Name: synthesis of a deuterated standard for the quantification of 2-arachidonoylglycerol in caenorhabditis elegans
Uploaded: 2019-09-21T14:00:00
Description: Watch this Scientific Journal Video about Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans at JoVE.com

This work was supported by a research grant from the Agencia Nacional de Promoci&#243;n Cient&#237;fica y Tecnol&#243;gica (ANPCyT, PICT 2014-3693). J.F.d.L., G.P., and B.H.C. are fellows from CONICET. D.d.M. and G.R.L. are members of the Research Career of CONICET. We are thankful to Gonzalo Lamberto (INMET) for LC-MS/MS analysis and helpful discussion. Video shooting and editing has been done by Ramiro Ortega and Mar&#237;a Soledad Casasola from Direcci&#243;n de Comunicaci&#243;n de la Ciencia, Facultad de Ciencia Pol&#237;tica y Relaciones Internacionales, Universidad Nacional de Rosario in Rosario, Argentina.

1. 1-AG-d5 preparation
NOTE: For obtaining 1-AG-d5 as a deuterated internal standard for quantification assays, follow the protocol as detailed below.
<ol>
	<li>Differential protection
	<ol>
		<li>To only protect primary alcohols, first add 38 mg of glycerol-d8 to a 10 mL reaction tube using a Pasteur pipette and add a magnetic stirrer.</li>
		<li>Add 5 mL of anhydrous dichloromethane (DCM) using a 5 mL Hamilton syringe, and fill the tube with dry N2</su...

<ol>
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J., Komuniecki, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabinoids+Stimulate+the+TRP+Channel-Dependent+Release+of+Both+Serotonin+and+Dopamine+to+Modulate+Behavior+in+C.+elegans.">Cannabinoids Stimulate the TRP Channel-Dependent Release of Both Serotonin and Dopamine to Modulate Behavior in C. elegans.</a> The Journal of Neuroscience. 39 (21), 4142-4152 (2019).</li><li>Batugedara, H. M., 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=Helminth-Derived+Endocannabinoids+That+Have+Effects+on+Host+Immunity+Are+Generated+during+Infection.">Helminth-Derived Endocannabinoids That Have Effects on Host Immunity Are Generated during Infection.</a> Infection and Immunity. 86 (11), e00441 (2018).</li><li>Zhang, M. 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=Simultaneous+determination+of+2-arachidonoylglycerol,+1-arachidonoylglycerol+and+arachidonic+acid+in+mouse+brain+tissue+using+liquid+chromatography/tandem+mass+spectrometry.">Simultaneous determination of 2-arachidonoylglycerol, 1-arachidonoylglycerol and arachidonic acid in mouse brain tissue using liquid chromatography/tandem mass spectrometry.</a> Journal of Mass Spectrometry. 45 (2), 167-177 (2010).</li><li>Zoerner, A. A., 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=Simultaneous+UPLC-MS/MS+quantification+of+the+endocannabinoids+2-arachidonoyl+glycerol+(2AG),+1-arachidonoyl+glycerol+(1AG),+and+anandamide+in+human+plasma:+Minimization+of+matrix-effects,+2AG/1AG+isomerization+and+degradation+by+toluene+solvent+extraction.">Simultaneous UPLC-MS/MS quantification of the endocannabinoids 2-arachidonoyl glycerol (2AG), 1-arachidonoyl glycerol (1AG), and anandamide in human plasma: Minimization of matrix-effects, 2AG/1AG isomerization and degradation by toluene solvent extraction.</a> Journal of Chromatography B. 883-884, 161-171 (2012).</li><li>Ivanov, I., Borchert, P., Hinz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+simultaneous+determination+of+N-arachidonoylethanolamine,+N-oleoylethanolamine,+N-palmitoylethanolamine+and+2-arachidonoylglycerol+in+human+cells.">A simple method for simultaneous determination of N-arachidonoylethanolamine, N-oleoylethanolamine, N-palmitoylethanolamine and 2-arachidonoylglycerol in human cells.</a> Analytical and Bioanalytical Chemistry. 407 (6), 1781-1787 (2015).</li><li>Keereetaweep, J., Chapman, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipidomic+Analysis+of+Endocannabinoid+Signaling:+Targeted+Metabolite+Identification+and+Quantification.">Lipidomic Analysis of Endocannabinoid Signaling: Targeted Metabolite Identification and Quantification.</a> Neural Plasticity. , (2016).</li><li>Folch, J., Lees, M., Sloane Stanley, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+the+isolation+and+purification+of+total+lipides+from+animal+tissues.">A simple method for the isolation and purification of total lipides from animal tissues.</a> Journal of Biological Chemistry. 226 (1), 497-509 (1957).</li><li>Zoerner, A. 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Endocannabinoids are a class of lipids that have been implicated in the regulation of dauer formation in C. elegans7. More specifically, the synthesis of polyunsaturated fatty acids (PUFAs) is important for cholesterol trafficking and the reproductive development of worms. It is revealed here that 2-AG, an arachidonic acid containing endocannabinoid, is responsible for restituting the dauer larva to its normal cycle in worms that have impaired cholesterol metabolism7</su...

