Name: Prostaglandin Extraction and Analysis in Caenorhabditis elegans (Video) | JoVE Journal
Uploaded: 2013-06-25T13:00:00.000+00:00
Duration: 8 min 50 s
Description: Learn prostaglandin extraction and analysis in Caenorhabditis elegans using LC-MS/MS. This optimized method enables sensitive, specific identification of eicosanoids for metabolic research.

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5 minutes

We thank the UAB Targeted Metabolomics and Proteomics Laboratory, which has been supported in part by the UAB Skin Disease Research Center (P30 AR050948 to C. Elmets), the UAB-UCSD O'Brien Acute Kidney Injury Center (P30 DK079337 to A. Agarwal) and the UAB Lung Health Center (R01 HL114439, R01 HL110950 to J.E. Blalock). Support for the mass spectrometer was from a NCRR Shared Instrumentation grant (S10 RR19261 to S. Barnes). We also thank Ray Moore for technical support. C. elegans strains were provided by the Caenorhabditis Genetics Center, which is funded by the NIH. This work was supported by the NIH (R01GM085105 to MAM, including an ARRA administrative supplement).

The protocol as described below is divided into three sections: worm culture, prostaglandin extraction, and prostaglandin analysis. As noted, there are multiple points when samples can be temporarily stored at -80 &deg;C or -20 &deg;C.
1. Worm Culture
We recommend approximately 6 g of mixed stage worms for comprehensive LC-MS/MS. This should provide enough material for detection at least six separate injections. For quantification of known PGs by MRM in a single injection or two, 1-2 g are sufficient. Analysis of extracts from synchronized cultures consisting of a specific stage is also possible. Up to 4 different strains can be grown simultaneously. For example, a wild type control and three different mutant strains.
<ol>
	<li>Prepare 65 X-large (16 cm) NGM plates and concentrated bacteria for supplemental feeding. Use NA22 bacteria for seeding. To make concentrated bacteria for supplemental feeding, grow at least four liters of NA22 in LB medium for 16-24 hr. Spin down, remove the supernatant, and resuspend bacteria in 100 ml of M9 buffer. 200 to 400 ml of this concentrated bacterial solution may be required for each worm strain. Store at 4 &deg;C.</li>
	<li>Start worm cultures on 5 X-large plates. Grow worms for approximately 3 to 4 generations until plates are full of gravid adults. Concentrated bacteria should be added to plates to prevent starvation, as necessary (~ 1 ml/plate and let dry completely before replacing lid).</li>
	<li>Wash gravid hermaphrodites off plates with M9 buffer and collect the worms in a 50 ml conical polypropylene tube.</li>
	<li>Allow gravid hermaphrodites to settle to the bottom (usually about 3-5 min). Remove supernatant, including bacteria, eggs, and larval stage worms. Resuspend in 15 ml M9 buffer and gently mix.</li>
	<li>Transfer 200 &mu;l of worm suspension to each of 60 seeded NGM plates. Disperse worms across the plates. Keep worm solution mixed well until all plates have been seeded to ensure that they receive roughly equal numbers of worms.</li>
	<li>Supplement with concentrated bacteria as needed (~1 ml/plate/12 to 24 hr period) to prevent starvation. Allow plates to air dry before replacing lids. Grow worms for 2-4 generations, depending on the original number of seeded worms, or until plates are full of gravid adults.</li>
	<li>Harvest worm plates one strain at a time. Wash worms off the plates with M9 buffer and collect in 50 ml polypropylene tubes (e.g. 6 tubes). Use M9 buffer to wash a plate, then transfer the worm-filled buffer to the next plate. After washing a number (e.g. 10th) of plates, transfer the worm-filled buffer to the 50 ml tube. Fill the tube to the top with M9 buffer. Repeat the wash for the rest of the plates.</li>
	<li>As the gravid adults settle to the bottom in 5-6 min, remove the supernatant (~ 35 ml) and consolidate into one or two tubes. Fill the tubes to 50 ml with fresh M9 buffer, let stand for 3-5 min, and remove supernatant leaving gravid adults. Repeat 3 times or until the supernatant is transparent.</li>
	<li>Using a large bore Pasteur pipette, transfer as many worms as possible to two 15 ml polypropylene conical tubes. Spin down at 1,000 rcf (~3,500 rpm in CentraSL2) for 5 min in a clinical centrifuge. Remove supernatant and repeat until all worms are transferred and pelleted in the same tube. Remove as much supernatant as possible and store worms at -80 &deg;C. Repeat for all strains.</li>
</ol>
2. Prostaglandin Extraction
The reagents needed for extraction are shown below. The method is modified from that of Golovko and Murphy17 and includes acetone extraction followed by liquid-liquid purification, which enhances LC-MS/MS sensitivity. A Bullet Blender 5 homogenizer is used in the procedure, although similar results can be obtained with a Dounce homogenizer. Butylated hydroxytoluene (BHT) and evaporation under Nitrogen (N2) gas are used to prevent oxidation. All glassware should be siliconized (Sigmacote) to reduce lipid binding. Extraction with organic solvents should be performed in a chemical hood.
<ol>
	<li>Weigh a 50 ml polypropylene conical tube. Transfer approximately 6.0 g of frozen worms from the 15 ml conical tube stored at -80 &deg;C by cutting through the tube with a hot razor blade near the 6.5 ml mark. A methanol-cleaned spatula can be used to remove the frozen worm pellet from the bottom of the tube. By cutting through the pellet to retrieve the lower portion, less dense larval worms and residual bacteria from the top of the pellet are left behind.</li>
	<li>Transfer the frozen worms to the pre-weighed 50 ml conical tube. If multiple samples are being processed, use the same amount (6.0 g) of tissue for comparative studies. As the worms thaw into a paste, add or remove worms with the spatula to obtain the desired mass. Optional: add 1.00 ng of PGF2&alpha;-d4 standard as an internal control for extraction efficiency.</li>
