pathways and their functions with greater confidence for both basic and applied research.

A Multi-Omics Extraction Method for the In-Depth Analysis of Synchronized Cultures of the Green Alga Chlamydomonas reinhardtii

System-wide analysis of multiple biomolecules is crucial to gain functional and mechanistic insights into biological processes. Hereby, an extensive protocol is described for high throughput extraction of lipids, metabolites, proteins and starch from a single sample harvested from synchronized Chlamydomonas culture.

Microalgae have been the focus of research for their applications in the production of high value compounds, food and fuel. Moreover, they are valuable ...

Developmental Biology

Large-Scale Multi-Omics Genome-Wide Association Studies (Mo-GWAS): Guidelines for Sample Preparation and Normalization

Both gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) are widely used metabolomics approaches to detect and quantify hundreds of thousands of metabolite features. However, the application of these techniques to a large number of samples is subject to more complex interactions, particularly for genome-wide association studies (GWAS). This protocol describes an optimized metabolic workflow, which combines an efficient and fast sample preparation with the analysis of a large number of samples for legume crop species. This slightly modified extraction method was initially developed for the analysis of plant and animal tissues and is based on extraction in methyl tert-butyl ether: methanol solvent to allow the capture of polar and lipid metabolites. In addition, we provide a step-by-step guide for reducing analytical variations, which are essential for the high-throughput evaluation of metabolic variance in GWAS.

Large-Scale Multi-Omics Genome-Wide Association Studies Mo-GWAS: Guidelines for Sample Preparation and Normalization

In this protocol, we present an optimized workflow, which combines an efficient and fast sample preparation of many samples. In addition, we provide a step-by-step guide to reduce analytical variations for high-throughput evaluation of metabolic GWAS studies.

Both gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) are widely used metabolomics approaches to detect ...

The MPLEx Protocol for Multi-omic Analyses of Soil Samples

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Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

A Method to Define the Effects of Environmental Enrichment on Colon Microbiome Biodiversity in a Mouse Colon Tumor Model

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Nanopore DNA Sequencing for Metagenomic Soil Analysis

Isolation and Analysis of Microbial Communities in Soil, Rhizosphere, and Roots in Perennial Grass Experiments

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures

A Multi-Omics Extraction Method for the In-Depth Analysis of Synchronized Cultures of the Green Alga Chlamydomonas reinhardtii

Large-Scale Multi-Omics Genome-Wide Association Studies (Mo-GWAS): Guidelines for Sample Preparation and Normalization