Endocannabinoids regulate multiple biological processes in a variety of organisms and are conserved lipid mediators1. The first discovered and most well-characterized endocannabinoids are anandamide (arachidonoylethanolamide, AEA) and 2-arachidonoyl glycerol (2-AG). Endocannabinoids play many critical roles, including those involved in brain reward systems as well as drug addiction, memory, mood, and metabolic processes2. AEA and 2-AG are only synthesized when needed and have short life spans, and they are degraded through transport protein reuptake and hydrolysis3.
The use of animal models like Caenorhabditis elegans (C. elegans) has become important to study the large variety of biological processes including apoptosis, cell signaling, cell cycle, cell polarity, gene regulation, metabolism, ageing, and sex determination4,5. Additionally, C. elegans is an excellent model for studying the physiological roles of polyunsaturated fatty acids (PUFAs). AEA has been identified in C. elegans and is reduced under dietary restriction6. This deficiency extends the lifespan of the nematode through a dietary restriction mechanism that can be suppressed by supplementation with the endocannabinoid. Recently, it was discovered that 2-AG and AEA play fundamental roles in the regulation of cholesterol trafficking in C. elegans7. More importantly, it was determined that supplementation with exogenous 2-AG can rescue dauer arrest, which is caused by the impaired cholesterol trafficking in Niemann-Pick type C1 C. elegans mutants.
To gain a better understanding of 2-AG&#39;s relationship with cholesterol trafficking and other biological processes in the nematode (i.e., monoaminergic signaling, nociception and locomotion), it is crucial to study this endogenous metabolite and how it is affected under certain environmental and dietary conditions8,9,10,11,12,13. Therefore, it is imperative to design and optimize a method to detect and quantify endogenous 2-AG in C. elegans that is simple to use for scientists of different fields, especially those who study the nematode&#39;s behavior in relation to this endocannabinoid.
In 2008, Lethonen and coworkers succeeded in identifying 2-AG and AEA in C. elegans using LC-MS analytical methods14. In 2011, they managed to expand this technique to other endocannabinoids15. More recent work has shown other analytical methods that have been successful in detecting and quantifying endocannabinoids in C. elegans, including mass spectrometry and GC-MS16,17,18, and it has also been reported that similar analytical methods can be expanded to other models19.
Previously reported analytical methods used for quantifying 2-AG in biological samples usually involve the use of deuterated standards that are commercially acquired and require availability for the purchase20,21. Many analytical standards for LC-MS/MS quantification of endocannabinoids are commercially available from different providers. Nevertheless, they are expensive, are sensitive, and become oxidized over time, due to the presence of multiple double bonds. The most common versions of these standards are based on the octa-deuterated arachidonic acid and are suitable for quantification by isotope dilution LC-MS/MS14,22. Also, most of these standards are substituted in position 2 of the glycerol, making them unstable under most conditions since they are prone to acyl migration19,23.
To overcome the difficulties associated with the costs and stability of these deuterated standards, a convenient and simple method is presented to prepare an analytical standard based on glycerol-d5. The sequence to prepare the penta-deuterated standard requires a three-step procedure that results in the standard 1-AG-d5, which is stable and does not undergo acyl migration (the main issue when aiming to synthesize 2-monoacylglycerols).
The main objective here is to show a simple and reproducible method to study 2-AG in C. elegans, including the synthesis of the analytical deuterated standard, preparation and extraction of the nematode samples, and analysis by LC-MS/MS (Figure 1). This synthetic procedure is achievable without the sophisticated organic synthesis knowledge or special equipment, making it suitable for scientists from different fields who are studying C. elegans behavior under endocannabinoid influence. The method is also expandable to other study models, making it useful for different targets. The standard, prepared as reported here, has been applied to successfully develop a fast and reliable chromatographic method that allows for effective detection and quantification of 2-AG in a reproducible manner.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4-dimethylaminopyridine</td>
			<td>Sigma-Aldrich</td>
			<td>107700</td>
			<td>reagent grade, 99%</td>
		</tr>
		<tr>
			<td>antioxidant BHT</td>
			<td>Sigma-Aldrich</td>
			<td>W21805</td>
			<td></td>
		</tr>
		<tr>
			<td>Arachidonic acid</td>
			<td>Sigma-Aldrich</td>
			<td>10931</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol-d8</td>
			<td>Sigma-Aldrich</td>
			<td>447498</td>
			<td></td>
		</tr>
		<tr>
			<td>Mass detector Triple Quadrupole</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>TSQ Quantum Access Max</td>
		</tr>
		<tr>
			<td>N,N&#8217;-diisopropylcarbodiimide</td>
			<td>Sigma-Aldrich</td>
			<td>D125407</td>
			<td></td>
		</tr>
		<tr>
			<td>NMR spectrometer</td>
			<td>Bruker</td>
			<td></td>
			<td>Avance II 300 MHz</td>
		</tr>
		<tr>
			<td>reversed-phase HPLC column</td>
			<td>Thermo Fisher</td>
			<td>25003-052130</td>
			<td>C18 Hypersil-GOLD (50 x 2.1 mm)</td>
		</tr>
		<tr>
			<td>tert-Butyldimethylsilyl chloride</td>
			<td>Sigma-Aldrich</td>
			<td>190500</td>
			<td>reagent grade, 97%</td>
		</tr>
		<tr>
			<td>tetrabutylammonium fluoride</td>
			<td>Sigma-Aldrich</td>
			<td>216143</td>
			<td>1.0M in THF</td>
		</tr>
		<tr>
			<td>UHPLC System</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>Ultimate 3000 RSLC Dionex</td>
		</tr>
		<tr>
			<td>worm strain N2 Bristol</td>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