	<li>Add 12 ml of ice-cold 2:1 acetone/saline with 0.005% BHT to the worm suspension and mix well by vortexing. Dispense 1.5 ml of the worm slurry to each of twelve 5 ml self-standing plastic tubes for use in the Bullet Blender.</li>
	<li>Add 0.7-0.8 ml of 0.5 mm diameter Ceria stabilized zirconium oxide beads to each 5 ml tube. Close caps tightly and load the 12 tubes into the Bullet Blender 5 homogenizer. Run the blender for 3 min at speed 8-9 and place the tubes on ice. Be sure to close the caps very tightly or the content may spill out. Check 10 &mu;l of homogenate from one tube on a slide using a stereoscope. There should be very few intact worms remaining. Repeat at the same speed for another minute, if necessary.</li>
	<li>Evenly transfer the homogenates to four 10 ml conical glass tubes using a 9" Pasteur pipette. Avoid transferring beads. Wash the beads from three tubes sequentially with 1 ml of 1:2 acetone/saline containing BHT. Repeat for other tubes until all beads are washed. Transfer the remaining solution (~ 4 ml) to the 10 ml glass tubes and place them on ice.</li>
	<li>Centrifuge the 10 ml glass tubes at 4 &deg;C in a clinical centrifuge at ~1,000 x g for 10 min. Transfer the supernatant from each tube to a clean 10 ml glass conical tube.</li>
	<li>In a chemical hood, add an equal volume of hexane to each 10 ml glass tube. For example, add 3.5 ml hexane to 3.5 ml acetone/saline solution. Vortex at maximum speed for 30 sec.</li>
	<li>Centrifuge the 10 ml glass tubes at 4 &deg;C in a clinical centrifuge at ~1,000 x g for 10 min. Discard the upper phase containing hexane and neutrally charged lipids.</li>
	<li>Acidify the lower phase to pH 3.5 using 2 M formic acid (approximately 100 - 150 &mu;l). Test pH using pH strips.</li>
	<li>Add an equal volume of chloroform to each tube. Vortex at maximum speed for 30 sec and centrifuge the tubes at 4 &deg;C in a clinical centrifuge at ~1,000 x g for 10 min.</li>
	<li>Remove and discard the upper aqueous phase. Insert a pipette tip through any residual milky interphase to the lower chloroform phase, avoiding the interphase. Collect the lower phase from each of the 4 tubes to a clean 15 ml conical glass tube. Incubate at -20 &deg;C for at least 24 hr. At this point, the extract can be stored at -20 &deg;C for up to two weeks.</li>
	<li>Take the extract out of the freezer and discard the remaining milky aqueous top layer, if present. Transfer the chloroform phase to a labeled Teflon-lined &frac12;-Dram glass vial using a new Pasteur pipet. In a chemical hood, evaporate the chloroform soluble fraction to dryness under a gentle flow of N2 gas. The dried extract can be stored at -20 &deg;C for up to 2 weeks. An overall scheme used for PG extraction and analysis is shown in Figure 1.</li>
</ol>
3. Prostaglandin Analysis
<ol>
	<li>Prepare stock solutions of individual reference prostaglandins, such as PGF2&alpha; or PGF2 &alpha;-d4 (1 &mu;g/ml) in methanol and dilute with methanol:water (8:2 v/v) to obtain a working solution. Prepare a series of dilutions (100, 10, 1, 0.1, 0.01 ng/ml) to generate a standard curve.</li>
	<li>Add 200 &mu;l methanol:water (8:2 v/v) to the dried C. elegans lipid extract(s) and mix thoroughly. If less than 6 g of worm tissue was extracted, the dried extract should be resuspended in less volume of methanol:water solution.</li>
	<li>Prepare the mobile phase consisting of 0.1% formic acid in water [A] and acetonitrile containing 0.1% formic acid [B].</li>
	<li>Set up the autosampler at 4 &deg;C and place the sample vials in a 70-count 1.5 ml vial rack.</li>
	<li>Equilibrate the Synergy hydro RP-C18 column with 0.1% formic acid at a flow rate of 0.2 ml/min for about 5 min.</li>
	<li>Inject the sample (20-50 &mu;l) into the column. Perform gradient elution starting with 10% B and going up to 80% B from 0-11 min, 80-100% B from 11-14 min, and returning back to 10% B at 16 min. The total run time is 20 min. The retention times of 13 authentic standards are shown in Table 1 for reference3.</li>
	<li>Introduce the column effluent into the mass spectrometer using an ESI interface operating in the negative ion mode. Nitrogen is used as a nebulizer and curtain gas (CUR = 10). The collision gas, collision energy, and temperature are set at 10, -35 eV and 600 &deg;C, respectively. Declustering potential (DP), collision energy (CE), and cell exit potential (CXP) are set at -90, -35 and -10, respectively.</li>
	<li>For survey scans (Q1 scans), use negative ion mode in the mass range of m/z 315-360 for most PGs (Figure 2). The range can be altered, depending on the mass of the compound(s) of interest. Extract ions from total ion current (TIC) of LC-MS ion chromatograms and determine whether the extracted ions correspond to deprotonated or adduct ions, or to fragment ions.</li>
	<li>For MRM experiments, mass transition m/z 355/311 is used to detect the F1 class, m/z 353/193 is used for the F2 class, and m/z 351/193 or 351/191 is used for the F3 class (Figure 3). MRM is also used to detect the internal standard (for example, m/z 357/197 for PGF2&alpha;-d4) and to generate the standard curve. See Murphy et al. for additional information on PG mass transitions18.</li>
	<li>For MS/MS experiments, use m/z 355 for F1 class, m/z 353for F2 class, and m/z 351for F3 class PGs. C. elegans MS/MS data for F-series PGs is available in Figure 4 and Table 23,16. MS/MS data for mammalian eicosanoids is available at <a href="http://www.lipidmaps.org" target="_blank">www.lipidmaps.org</a>.</li>
	<li>Process the analytical data using Analyst software(Version 1.4.2, Applied Biosystems).</li>
</ol>

