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 to yield an inert atmosphere.</li>
		<li>Prepare a bath using a shallow Dewar flask filled with distilled ethyl acetate.</li>
		<li>Fit the hermetically closed reaction tube inside the bath and cool it by slowly adding liquid N2 to the ethyl acetate until the solvent is frozen. 
		CAUTION: Liquid violently boils at room temperature (RT) and can cause severe burns when contacting eyes and skin.</li>
		<li>Add 54 mg of anhydrous collidine using a Hamilton syringe. 
		CAUTION: Collidine is volatile and has a very strong and unpleasant scent.</li>
		<li>Add 70 mg of tert-butyldimethylsilyl chloride and stir the entire solution for 3 h at -78 &#176;C on a magnetic stirrer.</li>
		<li>After 3 h, leave the reaction to warm at RT and keep stirring for an additional 12 h.</li>
		<li>Add 2 mL of brine to quench the reaction.</li>
		<li>Extract the solution 3x with 2 mL of distilled dichloromethane using a separating funnel, saving the organic extract each time.</li>
		<li>Combine the three organic extracts and dry them over sodium sulfate.</li>
		<li>Evaporate the dichloromethane under reduced pressure in a vacuum rotary evaporator carefully to avoid solvent projections.</li>
		<li>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.</li>
		<li>Combine the product-containing fractions and remove the solvent under reduced pressure in a vacuum rotary evaporator to obtain the pure 1-O,3-O-bis-(TBDMS) glycerol-d5 as a colorless liquid.</li>
	</ol>
	</li>
	<li>Esterification
	<ol>
		<li>Add 10 mg of the 1-O,3-O-bis(TBDMS)-glycerol-d5 (previously synthesized) to a 10 mL reaction tube using a Pasteur pipette and add a magnetic stirrer.</li>
		<li>Add 2 mL of anhydrous dichloromethane using a 5 mL Hamilton syringe, and fill the tube with dry N2 to yield an inert atmosphere.</li>
		<li>Cool the solution to 0 &#176;C using an ice bath.</li>
		<li>Add 36 mg of arachidonic acid using a multi-volume adjustable micropipette and stir.</li>
		<li>Add 15 mg of 4-dimethylaminopyridine and stir.</li>
		<li>Add 15 mg of N,N&#39;-diisopropylcarbodiimide using a multi-volume adjustable micropipette and stir.</li>
		<li>Let the mixture react at 0 &#176;C for 3 h.</li>
		<li>After 3 h, leave the reaction to warm at RT and keep stirring for an additional 12 h.</li>
		<li>Add 2 mL of water to quench the reaction.</li>
		<li>Extract the organic solution 3x with 2 mL of distilled dichloromethane (DCM) using a separating funnel.</li>
		<li>Place the three organic extracts in the same tube and dry them over sodium sulfate.</li>
		<li>Evaporate the dichloromethane under reduced pressure in a vacuum rotary evaporator carefully to avoid solvent projections.</li>
		<li>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 50% hexane/50% ethyl acetate.</li>
		<li>Combine the product-containing fractions and remove the solvent under reduced pressure in a vacuum rotary evaporator to obtain the pure 1-O, 3-O-bis(TBDMS)-2-AG-d5 as a yellowish liquid.</li>
	</ol>
	</li>
	<li>Deprotection
	<ol>
		<li>Add 15 mg of the 1-O,3-O-bis(TBDMS)-2-AG-d5 (previously synthesized) to a 10 mL reaction tube using a Pasteur pipette and add a magnetic stirrer.</li>
		<li>Add 2 mL of anhydrous THF using a 5 mL Hamilton syringe, and fill the tube with dry N2 to yield an inert atmosphere.</li>
		<li>Cool the solution to 0 &#176;C using an ice bath.</li>
		<li>Add 150 &#181;L dropwise of 1 M tetrabutylammonium fluoride solution in THF using a Hamilton syringe.</li>
		<li>Let the reaction warm to RT and stir for 1 h.</li>
		<li>After 1 h, add 2 mL of water to quench the reaction.</li>