synthesis of a deuterated standard for the quantification of 2-arachidonoylglycerol in caenorhabditis elegans

This work presents a method to prepare an analytical standard to analyze 2-arachidonoyl glycerol (2-AG) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass spectrometry (LC-ESI-MS/MS). Endocannabinoids are conserved lipid mediators that regulate multiple biological processes in a variety of organisms. In C. elegans, 2-AG has been found to possess different roles, including modulation of dauer formation and cholesterol metabolism. This report describes a method to overcome the difficulties associated with the costs and stability of deuterated standards required for 2-AG quantification. The procedure for the synthesis of the standard is simple and can be performed in any laboratory, without the need for organic synthesis expertise or special equipment. In addition, a modification of Folch&#39;s method to extract the deuterated standard from C. elegans culture is described. Finally, a quantitative and analytic method to detect 2-AG using the stable isotopically labeled analog 1-AG-d5 is described, which provides reliable results in a fast-chromatographic run. The procedure is useful for studying the multiple roles of 2-AG in C. elegans while also being applicable to other studies of metabolites in different organisms.

An isotopically labeled analog was successfully synthesized from commercially available d8-glycerol and arachidonic acid using a 3-step synthetic method (Figure 2, Figure 3). These steps are straightforward and do not require sophisticated equipment, specially controlled conditions, or expensive reagents. Thus, this method is robust and may be successfully extended to synthesize monoacylglycerides containing different ...

Watch this Scientific Journal Video about Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans at JoVE.com

Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans

This work describes a robust and straightforward method to detect and quantify the endocannabinoid 2-arachidonoylglycerol (2-AG) in C. elegans. An analytical deuterated standard us prepared and used for the quantification of 2-AG by isotopic dilution and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS).

Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans

This work presents a method to prepare an analytical standard to analyze 2-arachidonoyl glycerol (2-AG) qualitatively and quantitatively by liquid ...