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</ol>

The authors have no conflicts of interest.

We describe a procedure for eicosanoid extraction and analysis, focusing on F-series PGs. There are several parts of the procedure that can be problematic. First, it is critical that the worm cultures do not starve, as starvation can alter PG metabolism. For supplemental feeding, NA22 bacteria are recommended instead of the more commonly used OP50 bacteria because NA22 reaches higher density. However, the NA22 strain lacks antibiotic resistance and is more susceptible to contamination. Second, worms synthesize individual PG isomers in low abundance relative to mammalian tissues. This may be due in part to redundancy among the numerous isomers. We recommend about 6 g of tissue for comprehensive analysis and stronger signals. Less tissue (1-2 g) can be used if the dried extract is resuspended in less methanol:water solution to increase PG concentration. However, only one to two injections can be performed. The detection limit of our LC-MS/MS system is about 10 pg PGF2&alpha; /ml. One ml of densely packed mixed stage worms (about 1 g) yields roughly 25-50 pg of CePGF2. More sensitive mass spectrometry systems can reduce the amount of worm tissue required for analysis. Third, differences in PG extraction efficiency among samples can cause variable results. We have found that extraction efficiency is very similar when tissues are extracted in parallel. To determine the efficiency, add 1.0 ng of PGF2&alpha;-d4 at the homogenization step as an internal control. MRM using mass transition m/z 357/197 is used to measure PGF2&alpha;-d4 concentration relative to a 1 ng/ml standard solution. The amount of PGF2&alpha;-d4 lost during extraction is then calculated.
The chromatography parameters and mass transitions we include can be altered to detect other PGs and eicosanoids. Despite considerable effort, we have not been able to identify D-series or E-series PGs in the extracts17. Furthermore, we have not detected 8-iso PGF2&alpha; and 8-iso PGE2 that are characteristic of free radical-initiated peroxidation, which generates a nonselective mixture of PG stereoisomers19. Whether worms synthesize other PG types is not known. Endocannabinoids and various epoxy and hydroxy metabolites of arachidonic and eicosapentaenoic acids have been found in C. elegans extracts5,7,20,21. We recommend the use of both MRM and MS/MS compared to authentic standards for quantification and identification, respectively. For novel eicosanoids, these analyses should be combined with a comparative study of fat mutants, which are deficient in PUFA synthesizing enzymes22, as well as a functional assay, if possible. A caveat to analyzing fat mutants is that minute quantities of PUFAs present in worms are sufficient for PG synthesis. For example, fat-2(wa17) mutants have a small amount of D12 desaturase activity (~5% of wild type22) and these mutants still produce PGs6. fat-3(wa22) and fat-4(wa14) mutants fail to synthesize PGs derived from arachidonic and eicosapentaenoic acids16. We also have found that fat mutants compensate for the loss of PUFA classes by up-regulating PG synthesis from remaining classes. For example, fat-3(wa22) mutants fail to synthesize most PGs derived from 20-carbon PUFAs. Instead, they appear to up-regulate novel PGs derived from 18-carbon PUFAs16. To date, we have not identified a C. elegans strain that is completely deficient in PG synthesis. It is possible that PGs are essential for growth or development.

