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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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+proteome+and+lipidome+profiles+reveal+hepatitis+C+virus-associated+reprogramming+of+hepatocellular+metabolism+and+bioenergetics.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+GPR40+impairs+glucose-induced+insulin+secretion+in+vivo+in+mice+without+affecting+intracellular+fuel+metabolism+in+islets.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+the+accurate+mass+and+time+tag+approach+in+studies+of+the+human+blood+lipidome.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systems+virology+identifies+a+mitochondrial+fatty+acid+oxidation+enzyme,+dodecenoyl+coenzyme+A+delta+isomerase,+required+for+hepatitis+C+virus+replication+and+likely+pathogenesis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+preparation+and+digestion+for+proteomic+analyses+using+spin+filters.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+sample+preparation+method+for+proteome+analysis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+sodium+dodecyl+sulfate-assisted+sample+preparation+method+for+LC-MS-based+proteomics+applications.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+abundance+of+type+III+secretion+system-inducing+signals+in+Arabidopsis+mkp1+enhances+resistance+against+Pseudomonas+syringae.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+cellular+lysis/protein+extraction+protocol+for+soil+metaproteomics.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+probabilities+and+generating+functions+of+tandem+mass+spectra:+a+strike+against+decoy+databases.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MS-GF++makes+progress+towards+a+universal+database+search+tool+for+proteomics.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phototrophic+biofilm+assembly+in+microbial-mat-derived+unicyanobacterial+consortia:+Model+systems+for+the+study+of+autotroph-heterotroph+interactions.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+extraction+techniques+for+enhanced+proteomic+analysis+of+plant+tissues.">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...

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&#176;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&#176;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&#176;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&#181;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 &#8211; 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&#8211;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 &#956;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&#181;L and 250&#181;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 colspan="2">Labconco Centrivap Acid-Resistant Concentrator System</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,<sup...

Watch this Scientific Journal Video about The MPLEx Protocol for Multi-omic Analyses of Soil Samples at JoVE.com

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 ...

the-mplex-protocol-for-multi-omic-analyses-of-soil-samples

Research

JoVE Journal

Chemistry

10.8K 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

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control self-assembly and mimicking this process in vitro will bring about major advances in the areas of materials science and nanotechnology. Among the available biological building blocks, peptides have several advantages as they present substantial diversity, their synthesis in large scale is straightforward, and they can easily be modified with biological and chemical entities1,2. Several classes of designed peptides such as cyclic peptides, amphiphile peptides and peptide-conjugates self-assemble into ordered structures in solution. Homoaromatic dipeptides, are a class of short self-assembled peptides that contain all the molecular information needed to form ordered structures such as nanotubes, spheres and fibrils3-8. A large variety of these peptides is commercially available.
This paper presents a procedure that leads to the formation of ordered structures by the self-assembly of homoaromatic peptides. The protocol requires only commercial reagents and basic laboratory equipment. In addition, the paper describes some of the methods available for the characterization of peptide-based assemblies. These methods include electron and atomic force microscopy and Fourier-Transform Infrared Spectroscopy (FT-IR). Moreover, the manuscript demonstrates the blending of peptides (coassembly) and the formation of a &#34;beads on a string&#34;-like structure by this process.9 The protocols presented here can be adapted to other classes of peptides or biological building blocks and can potentially lead to the discovery of new peptide-based structures and to better control of their assembly.

This paper describes the formation of highly ordered peptide-based structures by the spontaneous process of self-assembly. The method utilizes commercially available peptides and common lab equipment. This technique can be applied to a large variety of peptides and may lead to the discovery of new peptide-based assemblies.

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control ...

Transport of Surface-modified Carbon Nanotubes through a Soil Column

Carbon nanotubes (CNTs) are widely manufactured nanoparticles, which are being utilized in a number of consumer products, such as sporting goods, electronics and biomedical applications. Due to their accelerating production and use, CNTs constitute a potential environmental risk if they are released to soil and groundwater systems. It is therefore essential to improve the current understanding of environmental fate and transport of CNTs. The transport and retention of CNTs in both natural and artificial media have been reported in literature, but the findings widely vary and are thus not conclusive. There are a number of physical and chemical parameters responsible for variation in retention and transport. In this study, a complete procedure of selected multiwalled carbon nanotubes (MWCNTs) is presented starting from their surface modification to a complete set of laboratory column experiments at critical physical and chemical scenarios. Results indicate that the stability of the commercially available MWCNTs are critical with their attached surface functional group which can also influence the transport and retention of MWCNT through the surrounding medium.