		<li>Extract the solution 3x with 2 mL of distilled dichloromethane using a separating funnel.</li>
		<li>Combine the three organic extracts and dry them over sodium sulfate.</li>
		<li>Evaporate the dichloromethane under reduced pressure in a vacuum rotary evaporator to obtain the pure 1-AG-d5 as a yellowish liquid.</li>
	</ol>
	</li>
	<li>Monitor all reactions by thin layer chromatography performed on silica gel 60 F254 pre-coated aluminum sheets. Visualize the bands under a 254 nm UV lamp after staining with an ethanolic solution of 4-anisaldehyde.</li>
</ol>
2. Preparation of standard stock and measuring solutions
<ol>
	<li>Dissolve 1 mg of the internal standard 1-AG-d5 in 1 mL of ACN and sonicate for 1 min to obtain the 1,000 ppm standard stock solution.</li>
	<li>To prepare the 1,000 ppb solution used for quantification in worms, first prepare a 10 ppm solution: take 10 &#181;L of the stock solution using a Hamilton syringe and dilute it to a final volume of 1 mL by adding 990 &#181;L of ACN.</li>
	<li>Take 100 &#181;L from the solution produced in step 2.2 using a Hamilton syringe, and dilute it to a final volume of 1 mL by adding 900 &#181;L of ACN to obtain the 1,000 ppb solution used for the quantification.</li>
	<li>Sonicate for 1 min between each step to ensure complete solubilization. Store the solutions at -78 &#176;C to maintain the concentrations and integrity of the standards. After the standard solution is used, flow some nitrogen before closing the vial to prevent oxidation.</li>
</ol>
3. Growth and maintenance of C. elegans
NOTE: Seed the nematode growth medium (NGM) agar plates with E. coli OP50 and propagate the worms on these plates.
<ol>
	<li>Mix 3 g of NaCl with 17 g of agar.</li>
	<li>Add 2.5 g of peptone, then add 975 mL of H2O.</li>
	<li>Autoclave for 50 min, then cool the flask to 55 &#176;C.</li>
	<li>Mix the following: 1 mL of 1 M CaCl2, 1 mL of 1 M MgSO4, 25 mL of 1 M KH2PO4 buffer (all of which have been previously autoclaved), and 1 mL of 5 mg/mL cholesterol in ethanol.</li>
	<li>While maintaining a sterile environment, dispense the NGM solution into 60 mm Petri plates, filling the plates to two-thirds of their volume. Store the plates at 4 &#176;C.</li>
	<li>Streak the E. coli bacterial culture from a -80 &#176;C glycerol stock onto the LB agar plate. Let it grow on the plate overnight at 37 &#176;C.</li>
	<li>Pick up a single colony to inoculate 100 mL of liquid LB overnight at 37 &#176;C with agitation. 
	NOTE: It is not necessary to check the O.D. because this strain can reach stationary phase over this time.</li>
	<li>Remove the stored NGM plates, remove the lids in the laminar flow hood, and leave open to allow evaporation of excess moisture from the plates.</li>
	<li>Once the plates are dried, use a Pasteur pipette to add 100 &#181;L of OP50 E. coli to the center of the plate without spreading.</li>
	<li>Leave the OP50 E. coli lawn to grow overnight at RT or at 37 &#176;C for 8 h.</li>
	<li>Add the desired number worm embryos obtained by hypochlorite treatment or &#34;bleaching&#34; (Section 4). 
	NOTE: Cool the plates to RT before the addition of worms.</li>
</ol>
4. Bleaching technique for synchronizing C elegans cultures
<ol>
	<li>Seed and chunk worms onto 6 cm NGM plates.</li>
	<li>Leave the worms growing for 2-3 days to obtain sufficient numbers of eggs and gravid adults on the plate.</li>
	<li>Once there are enough eggs/adults, pour 5 mL of M9 onto the plate.</li>
	<li>Transfer the worms to a 15 mL centrifuge tube using a glass pipette.</li>
	<li>Centrifuge the tube for 2 min at 2,000 x g and pellet the worms.</li>
	<li>Suction out most of the M9, avoiding disturbance of the worm pellet.</li>