Prostaglandins (PGs) are an important class of lipid hormones that have been extensively investigated and implicated in regulating reproduction, immunity, and development in a wide array of organisms12-14. PGs are ideally suited for comprehensive systems biology analyses because they comprise a series of lipids derived from a common precursor(s). The nematode C. elegans is one of the most widely used model organisms to address fundamental questions in genetics and systems biology.
We have demonstrated that C. elegans synthesizes F-series PGs that guide motile sperm to oocytes3,6,16. F-series PGs derived from 20-carbon PUFAs with three, four, and five double bonds, comprising the F1, F2, and F3 classes, respectively have been identified3,16. These PGs are synthesized independent of cyclooxygenase enzymes, yet PGF1&alpha; and PGF2&alpha; stereoisomers are still generated. For qualitative and quantitative analyses of C. elegans PGs, LC-MS/MS is a powerful and sensitive analytical technique. Before performing this analysis, it is important to develop an optimized extraction method because the performance of the analytical process heavily depends on the extract quality. Liquid chromatography and mass spectrometry can only provide anticipated mass to charge ratio (m/z), as well as desirable peak shape and retention time of the PG parent ion. Metabolic profiling of PGs in C. elegans is a challenging task requiring various MS acquisition strategies.
LC-MS full scan or survey scanning (Q1 scanning) has the advantage of ensuring that most ionizable PGs will generate a mass spectrometric response. While the survey scan provides useful information on total profiling of PGs with respect to wild-type and mutant C. elegans, detection of minor PGs is compromised due to low sensitivity. Nevertheless, LC-MS survey scanning can give a global view of PGs and is particularly useful for analyzing mutants predicted to affect metabolism of a large PG population.
In metabolite identification, LC-MS/MS analysis of chemically synthesized PG standards is first performed to obtain their product ion spectra as reference compounds. These spectra can be compared to the spectrum of an unknown metabolite. Multiple reaction monitoring (MRM) mode is used for enhanced sensitivity and specificity. An MRM experiment is accomplished by specifying the parent ion/product ion mass transition of a compound. By knowing the mass and structure of the target analytes, it is possible to predict theoretical MRM transitions for many unknown metabolites.
An optimized LC-MS/MS method for profiling isomeric PGs in C. elegans has not been reported. Here we describe an extraction and integrated mass spectrometry approach consisting of LC-MS and LC-MS/MS methods for detecting, quantifying, and investigating C. elegans PG metabolites. This approach can be applied to other eicosanoids.

<table><tbody><tr><td colspan="4">&lt;strong&gt;REAGENTS&lt;/strong&gt;</td></tr><tr><td>X-large (16 cm) plates</td><td>Fisher Scientific</td><td>0875714</td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; strain NA22</td><td>Caenorhabditis Genetics Center (CGC)</td><td>NA22</td><td>&lt;a href=&quot;http://www.cgc.cbs.umn.edu&quot; target=&quot;_blank&quot;&gt;http://www.cgc.cbs.umn.edu&lt;/a&gt;</td></tr><tr><td>Acetone, &lt;sup&gt;3&lt;/sup&gt;99.5%</td><td>Fisher Scientific</td><td>A928-4</td><td></td></tr><tr><td>Butylated Hydroxytoluene</td><td>MP Biomedicals</td><td>101162</td><td></td></tr><tr><td>Hexane, HPLC grade</td><td>Fisher Scientific</td><td>H302-1</td><td></td></tr><tr><td>Formic acid, &lt;sup&gt;3&lt;/sup&gt;99%</td><td>Acros</td><td>AC27048-0010</td><td>Available through Fisher Scientific</td></tr><tr><td>Chloroform, HPLC grade</td><td>Acros</td><td>AC26832-0010</td><td>Available through Fisher Scientific</td></tr><tr><td>PGF2&lt;sub&gt;&amp;alpha;&lt;/sub&gt;-d4</td><td>Cayman Chemicals</td><td>316010</td><td>Cayman has a wide array of standards available for purchase</td></tr><tr><td>Sterile screw cap self-standing 5 ml tube</td><td>Fisher Scientific</td><td>14-222-651</td><td>For use with Bullet Blender</td></tr><tr><td>0.5 mm zirconium oxide beads</td><td>Next &amp;gt;&amp;gt;&amp;gt; Advance</td><td>ZrOB05</td><td></td></tr><tr><td>10 ml glass conical heavy-duty graduated centrifuge tubes</td><td>Kimble Kimax</td><td>45200-10</td><td>Available through Fisher Scientific</td></tr><tr><td>15 ml glass conical tubes</td><td>Corning Pyrex</td><td>8082-15</td><td>Available through Fisher Scientific</td></tr><tr><td>Sigmacote (Siliconizing reagent)</td><td>Sigma-Aldrich</td><td>SL2-100ML</td><td></td></tr><tr><td>Teflon lined capped 1/2 Dram glass vial</td><td>Fisher Scientific</td><td>V1235C-TFE</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td></tr><tr><td>CentraSL2 Clinical Centrifuge</td><td>Thomas Scientific</td><td>2517N92-TS</td><td></td></tr><tr><td>Bullet Blender 5 blue homogenizer</td><td>Next &amp;gt;&amp;gt;&amp;gt; Advance</td><td>BBY5MB</td><td>A 40 ml Dounce homogenizer can be used as an alternative</td></tr><tr><td>Synergi 4 &amp;mu;m Hydro-RP 80 &amp;#197; LC Column 250 x 2.0 mm</td><td>Phenomenenex</td><td>00G-4375-B0</td><td>HPLC Column</td></tr><tr><td>SecurityGuard Cartridges AQ C18 4 x 2.0 mm</td><td>Phenomenenex</td><td>AJ0-7510</td><td></td></tr><tr><td>SecurityGuard Guard Cartridges Kit</td><td>Phenomenenex</td><td>KJ0-4282</td><td></td></tr><tr><td>Shimadzu Prominence HPLC with refrigerated auto sampler</td><td>Shimadzu Scientific Instruments, Inc.</td><td></td><td>Any standard HPLC system coupled to a triple quadrupole mass spectrometer should be sufficient</td></tr><tr><td>API 4000 triple quadrupole mass spectrometer</td><td>Applied Biosystems/MDS SCIEX</td><td></td><td></td></tr><tr><td colspan="4"> &lt;table border=&quot;1&quot;&gt; &lt;tbody&gt; &lt;tr&gt; &lt;td colspan=&quot;2&quot;&gt;&lt;strong&gt;M9 buffer recipe (4 liter)&lt;/strong&gt;&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;12 g&lt;/td&gt; &lt;td&gt;KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;24 g&lt;/td&gt; &lt;td&gt;Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;20 g&lt;/td&gt; &lt;td&gt;NaCl&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;Up to 4 liter&lt;/td&gt; &lt;td&gt;Deionized water&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td colspan=&quot;2&quot;&gt;Aliquot to eight 1 liter bottles. Autoclave and cool.&lt;/td&gt; &lt;/tr&gt; &lt;tr&gt; &lt;td&gt;0.5 ml/bottle&lt;/td&gt; &lt;td&gt;1 M MgSO&lt;sub&gt;4&lt;/sub&gt;&lt;/td&gt; &lt;/tr&gt; &lt;/tbody&gt; &lt;/table&gt; </td></tr></tbody></table>