Surface properties of a nanoparticle are important for their interaction with the surrounding medium. Therefore the surface modification of carbon nanotubes can be critical for their transport and retention through porous media. Here, lab scale column experiments are used to understand the possible transport and retention of these nanoparticles.

Carbon nanotubes (CNTs) are widely manufactured nanoparticles, which are being utilized in a number of consumer products, such as sporting goods, ...

A Direct, Early Stage Guanidinylation Protocol for the Synthesis of Complex Aminoguanidine-containing Natural Products

The guanidine functional group, displayed most prominently in the amino acid arginine, one of the fundamental building blocks of life, is an important structural element found in many complex natural products and pharmaceuticals. Owing to the continual discovery of new guanidine-containing natural products and designed small molecules, rapid and efficient guanidinylation methods are of keen interest to synthetic and medicinal organic chemists. Because the nucleophilicity and basicity of guanidines can affect subsequent chemical transformations, traditional, indirect guanidinylation is typically pursued. Indirect methods commonly employ multiple protection steps involving a latent amine precursor, such as an azide, phthalimide, or carbamate. By circumventing these circuitous methods and employing a direct guanidinylation reaction early in the synthetic sequence, it was possible to forge the linear terminal guanidine containing backbone of clavatadine A to realize a short and streamlined synthesis of this potent factor XIa inhibitor. In practice, guanidine hydrochloride is elaborated with a carefully constructed protecting array that is optimized to survive the synthetic steps to come. In the preparation of clavatadine A, direct guanidinylation of a commercially available diamine eliminated two unnecessary steps from its synthesis. Coupled with the wide variety of known guanidine protecting groups, direct guanidinylation evinces a succinct and efficient practicality inherent to methods that find a home in a synthetic chemist&#39;s toolbox.

Here, we present a protocol for direct, early stage guanidinylation that enables rapid total synthesis of aminoguanidine-containing small organic molecules. An advanced synthetic intermediate used in the synthesis of a blood coagulation factor XIa inhibitor was prepared using this protocol.

The guanidine functional group, displayed most prominently in the amino acid arginine, one of the fundamental building blocks of life, is an important ...

Protocol for the Synthesis of Ortho-trifluoromethoxylated Aniline Derivatives

Molecules bearing trifluoromethoxy (OCF3) group often show desired pharmacological and biological properties. However, facile synthesis of trifluoromethoxylated aromatic compounds remains a formidable challenge in organic synthesis. Conventional approaches often suffer from poor substrate scope, or require use of highly toxic, difficult-to-handle, and/or thermally labile reagents. Herein, we report a user-friendly protocol for the synthesis of methyl 4-acetamido-3-(trifluoromethoxy)benzoate using 1-trifluoromethyl-1,2-benziodoxol-3(1H)-one (Togni reagent II). Treating methyl 4-(N-hydroxyacetamido)benzoate (1a) with Togni reagent II in the presence of a catalytic amount of cesium carbonate (Cs2CO3) in chloroform at RT afforded methyl 4-(N-(trifluoromethoxy)acetamido)benzoate (2a). This intermediate was then converted to the final product methyl 4-acetamido-3-(trifluoromethoxy)benzoate (3a) in nitromethane at 120 &#176;C. This procedure is general and can be applied to the synthesis of a broad spectrum of ortho-trifluoromethoxylated aniline derivatives, which could serve as useful synthetic building blocks for the discovery and development of new pharmaceuticals, agrochemicals, and functional materials.

Protocol for the Synthesis of Ortho-trifluoromethoxylated Aniline Derivatives

An operationally simple procedure for the synthesis of ortho-trifluoromethoxylated aniline derivatives via a two-step sequence of O-trifluoromethylation of N-aryl-N-hydroxyacetamide followed by thermally induced intramolecular OCF3-migration is reported.

Molecules bearing trifluoromethoxy (OCF3) group often show desired pharmacological and biological properties. However, facile synthesis of ...