Endocannabinoids are conserved lipid mediators that regulate multiple biological processes in a variety of organisms. The first discovered endocannabinoids are anandamide and 2-arachidonoylglycerol, 2-AG. The nematode C.elegans is a powerful animal model to study a large variety of biological processes.Recently, we discovered that 2-AG plays a fundamental role in regulation of cholesterol trafficking in C.elegans. This is why it became imperative to design and optimize a method to study endogenous 2-AG by detection and quantification in C.elegans. Previously reported analytical methods to quantify 2-AG in biological samples usually involve the use of deuterated standards that are commercial acquired are required to have them available for purchase.Nevertheless, they are expensive, and due to the presence of multiple double bonds, are sensitive to get oxidized over time. The most common version of the standards are based on the octadeuterated arachidonic acid being suitable for quantification by isotope dilution, by liquid chromatography, and posterior mass spectroscopy. To overcome the difficulties associated to the cost and stability of the deuterated standards to quantify 2-AG, we used a convenient and simple method to prepare an analytical standard based on deuterium-5-glycerol.The sequence to prepare the pendeuterated standard requires a three steps procedure. Being possible to be kept in the protected form until it is needed. The main objective of this work is to show a complete method to study 2-AG in C.elegans from the synthesis of the analytical deuterated standard to the preparation and extraction of the nematode samples and finally, the analysis by liquid chromatography and mass spectroscopy.For obtaining deuterated 1-AG as a deuterated internal standard for quantification assays follow a three step protocol. In order to only protect primary alcohols, first add 38 milligrams of glycerol deuterated to a 10 milliliter reaction tube using a Pasteur pipette and add a magnetic stirrer. Next, add five milliliter of anhydrous DCM using a five milliliters Hamilton syringe and fill the tube with dry nitrogen to give an inert atmosphere.Prepare a bath using a shallow Dewar flask filled with distilled ethyl acetate. Fill the hermetically closed reaction tube inside the bath and cool it by slowly the adding liquid nitrogen to ethyl acetate until the solvent is frozen. Caution, liquid violently boils at room temperature and might cause severe burns if in contact with eyes and skin.Next, add 54 milligrams of anhydrous collidine using a Hamilton syringe. Caution, collidine is volatile and has a very strong and unpleasant smell. At 70 milligrams of terbutile-dimethyl-cetylchloride and stir everything for three hours at minus 78 degrees on a magnetic stirrer.Add two milliliters of brine to quench the reaction. Extract the solution with two milliliters of distilled dichloromethane three times using a separated funnel keeping the organic extracts each time. Then, combine the three organic extracts and dry over sodium sulfite.Purify the crude mixture by column chromatography using silica gel as the stationary phase and a 10%increasing hexane ethyl acetate gradient starting from 100%hexane and finishing with 100%ethyl acetate. For this esterification step, add 10 milligrams of the product previously stated to a 10 milliliter reaction tube using a Pasteur pippette and add a magnetic stirrer. Add two milliliter of anhydrous DCM using a five milliliters Hamilton syringe and fill the tube with dry nitrogen to give an inert atmosphere.Cool the solution to zero degrees using an ice bath. Ad 36 milligrams of arachidonic acid using a multi-volume adjustable micropipette and stir. Then, add 15 milligrams of 40-methyl-aminopyridine and stir.Add 15 milligrams of diacylpropylcarbodiimid using a multi-volume adjustable micropipette and stir. Add two milliliters of water to quench the reaction and then extract the solution with two milliliters of distilled DCM three times using a separating funnel. Combine the three organic extracts and dry over sodium sulfite, purify the crude mixture by column chromatography using silica gel as a stationary phase an then 10%increasing hexane ethyl acetate gradient starting from 100%hexane and finishing with 50 50%hexane ethyl acetate.Finally, for the deprotection step, add 15 milligrams of the previously synthesized product to a 10 milliliters reaction tube using a Pasteur pipette and add a magnetic stirrer. Add two milliliters of anhydrous tetrahydrofuran using a five milliliters Hamilton syringe and fill the tube with dry nitrogen to give an inert atmosphere. Then cool the solution to zero degrees using an ice bath.Add 150 microliters drop-wise of one molar solution of tetrabutylammonium fluoride in THF using a Hamilton syringe. After one hour, add two milliliters of water to quench the reaction. Extract the solution with two milliliters distilled DCM three times using a separating funnel.Combine the three organic extracts and dry over sodium sulfite. For the bleaching procedure, turn worms onto seeded six centimeters NGM plates. Allow the worm to grow from two to three days so that there are lots of