prostaglandin extraction and analysis in caenorhabditis elegans

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Sample preparation was carried out by liquid-liquid extraction adapted from Golovko and Murphy17. It provided excellent recovery of the internal standard (PGF2α-d4). The general scheme for PG extraction from C. elegans is shown in Figure 1. Chromatographic conditions were optimized to provide baseline separation of &gt;30 eicosanoid standards (Table 1 shows 13 standards). A Hydro-Rp column (250 x 2.0 mm i.d) with water and acetonitrile containing 0.1% formic acid provided the best separation and sensitivity. Survey scans of wild-type and fat-3 mutant extracts shown in Figure 2 document global levels of ions within the mass range of 315-360 atomic mass units. fat-3(wa22) mutants lack most 20-carbon PUFAs. An F3 class PG is highlighted. In Figure 3, MRM analyses of a wild-type extract show multiple PG isomers of the F1 (Figure 3A), F2 (Figure 3B), and F3 (Figures 3C and 3D) classes. The F3 class can be detected with either of two mass transitions, m/z 351/193 or m/z 351/191. CePGF2 is predominantly comprised of the PGF2αenantiomer. CePGF1 is likely to be PGF1α or its enantiomer. Figure 4 shows the collision-induced decomposition of CePGF2 (RT=11.8) compared to the PGF2α standard (RT=11.8). Minor differences in product ions are likely due to low CePGF2 abundance and co-eluting compounds in the extract.
<img alt="Lipid extraction and purification; chloroform partition diagram; centrifugation; liquid chromatography; MS/MS analysis." src="/files/ftp_upload/50447/50447fig1.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
 Figure 1. Schematic diagram of C. elegans PGs extraction and LC-MS analyses. Note that acetone/saline and chloroform both have 0.005% BHT to minimize lipid oxidation. Refer to the text for more details. <a href="https://www.jove.com/files/ftp_upload/50447/50447fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Chromatography mass spectra diagram; intensity vs retention time for wild type, fat-3(wa22) comparison." fo:content-width="4.75in" fo:src="/files/ftp_upload/50447/50447fig2highres.jpg" src="/files/ftp_upload/50447/50447fig2.jpg" data-altupdatedat="1787630408000" data-alt="Figure 2"> 
 Figure 2. Survey scans of m/z 315-360 of wild-type and fat-3(wa22) mutant extracts. Liquid chromatography retention time (RT) is shown in panel [A]. Extracted ions at RT = 11.3 are shown for wild-type [B] and fat-3(wa22) [C] extracts. An F3 class PG with mass m/z 351 is highlighted. Cps, counts/sec; a.m.u., atomic mass unit.
<img alt="Mass spectrometry chromatograms; intensity vs. retention time; compound identification peaks; analysis." fo:content-width="4.25in" fo:src="/files/ftp_upload/50447/50447fig3highres.jpg" src="/files/ftp_upload/50447/50447fig3.jpg" data-altupdatedat="1787630408000" data-alt="Figure 3"> 
 Figure 3. MRM analyses of wild-type extracts. F1 class PGs are detected with mass transition m/z 355/311 [A]. Notice that several hydrophobic compounds (RT &gt; 14 min), which are unlikely to be PGs, are also detected with this transition. F2 class PGs are detected with mass transition m/z 353/193 [B]. F3 class PGs are detected with either mass transition m/z 351/193 [C] or m/z 351/191 [D]. Notice that the extracts contain multiple PG isomers of each class. Numbers correspond to PGs shown in Table 2. The concentration of CePGF2 is 1.8 ng/ml. RT, retention time; cps, counts/sec.
<img alt="Mass spectrometry data and chemical structure analysis diagram; lipid identification process." fo:content-width="5.5in" fo:src="/files/ftp_upload/50447/50447fig4highres.jpg" src="/files/ftp_upload/50447/50447fig4.jpg" data-altupdatedat="1787630408000" data-alt="Figure 4"> 