	<li>Add 3 mL of bleaching solution (2:1:1 ratio of NaOH:NaOCl:H2O).</li>
	<li>Invert gently to mix the solution for 5 min or until the number of intact adult worms decreases. 
	CAUTION: Do not bleach for more than 5 min.</li>
	<li>Centrifuge for 1 min at 2,000 x g and suction most of the bleaching solution without disturbing the worm pellet.</li>
	<li>Add 15 mL of M9 and mix well.</li>
	<li>Centrifuge again at 2000 x g for 1 min.</li>
	<li>Suction out most of the M9 without disturbing the worm pellet.</li>
	<li>Repeat steps 4.10-4.12 one or two more times.</li>
	<li>Add 5 mL of fresh M9 and agitate.</li>
	<li>Let the eggs hatch overnight with gentle rocking.</li>
</ol>
5. Worm sample preparation
<ol>
	<li>Let the N2 embryos obtained by the bleaching procedure hatch overnight in M9 buffer (5 mL in a 15 mL centrifuge tube) at 20 &#176;C.</li>
	<li>Harvest the synchronized L1s by centrifuging the tube for 2 min at 2,000 x g.</li>
	<li>Wash the worms with M9 buffer 1x, then quantify the number of live L1 worms.</li>
	<li>Seed approximately 10,000 worms into NGM plates (10 cm diameter) with 1 mL of OP50 E. coli (previously dried).</li>
	<li>Incubate the plates for 48 h at 20 &#176;C until worms reach the L4 stage.</li>
	<li>Harvest the worms using cold M9 buffer in a 15 mL centrifuge tube, wash them 1x, then and transfer them to a 1.5 mL tube.</li>
	<li>Pellet the worms by centrifugation at 2,000 x g for 1 min, eliminate most of the supernatant, immerse the tubes in liquid nitrogen, and store at -80 &#176;C.</li>
</ol>
6. Lipid extraction
<ol>
	<li>Thaw approximately 100 &#181;L of frozen worm pellets belonging to N2 on ice, add 1.3 mL of methanol, and sonicate the sample for 4 min.</li>
	<li>Add 2.6 mL of chloroform, and 1.3 mL of 0.5 M KCl/0.08 M H3PO4 to a final ratio of 1:2:1, 1,000 ppb of the internal standard 1-AG-d5, and butylated hydroxytoluene as an antioxidant agent at a final concentration of 50 &#181;g/mL.</li>
	<li>Vortex the samples for 1 min and sonicate in an ultrasonic water bath for 15 min on ice.</li>
	<li>Vortex the samples 2x for 1 min and centrifuge for 10 min at 2,000 x g to induce the phase separation.</li>
	<li>Collect the lower phase and collect it in a clean tube, dry it under nitrogen, and resuspend the solid residue in 100 &#181;L of ACN.</li>
</ol>
7. Endocannabinoid analysis by HPLC-MS/MS
<ol>
	<li>Use liquid chromatography coupled with an ESI triple quadrupole mass spectrometer to detect and quantify 2-AG from nematode samples.</li>
	<li>Use the following ratio for reversed-phase HPLC: from 0.0-0.5 min H2O:ACN (40:60), 0.5-6.5 min H2O:ACN (40:60) to (25:75), 6.5-7.5 min H2O:ACN (25:75), 7.5-8.0 min H2O:ACN (25:75) to (40:60),&#160;8.0-12.0 min H2O:ACN (40:60).</li>
	<li>Maintain the column temperature at 40 &#176;C and set the autosampler tray temperature to 10 &#176;C.</li>
	<li>Set the following ionization conditions: positive-ion mode; drying gas (N2) temperature = 300 &#176;C; drying gas flow rate = 10 L/min; nebulizer pressure = 10 UA; and cap. voltage = 4 kV.</li>
	<li>For the analyte detection, use MRM with the following transitions: 379.2 m/z to 289.2 m/z for 2-AG; and 384.2 m/z to 289.2 m/z for 1-AG-d5.</li>
</ol>
8. Endocannabinoid quantification in worms
<ol>
	<li>Use deuterated internal standard 1-AG-d5 and calculate the peak area ratios of the analyte to the internal standard.</li>
	<li>Use the following transitions: 384.2 m/z to 287.2 m/z for 2-AG; and 379.2 m/z to 287.2 m/z for 1-AG-d5.</li>
	<li>Calculate the concentration of the endogenous 2-AG by comparing to the peak area ratios of the deuterated standard using the concentration value of the standard.</li>
</ol>