eggs and gravid adults on the plate.Once you have plenty of eggs and adults, pour five milliliters of M9 onto the plate. Using a glass pipette, transfer the worms to a 15 milliliters falcon tube. Centrifuge the tube for two minutes at 2, 000 G and pellet the worms.Suction most of the M9 without disturbing the worm pellet. Add three milliliters of bleaching solution and mix the solution gently by inverting for approximately five minutes or until you see a decrease in the number of intact adult worms. Once most of the bodies have dissolved, centrifuge at 2, 000 G for one minute.Suction most of the bleaching solution without disturbing the worm pellet. Add 15 milliliters of M9 to the tube and mix well. Centrifuge again at 2, 000 G for one minute.Suction most of the M9 without disturbing the worm pellet. For worm sample preparation, let N2 embryos obtained by bleaching procedure had in overnight in M9 buffer at 20 degrees. Then, harvest synchronized L1s by centrifuging the tube two minutes at 2, 000 G.Wash the worms with M9 buffer one more time and quantify the number of live L1.Seed approximately 10, 000 worms into NGM plates with one milliliter of OP 50 Escherichia coli previously dried.Incubate the plates at least for 48 hours at 20 degrees until the worms reach the L4 stage. Harvest the worms using cold M9 buffer in a 50 milliliters falcon tube. Wash them one more time and transfer them into a 1.5 milliliters tube.Pellet the worms by centrifugation at 2, 000 G for one minute. Eliminate most of the supernatant. Then, immerse the tube in liquid nitrogen and stir at minus 80 degrees.Thaw approximately 100 microliters of frozen worm pellets belonging to N2 and then add 1.3 milliliters of methanol. After that, sonicate the sample for four minutes. After sonication, add 1, 000 ppb of the internal deuterated standard, 2.6 milliliters of chloroform, and 1.3 milliliters of 0.5 molar of potassium chloride, 0.08 molar of phosphoric acid to a final ratio of one to one.Vortex the samples for one minute and then sonicate them in an ultrasonic water bath for 15 minutes on ice. Vortex the samples again twice for one minute and centrifuge them for 10 minutes at 2, 000 G to induce phase separation. Collect the lower phase into a clean glass tube, dry it under nitrogen, and resuspend it in 100 microliters of acetonitrile.To achieve a successful quantification of the endogenous 2-AG, it was necessary to synthesize it's saturated analog using a three step method. Afterwards, it was added to worm samples, extracted and analyzed by high performance liquid chromatography, coupled to mass spectrometry. Used as an internal standard, the synthetic deuterated 1-AG was the tool to quantify the endogenous metabolite.The method was optimized using the transitions for 2-AG and for deuterated 1-AG where glycerol molecules are lost. 2-AG endogenous from the C.elegans samples was successfully detected and quantified by isotopic dilution with the chemically synthesized deuterized 1-AG using high performance liquid chromatography coupled to mass spectrometry. Sample one was prepared with the deuterated standard 1-AG, while sample two without the standard.Since the original concentration of deutrated standard in sample one and three was of 1, 000 ppb each, from the peak area ratio it is possible to calculate the endogenous concentration of 2-AG in the sample of 340 ppb for sample one and 360 ppb for sample three, giving an average of 350 ppb. The ratios were calculated as a quality between the peak areas of 2-AG and deuterated 1-AG, respectively, for two isolated samples. Both with the deuterated standard added previous to extraction.Given the recently discovered importance of 2-AG in the enhancement of cholesterol trafficking and lack of information of how lipids influence that process, it was necessary a reliable detection method for this endocannabinoid. The successful development of a simple and robust synthetic method reported here to obtain deuterated analog 2-AG was a key step of this protocol. Most of the reported methods to quantify monoacylglycerols involve the use of commercially available analytical standards, which are usually expensive and unstable under regular storage conditions, making them a unreachable for lower budget laboratories.However, this method solves this by proposing a synthesis of the standard using more accessible starting materials. The synthetic method is straight forward without sophisticated conditions needed, making it ideal to be performed in any laboratory with minimal equipment and access to reactants, even in a reduced budget. In summary, this new procedure describes a straight forward and reproducible way of detecting and quantifying 2-AG that will help to answer some of the questions raised after the description of the role of these endocannabinoids in cholesterol trafficking in C.elegans.

Research

JoVE Journal

Biochemistry

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine ...

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

Quantification of Coenzyme A in Cells and Tissues

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. ...

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, ...