 Figure 4. Collision-induced decomposition of chemically synthesized PGF2α and CePGF2. Arrows in panel [A] indicate product or fragmentation ions shared between PGF2α and CePGF2 (RT=11.8). The product ions at m/z 309 and m/z 193 are generated from indicated cleavage sites (highlighted a and b), correspond to the structures shown, and are characteristic of F-series PGs [B]. Cps, counts/sec. <a href="https://www.jove.com/files/ftp_upload/50447/50447fig4large.jpg" target="_blank">Click here to view larger figure</a>.
<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>Standard</td>
			<td>RT (min)</td>
			<td>[M-H]- m/z</td>
			<td>Key product ions in MS/MS</td>
		</tr>
		<tr>
			<td>20-hydroxy PGE2</td>
			<td>9.43</td>
			<td>367</td>
			<td>349, 331, 287, 234, 189, 129, 109</td>
		</tr>
		<tr>
			<td>PGF3-</td>
			<td>11.26</td>
			<td>351</td>
			<td>333, 307, 289, 271, 245, 219, 209, 193, 191, 171, 165, 111</td>
		</tr>
		<tr>
			<td>Thromboxane B2</td>
			<td>11.66</td>
			<td>369</td>
			<td>289, 191, 177, 169, 151</td>
		</tr>
		<tr>
			<td>PGF2-</td>
			<td>11.80</td>
			<td>353</td>
			<td>335, 309, 291, 273, 263, 247, 235, 209, 193, 171, 165, 111</td>
		</tr>
		<tr>
			<td>PGF1-</td>
			<td>11.79</td>
			<td>355</td>
			<td>337, 319,311, 301, 293, 275, 265, 237, 211, 195</td>
		</tr>
		<tr>
			<td>Lipoxin B4</td>
			<td>12.38</td>
			<td>351</td>
			<td>201, 191,189, 165, 155, 115, 107, 71, 59</td>
		</tr>
		<tr>
			<td>PGD2</td>
			<td>12.56</td>
			<td>351</td>
			<td>315, 271,203, 189</td>
		</tr>
		<tr>
			<td>5(S), 6(R)- Lipoxin A4</td>
			<td>12.83</td>
			<td>351</td>
			<td>235, 217, 189, 144, 135, 115, 99, 59</td>
		</tr>
		<tr>
			<td>PGA2</td>
			<td>14.02</td>
			<td>333</td>
			<td>315, 297, 271, 235, 191, 189, 175, 163, 137, 113, 109</td>
		</tr>
		<tr>
			<td>Δ12-PGJ2</td>
			<td>14.20</td>
			<td>333</td>
			<td>271, 189, 123</td>
		</tr>
		<tr>
			<td>Leukotriene B4</td>
			<td>14.73</td>
			<td>335</td>
			<td>181, 109, 93, 71, 69, 59, 57</td>
		</tr>
		<tr>
			<td>15d-Δ12,14-PGJ2</td>
			<td>16.80</td>
			<td>315</td>
			<td>297, 271, 217, 203, 158</td>
		</tr>
		<tr>
			<td>5(S)-HpETE</td>
			<td>17.88</td>
			<td>335</td>
			<td>97, 83, 81, 57</td>
		</tr>
	</tbody>
</table>
Table 1. Retention times and key product ions of 13 eicosanoid standards from the LC-MS/MS method3 . Over 30 standards were used to develop the LC-MS/MS program. The chromatographic retention times (RTs) differ, even among very similar PGs. An exception is PGF1α and PGF2α. Nevertheless, these PGs are distinguished by their parent ion masses ([M-H]- m/z) and collision-induced decomposition spectra (MS/MS).
<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>Prostaglandin class</td>
			<td>No. in Figure 3</td>
			<td>[M-H]- m/z</td>
			<td>Key product ions in MS/MS</td>
		</tr>
		<tr>
			<td>F1 (CePGF1)</td>
			<td>1</td>
			<td>355</td>
			<td>319, 301, 311, 293, 275, 265, 237, 223, 211, 195, 157</td>
		</tr>
		<tr>
			<td>F1</td>
			<td>2</td>
			<td>355</td>
			<td>311, 301, 293, 275, 265, 211, 195, 167, 157</td>
		</tr>
		<tr>
			<td>F2 (CePGF2)</td>
			<td>3</td>
			<td>353</td>
			<td>309, 291, 273, 263, 247, 209, 193, 171, 165, 127</td>
		</tr>
		<tr>
			<td>F2</td>
			<td>4</td>
			<td>353</td>
			<td>309, 291, 273, 263, 255, 247, 219, 209, 193, 171, 113</td>
		</tr>
		<tr>
			<td>F3</td>
			<td>5</td>
			<td>351</td>
			<td>315, 307, 289, 275, 249, 205, 193, 191, 167, 153, 139</td>
		</tr>
		<tr>
			<td>F3</td>
			<td>6</td>
			<td>351</td>
			<td>333, 289, 271, 261, 245, 223, 193, 191, 163</td>
		</tr>
	</tbody>
</table>
Table 2. Collision-induced decomposition product ions for several major C. elegans F1, F2, and F3 PGs shown in Figure 3. MS/MS of other less abundant isomers are not shown. These data are from multiple analyses and not all product ions may be visible in a given run. Given the complexity of the extracts, some less abundant product ions may derive from another parent ion with similar retention time or result from interference. Peak 2 is likely a stereoisomer of CePGF1 and peak 4 is likely a stereoisomer of CePGF2. See Edmonds, et al. (2010) for additional data3.