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The authors declare no conflicts of interest.

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 ...

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Research

JoVE Journal

Biochemistry

7.2K Views.  Universidad Nacional de Rosario. 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).

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

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. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

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

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. The relative levels of different tRNAs, and the ratio of aminoacylated tRNA to total tRNA, known as the charging level, are important factors in determining the accuracy and speed of translation. Therefore, the abundance and charging levels of tRNAs are important variables to measure when studying protein synthesis, for example under various stress conditions. Here, we describe a method for harvesting tRNA and directly measuring both the relative abundance and the absolute charging level of specific tRNA species in Escherichia coli. The tRNA is harvested in such a way that the labile bond between the tRNA and its amino acid is preserved. The RNA is then subjected to gel electrophoresis and Northern blotting, which results in separation of the charged and uncharged tRNAs. The levels of specific tRNAs in different samples can be compared due to the addition of spike-in cells for normalization. Prior to RNA purification, we add 5% of E. coli cells that overproduce the rare tRNAselC to each sample. The amount of the tRNA species of interest in a sample is then normalized to the amount of tRNAselC in the same sample. Addition of spike-in cells prior to RNA purification has the advantage over addition of purified spike-in RNAs that it also accounts for any differences in cell lysis efficiency between samples.

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

Here we present a method for directly measuring transfer RNA charging levels from purified Escherichia coli RNA as well as a way to compare relative levels of transfer RNA, or any other short RNA, across different samples based on the addition of spike-in cells expressing a reference gene.

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

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

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 is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

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

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine whether there is an imbalance in age-related energy metabolism between mitochondrial oxidative phosphorylation and aerobic glycolysis. Here, we show the utilization of commercial colorimetric assay kits for lactate and pyruvate in the model organism C. elegans. Recently, the sensitivity and accuracy of the colorimetric/fluorimetric assay kits have been improved greatly by the research and development conducted by reagent manufacturers. The improved reagents have enabled the use of small-scale assays with a 96-well plate in C. elegans. In general, a fluorimetric assay is superior in sensitivity to a colorimetric assay; however, the colorimetric approach is more suitable for the use in common laboratories. Another important issue in these assays for quantitative determination is protein precipitation of homogenized C. elegans samples. In our protein precipitation method, common precipitants (e.g., trichloroacetic acid, perchloric acid and metaphosphoric acid) are used for sample preparation. A protein-free assay sample is prepared by directly adding cold precipitant (final concentration of 5%) during homogenization.

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

We describe a modified small-scale extraction and colorimetric assays of lactate and pyruvate in the nematode C. elegans. When utilizing commercial assay kits, the technical development of their sensitivity and accuracy is important. Protein precipitation in extraction is the most critical step for the quantitative determination of intracellular metabolites.