A Protocol for the Production of Gliadin-cyanoacrylate Nanoparticles for Hydrophilic Coating

This article presents a protocol for the production of protein-based nanoparticles that changes the hydrophobic surface to hydrophilic by a simple spray coating. These nanoparticles are produced by the polymerization reaction of alkyl cyanoacrylate on the surface of cereal protein (gliadin) molecules. Alkyl cyanoacrylate is a monomer that instantly polymerizes at RT when it is applied to the surface of materials. Its polymerization reaction is initiated by the trace amounts of weakly basic or nucleophilic species on the surface, including moisture. Once polymerized, the polymerized alkyl cyanoacrylates show a strong affinity with the object materials because nitrile groups are in the backbone of poly (alkyl cyanoacrylate). Proteins also work as initiator for this polymerization because they contain amine groups that can initiate the polymerization of cyanoacrylate. If aggregated protein is used as an initiator, protein aggregate is surrounded by the hydrophobic poly(alkyl cyanoacrylate) chains after the polymerization reaction of alkyl cyanoacrylate. By controlling the experimental condition, particles in the nanometer range are produced. The produced nanoparticles readily adsorb to the surface of most materials including glass, metals, plastics, wood, leather, and fabrics. When the surface of a material is sprayed with the produced nanoparticle suspension and rinsed with water, the micellar structure of nanoparticle changes its conformation, and the hydrophilic proteins are exposed to the air. As a result, the nanoparticle-coated surface changes to hydrophilic.

This article presents a protocol for the production of protein-based nanoparticles that changes the hydrophobic surface to hydrophilic. The produced nanoparticle is an assembly of gliadin-cyanoacrylate diblock copolymers. Spray coating with the produced nanoparticle changes the surface of target material to a hydrophilic surface.

This article presents a protocol for the production of protein-based nanoparticles that changes the hydrophobic surface to hydrophilic by a simple spray ...

A Simple and Efficient Protocol for the Catalytic Insertion Polymerization of Functional Norbornenes

Norbornene can be polymerized by a variety of mechanisms, including insertion polymerization whereby the double bond is polymerized and the bicyclic nature of the monomer is conserved. The resulting polymer, polynorbornene, has a very high glass transition temperature, Tg, and interesting optical and electrical properties. However, the polymerization of functional norbornenes by this mechanism is complicated by the fact that the endo substituted norbornene monomer has, in general, a very low reactivity. Furthermore, the separation of the endo substituted monomer from the exo monomer is a tedious task. Here, we present a simple protocol for the polymerization of substituted norbornenes (endo:exo ca. 80:20) bearing either a carboxylic acid or a pendant double bond. The process does not require that both isomers be separated, and proceeds with low catalyst loadings (0.01 to 0.02 mol%). The polymer bearing pendant double bonds can be further transformed in high yield, to afford a polymer bearing pendant epoxy groups. These simple procedures can be applied to prepare polynorbornenes with a variety of functional groups, such as esters, alcohols, imides, double bonds, carboxylic acids, bromo-alkyls, aldehydes and anhydrides.

We describe the catalytic insertion polymerization of 5-norbornene-2-carboxylic acid and 5-vinyl-2-norbornene to form functional polymers with a very high glass transition temperature.

Norbornene can be polymerized by a variety of mechanisms, including insertion polymerization whereby the double bond is polymerized and the bicyclic ...

A Protocol for Safe Lithiation Reactions Using Organolithium Reagents

Organolithium reagents are powerful tools in the synthetic chemist&#39;s toolbox. However, the extreme pyrophoric nature of the most reactive reagents warrants proper technique, thorough training, and proper personal protective equipment. To aid in the training of researchers using organolithium reagents, a thorough, step-by-step protocol for the safe and effective use of tert-butyllithium on an inert gas line or within a glovebox is described. As a model reaction, preparation of lithium tert-butyl amide by the reaction of tert-butyl amine with one equivalent of tert-butyl lithium is presented.

The safe and proper use of organolithium reagents is described.

Organolithium reagents are powerful tools in the synthetic chemist&#39;s toolbox. However, the extreme pyrophoric nature of the most reactive reagents ...

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient radiolabeling of DBCO-group-functionalized gold nanoparticles using a copper-free click reaction. Radioiodination of the stannylated precursor (2) was carried out by using [125I]NaI and chloramine T as an oxidant at room temperature for 15 min. After HPLC purification of the crude product, the purified 125I-labeled azide (1) was obtained with high radiochemical yield (75 &plusmn; 10%, n = 8) and excellent radiochemical purity (&gt;99%). For the synthesis of radiolabeled 13-nm-sized gold nanoparticles, the DBCO-functionalized gold nanoparticles (3) were prepared by using a thiolated polyethylene glycol polymer. A copper-free click reaction between 1 and 3 gave the 125I-labeled gold nanoparticles (4) with more than 95% of radiochemical yield as determined by radio-thin-layer chromatography (radio-TLC). These results clearly indicate that the present radiolabeling method using a strain-promoted copper-free click reaction will be useful for the efficient and convenient radiolabeling of DBCO-group-containing nanomaterials.