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

This procedure aims to identify and measure prostaglandins and other eicosanoids in extracts of sea elegance. First, grow the worms in large scale cultures. Then using a liquid liquid extraction technique, extract the hydrophilic lipids from the worm tissue, inject the lipid extracts into an HPLC column coupled to a mass spectrometer.Then analyze the data from the liquid chromatography tandem mass spectrometry. Ultimately this lc MSMS approach to purified warm extracts can identify new metabolites and also measure known metabolites such as the S series prostaglandins. So the main advantage of this technique over existing methods, such as enzyme immunoassays, is that it is highly sensitive and specific, while at the same time, comprehensive and flexible demonstrating this technique will be Hugh Hong, a graduate student from my laboratory For supplemental feeding of the worms pellet and NA 22.Bacterial culture by centrifugation, discard the supernatant and resus suspend the bacterial pellet in 100 milliliters of M nine buffer. Store the concentrated bacterial solution at four degrees Celsius. Start the worm cultures on five extra large NGM plates.Continue to propagate the worms for three to four generations until the plates are full of gravity. Adults to prevent starvation at approximately one milliliter of the concentrated bacteria to the worm plates as needed. Air dry completely before placing the lid.Next, wash the grab hermaphrodites off the plates with M nine buffer. Harvest the worms in a 50 milliliter conical polypropylene tube. Then allow the worms to settle to the bottom.Discard the supernatant containing bacteria eggs and larval stage worms. Now Resus, suspend the remaining worms in 15 milliliters of M nine buffer and gently mix. Distribute 200 microliters of the worm suspension to each of 60 seeded N GM plates.Keep the worm solution mixed well until all plates have been seeded to ensure that they receive roughly equal numbers of worms. Disperse the worms across the plates. Grow the worms for two to four generations, supplementing with one milliliter of concentrated bacteria per plate per 12 to 24 hour period.When the plates are full of GR adults, harvest the worms one strain at a time. Wash the worms off the plates with M nine buffer and transfer to six 50 milliliter polypropylene tubes. Fill the tubes with M nine buffer to wash the worms.When the grab adult settle to the bottom, remove about 35 milliliters of the supernatant. Then pull the worms into one or two tubes. Repeat the M nine wash three times or until the supernatant is transparent with a large bore pasti pipette.Transfer as many worms as possible to a 15 milliliter polypropylene conical tube. Centrifuge at 1000 RCF for five minutes and discard the supernatant. Repeat the harvesting, washing, and consolidating steps for the other strains.Store stocks of the harvested worm strains at minus 80 degrees Celsius. Note the weight of a polypropylene conical tube using a red hot razor blade. Cut a frozen polypropylene tube containing harvested worms near the six milliliter mark.Under sterile conditions. Transfer about six grams of frozen worm pellet to the thought sample. Add one nanogram of the internal standard and 12 milliliters of ice cold acetone saline solution.With BHT mix, well distribute 1.5 milliliters of the worm slurry to 12 five milliliter self-standing plastic tubes to each tube. Add 0.7 milliliters of the serious stabilized zirconium oxide beads. Homogenize the worm suspension if necessary.Repeat the process for another minute to obtain less than 10%of intact worms using a nine inch pasture pipette. Carefully transfer the homogenate but not the beads into four 10 milliliter conical glass tubes. Combine the beads from three tubes and wash with one milliliter of acetone saline solution containing BHT.Repeat this wash for the remaining bead containing tubes. Transfer the wash solution to the glass tubes on ice. Next, centrifuge the homogenate at 1000 Gs for 10 minutes.At four degrees Celsius, transfer the supernatant from each tube to a clean 10 milliliter glass conical tube. Then in a chemical hood, add equal volume of hexane to each tube vortex at maximum speed for 30 seconds. After centrifusion at four degrees Celsius at 1000 Gs for 10 minutes.Discard the upper phase containing the hexane and neutrally charged lipids at two millimolar formic acid to acidify the lower aqueous phase. Testing with pH strips for a pH of 3.5. Now, add equal volumes of chloroform to each tube vortex at maximum speed for 30 seconds.Centrifuge the samples at four degrees Celsius at about 1000 GS per 10 minutes. Insert a glass pipette tip into the lower chloroform phase and try to avoid the solid interface by tilting the tube. Pull the extracts from four tubes into a single 15 milliliter conical glass tube.Purge the air in the glass tube with nitrogen gas and incubate at minus 20 degrees Celsius for 24 hours to two weeks. After thawing the extract, discard the remaining milky aqueous top layer if present. Using a new past pipette.Transfer the chloroform to a labeled Teflon lined half DRM glass vial in a chemical hood. Evaporate the organic solvent in a gentle stream of nitrogen gas store samples at minus 20 degrees Celsius for up to two weeks. First, prepare stock solutions of individual reference prostaglandins in methanol.Now generate a standard curve by serial dilution in 80%methanol. Next, dissolve the dried sea elegance lipid extracts in 200 microliters of 80%methanol for analysis. Inject 20 to 50 microliters of the sample into the reverse phase column and perform gradient analysis as detailed in the text protocol using negative ion mode ESI interface.Introduce the column effluent into the mass spectrometer. Set the collision gas at 10 electron volts. The collision energy at minus 35 electron volts.The temperature at 600 degrees Celsius. The decluttering potential at minus 90 and the cell exit potential at minus 11 generate a standard curve to analyze the different classes of prostaglandins present in the extracted samples using analyst software. Process the data to identify prostaglandins present in the worm extracts.Survey scans of the wild type and fat three mutant worm extracts show the global levels of ions within the mass range of 315 to 360 atomic mass units. Compared to the wild type worms, the fat three mutants lack most 20 carbon pfas as exemplified by an F three class prostaglandin. With master charge ratio 3 51 highlighted in gray, MRM analyses can identify the different classes of prostaglandins.For instance, the F1 class prostaglandins are present in wild type extracts as well as several other hydrophobic compounds with greater retention times varying the mass transition allows for further analysis of the F two and F three class prostaglandins indicating the worm extracts contain multiple prostaglandin isomers of each class. Comparison of the collision induced decomposition of the sea elegance prostaglandin to the standard prostaglandin can be used to map to product ions of expected cleavage sites. In order to prevent lipid oxidation.Remember to take the appropriate precautions. For example, add the antioxidant VHT to the indicated solution and purge extract with nitrogen gas.