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

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple and inexpensive method to obtain a large number of animals for the isolation of proteins to proceed with a western blot, mass spectrometry, or further proteomics analyses. Furthermore, an increase of nematode numbers for immunostainings and the integration of multiple analyses under the same culturing conditions can easily be achieved. Additionally, a transfer between plates with different experimental conditions is facilitated. Common techniques in plate culture involve the transfer of a single C. elegans using a platinum wire and the transfer of populated agar chunks using a scalpel. However, with increasing nematode numbers, these techniques become overly time-consuming. This protocol describes the large-scale culture of C. elegans including numerous steps to minimize the impact of the sample preparation on the physiology of the worm. Fluid and shear stress can alter the lifespan of and metabolic processes in C. elegans, thus requiring a detailed description of the critical steps in order to retrieve reliable and reproducible results. C. elegans is a model organism, consisting of neuronal cells for up to one-third, but lacking blood vessels, thus providing the possibility to investigate solely neuronal alterations independent of vascular control. Recently, early neurodegeneration in diabetic retinopathy was found prior to vascular alterations. Thus, C. elegans is of special interest for studying general mechanisms of diabetic complications. For example, an increased formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS) is observed, which are reproducibly found in C. elegans. Protocols to handle samples of adequate size for a broader spectrum of investigations are presented here, exemplified by the study of diabetes-induced biochemical alterations. In general, this protocol can be useful for studies requiring large C. elegans numbers and in which liquid culture is not suitable.

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

This protocol describes a method for the large-scale cultivation of Caenorhabditis elegans on solid media. As an alternative to liquid culture, this protocol allows obtaining parameters of different scales under plate-based cultivation. This increases the comparability of results by omitting the morphological and metabolic differences between liquid and solid media culture.

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 fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample 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 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. Measurement of cellular CoA is a challenge due to its relatively low abundance and the dynamic conversion of free CoA to CoA thioesters that, in turn, participate in numerous metabolic reactions. A method is described that navigates through potential pitfalls during sample preparation to yield an assay with a broad linear range of detection that is suitable for use in many biomedical laboratories.

This method describes sample preparation from cultured cells and animal tissues, extraction and derivatization of coenzyme A in the samples, followed by high pressure liquid chromatography for purification and quantification of the derivatized coenzyme A by absorbance or fluorescence detection.

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

Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, and protein-protein interactions. FPOP utilizes a KrF excimer laser at 248 nm for photolysis of hydrogen peroxide to generate hydroxyl radicals which in turn oxidatively modify solvent-accessible amino acid side chains. Recently, we expanded the use of FPOP of in vivo oxidative labeling in Caenorhabditis elegans (C. elegans), entitled IV-FPOP. The transparent nematodes have been used as model systems for many human diseases. Structural studies in C. elegans by IV-FPOP is feasible because of the animal&#8217;s ability to uptake hydrogen peroxide, their transparency to laser irradiation at 248 nm, and the irreversible nature of the modification. The assembly of a microfluidic flow system for IV-FPOP labeling, IV-FPOP parameters, protein extraction, and LC-MS/MS optimized parameters are described herein.

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

In vivo fast photochemical oxidation of proteins (IV-FPOP) is a hydroxyl radical protein footprinting technique that allows for mapping of protein structure in their native environment. This protocol describes the assembly and set-up of the IV-FPOP microfluidic flow system.

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

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Caenorhabditis elegans has been an important model system for biological research since it was introduced in 1963. However, C. elegans has not been fully utilized in the biochemical study of biological reactions using its nuclear extracts such as in vitro transcription and DNA replication. A significant hurdle for using C. elegans in biochemical studies is disrupting the nematode&#39;s thick outer cuticle without sacrificing the activity of the nuclear extract. While several methods are used to break the cuticle, such as Dounce homogenization or sonication, they often lead to protein instability. There are no established protocols for isolating active nuclear proteins from larva or adult C. elegans for in vitro reactions. Here, the protocol describes in detail the homogenization of larval stage 4 C. elegans using a Balch homogenizer. The Balch homogenizer uses pressure to slowly force the animals through a narrow gap breaking the cuticle in the process. The uniform design and precise machining of the Balch homogenizer allow for consistent grinding of animals between experiments. Fractionating the homogenate obtained from the Balch homogenizer yields functionally active nuclear extract that can be used in an in vitro method for assaying transcription activity of C. elegans.

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Here, we describe a detailed protocol for isolating active nuclear extract from larval stage 4 C. elegans and visualizing transcription activity in an in vitro system.

Caenorhabditis elegans has been an important model system for biological research since it was introduced in 1963. However, C. elegans has not been fully ...