An Optimized Protocol for the Efficient Radiolabeling of Gold Nanoparticles by Using a 125I-labeled Azide Prosthetic Group

A detailed procedure for the synthesis of a 125I-labeled azide and the radiolabeling of dibenzocyclooctyne (DBCO)-group-conjugated, 13-nm-sized gold nanoparticles using a copper-free click reaction is described.

Here, we demonstrate a detailed protocol for the radiosynthesis of a 125I-labeled azide prosthetic group and its application to the efficient ...

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular methodology for applications in various fields and for different research purposes. The procedure described herein focuses on the synthesis, purification, and characterization of dodecamers of RNA 5&#39;-[CUA CGG AAU CAU]-3&#39; containing zero, one, or two modifications located at the C2&#39;-O-position. The probes are based on 2-thiophenylmethyl groups, incorporated into RNA nucleotides via standard organic synthesis and introduced into the corresponding oligonucleotides via their respective phosphoramidites. This report makes use of phosphoramidite chemistry via the four canonical nucleobases (Uridine (U), Cytosine (C), Guanosine (G), Adenosine (A)), as well as 2-thiophenylmethyl functionalized nucleotides modified at the 2&#39;-O-position; however, the methodology is amenable for a large variety of modifications that have been developed over the years. The oligonucleotides were synthesized on a controlled-pore glass (CPG) support followed by cleavage from the resin and deprotection under standard conditions, i.e., a mixture of ammonia and methylamine (AMA) followed by hydrogen fluoride/triethylamine/N-methylpyrrolidinone. The corresponding oligonucleotides were purified via polyacrylamide electrophoresis (20% denaturing) followed by elution, desalting, and isolation via reversed-phase chromatography (Sep-pak, C18-column). Quantification and structural parameters were assessed via ultraviolet-visible (UV-vis) and circular dichroism (CD) photometric analysis, respectively. This report aims to serve as a resource and guide for beginner and expert researchers interested in embarking in this field. It is expected to serve as a work-in-progress as new technologies and methodologies are developed. The description of the methodologies and techniques within this document correspond to a DNA/RNA synthesizer (refurbished and purchased in 2013) that uses phosphoramidite chemistry.

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

This article provides a detailed procedure on the solid-phase synthesis, purification, and characterization of dodecamers of RNA modified at the C2&#39;-O-position. UV-vis and circular dichroism photometric analyses are used to quantify and characterize structural aspects, i.e., single-strands or double-strands.

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular ...

Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles (PPAs) and Related Biomaterials

Polyamine-based Peptide Amphiphiles (PPAs) are a new class of self-assembling amphiphilic biomaterials-related to the peptide amphiphiles (PAs). Traditional PAs possess charged amino acids as solubilizing groups (lysine, arginine), which are directly connected to a lipid segment or can contain a linker region made of neutral amino acids. Tuning the peptide sequence of PAs can yield diverse morphologies. Similarly, PPAs possess a hydrophobic segment and neutral amino acids, but also contain polyamine molecules as water solubilizing (hydrophilic) groups. As is the case with PAs, PPAs can also self-assemble into diverse morphologies, including small rods, twisted nano-ribbons, and fused nano-sheets, when dissolved in water. However, the presence of both primary and secondary amines on a single polyamine molecule poses a significant challenge when synthesizing PPAs. In this paper, we show a simple protocol, based on literature precedents, to achieve a facile synthesis of PPAs using solid phase peptide synthesis (SPPS). This protocol can be extended to the synthesis of PAs and other similar systems. We also illustrate the steps that are needed for cleavage from the resin, identification, and purification.

Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles PPAs and Related Biomaterials

The synthesis of polyamine-based peptide amphiphiles (PPAs) is a significant challenge due to the presence of multiple amine nitrogens, which requires judicious use of protecting groups to mask these reactive functionalities. In this paper, we describe a facile method for the preparation of these new class of self-assembling molecules.