prostaglandin-extraction-and-analysis-in-caenorhabditis-elegans

Research

JoVE Journal

Biology

Following exposure to specific environmental stressors, the nematode Caenorhabditis elegans undergoes extensive phenotypic plasticity to enter into a stress-resistant ‘dauer’ juvenile stage.  We present methods for the controlled induction and imaging of neuroplasticity during dauer.

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

We present an immunofluorescence imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

This protocol demonstrates how to track a protein nuclear translocation under heat stress by using a green fluorescence protein (GFP) fusion protein as a marker and 4',6-diamidino-2-phenylindole (DAPI) staining. The DAPI staining protocol is fast and preserves the GFP and protein subcellular localization signals.

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

Sperm must successfully navigate through the oviduct to fertilize an oocyte. Here, we describe an assay for measuring sperm migration within the C. elegans hermaphrodite uterus. This assay can provide quantitative data on sperm distribution within the uterus after mating, as well as on speed, directional velocity, and reversal frequency.

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis.

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

A protocol for imaging the dynamic microtubules in vivo using fluorescently labeled End binding protein has been presented. We described the methods to label, image, and analyze the dynamic microtubules in the Posterior lateral microtubule (PLM) neuron of C. elegans.

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

Presented here is a description of the straightforward and relatively rapid isolation of Caenorhabditis elegans genomic DNA from one or a few animals using a commercially available tissue kit. The resulting gDNA preparation is a suitable template for PCR.

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

This protocol describes an RNA interference and ChIP assay to study the epigenetic inheritance of RNAi-induced silencing and associated chromatin modifications in C. elegans.

Analysis of Transgenerational Epigenetic Inheritance in C. elegans Using a Fluorescent Reporter and Chromatin Immunoprecipitation (ChIP)

This protocol establishes methods for extracting and quantifying responses to the volatile sex pheromone in C. elegans, providing tools to study chemical communication and navigation trajectory.

Volatile Sex Pheromone Extraction and Chemoattraction Assay in Caenorhabditis elegans

Learn prostaglandin extraction and analysis in Caenorhabditis elegans using LC-MS/MS. This optimized method enables sensitive, specific identification of eicosanoids for metabolic research.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

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