Analysis of Ascarosides from Caenorhabditis elegans Using Mass Spectrometry and NMR Spectroscopy

Abstract

The nematode Caenorhabditis elegans secretes a family of water-soluble small molecules, known as the ascarosides, into its environment and uses these ascarosides in chemical communication. The ascarosides are derivatives of the 3,6-dideoxysugar ascarylose, modified with different fatty acid-derived side chains. C. elegans uses specific ascarosides, which are together known as the dauer pheromone, to trigger entry into the stress-resistant dauer larval stage. In addition, C. elegans uses specific ascarosides to control certain behaviors, including mating attraction, aggregation, and avoidance. Although in general the concentration of the ascarosides in the environment increases with population density, C. elegans can vary the types and amounts of ascarosides that it secretes depending on the culture conditions under which it has been grown and its developmental history. Here, we describe how to grow high-density worm cultures and the bacterial food for those cultures, as well as how to extract the culture medium to generate a crude pheromone extract. Then, we discuss how to analyze the types and amounts of ascarosides in that extract using mass spectrometry and NMR spectroscopy.

1 Introduction

C. elegans secretes ascarosides as chemical signals to coordinate its development and behavior. All ascarosides are derivatives of the 3,6-dideoxysugar ascarylose, with different fatty acid-derived side chains [1-9]. The ascarosides that are found in C. elegans culture medium can be divided into several structural classes (Table 1). In this chapter, we describe a general method for growing high-density worm cultures and extracting the culture medium in order to generate a crude pheromone extract that contains secreted ascarosides (Subheadings 3.1 and 3.2). This crude pheromone extract can be directly used in biological assays, such as the dauer formation assay (see Chapter 20), it can be fractionated chromatographically for bioactivity-guided purification of ascarosides [1-4 , 6], or the ascarosides in the extract can be analyzed using analytical techniques, such as liquid chromatography-mass spectrometry (LC-MS) (Subheading 3.3), LC-MS/MS (Subheading 3.4), and NMR spectroscopy (Subheading 3.5). Each of these techniques has its own advantages and disadvantages, as described below. The structural class of a specific ascaroside, as well as its concentration in the crude pheromone extract, can affect whether or not it can be detected in the extract using a specific analytical technique.

Table 1.

Summary of ascarosides found in wild-type (N2) C. elegans culture medium that have had their structures confirmed through chemical synthesis^a

Structure-based name	Other name(s)	Orig. ref.	Mol. formula	[M+Na]+	[M−H]−	n	R	Structure
Ascarosides with a saturated fatty acid side chain that is attached to the sugar at the penultimate (ω–1) carbon
asc-C4	ascr#11	[7]	C10H18O6	257.1001	233.1025	1	H
asc-C5	ascr#9	[7,8]	C11H20O6	271.1158	247.1182	2	H
IC-asc-C5	indolecarboxyl-  ascaroside C5;  ascaroside C5; icas#S	[6]	C20H25NO7	414.1529	390.1553	2	IC
OS-asc-C5	osas#9	[9]	C23H33NO10	506.2002	482.2026	2	OS
asc-C7	daumone 1; ascr#l;  ascaroside C7	[1]	C13H24O6	299.1471	275.1495	4	H
IC-asc-C7	icas#l	[7,8]	C22H29NO7	442.1842	418.1866	4	IC
asc-C9	ascr#10	[7,8]	C15H28O6	327.1784	303.1808	6	H
Ascarosides with an α,β-unsaturated (Δ) fatty acid side chain that is attached to the sugar at the penultimate (ω–1) carbon
asc-ΔC7	ascr#7	[5]	C13H22O6	297.1314	273.1339	2	H
IC-asc-ΔC7	icas #7	[7,8]	C24H33NO7	440.1685	416.1709	2	IC
asc-ΔC9	ascaroside C9;  ascr#3; daumone 3	[2]	C15H26O6	325.1627	301.1651	4	H
IC-asc-ΔC9	icas#3	[7,8]	C24H31NO7	468.1998	444.2022	4	IC
HB-asc-ΔC9	hbas#3	[7]	C22H30O8	445.1838	421.1862	4	HB
MB-asc-ΔC9	mbas#3	[7]	C20H32O7	407.2046	383.2070	4	MB
Ascarosides with a side chain that is attached to the sugar at the penultimate (ω–1) carbon and terminates in a methyl ketone (MK)b
asc-C6-MK	ascaroside C6;  ascr#2; daumone 2	[2]	C12H22O5	269.1365	n.a.	2	H
Glu-asc-C6-MK	ascr#4; daumone 4	[4]	C18H32O10	431.1893	n.a.	2	Glu
Ascarosides with a saturated fatty acid side chain that is attached to the sugar at the terminal (ω) carbon
asc-ωC3	ascaroside C3; ascr#5;  daumone 5	[3]	C9H16O6	243.0845	219.0869	1	–
asc-ωC5	oscr#9	[7]	C11H20O6	271.1158	247.1182	3	–
asc-ωC9	oscr#10	[7]	C15H28O6	327.1784	303.1808	7	–
Ascarosides with an α,β-unsaturated (Δ) side chain attached at the penultimate (ω–1) carbon and linked to p-aminobenzoic acid (PABA)
asc-ΔC7-PABA	ascr#8	[5]	C20H27NO7	n.a.	392.1709	2	-
R group definitions:

The structure-based name given in the first column assumes that the canonical ascaroside structure is an ascaroside with a saturated fatty acid side chain that is attached to the sugar at the penultimate (ω–1) carbon. Deviations from the canonical structure are indicated such that each name conforms to the following: (modifications to the sugar)-asc-(deviations in the fatty acid)C#-(modifications at the end of the fatty acid)

Modifications to the sugar and their abbreviations are listed at the bottom of the table. Deviations in the fatty acid include attachment of the fatty acid to the sugar at the terminal (to) carbon, instead of penultimate (ω-1) carbon, as well as α-β (Δ) unsaturation of the fatty acid. Modifications at the end of the fatty acid include the methyl ketone (MK) moiety and addition of p-aminobenzoic acid (PABA)

^aMany other ascarosides, such as asc-C6 (ascr#12), asc-C8 (ascr#14), IC-asc-C9 (icas#10), asc-Cll (ascr#18), asc-ωCll (oscr#18), asc-C12 (ascr#20), OS-asc-C9 (osas#10), and OS-asc-C6-MK (osas#2), have also been detected in wild-type C. elegans culture medium, but their structures have not yet been fully confirmed through chemical synthesis [7-9]

^bReduced versions of asc-C6-MK (ascr#6.1 and ascr#6.2) with a hydroxyl instead of a ketone have also been reported [5]

LC-MS analysis on a single quadrupole mass spectrometer (Subheading 3.3) is a rapid technique for analyzing the concentrations of specific known ascarosides in a sample [10 , 11]. In LC-ESI-MS, the molecules in the extract are separated on a column based on their polarity. As the molecules elute from the column, they are ionized by electrospray ionization (ESI), and the mass-to-charge (m/z) ratios of the ionized species are then detected. Most ascarosides will ionize most strongly as positively charged, sodium adducts. Thus, they can be detected by positive ion mode LC-MS, in which the ionized ascaroside is detected as [M+Na]⁺ , where M is the mass of the ascaroside (see Table 1 for which ascarosides ionize in positive ion mode). Many ascarosides, but not all, will also ionize as negatively charged, deprotonated species. Thus, they can also be detected by negative ion mode LC-MS, in which the ionized ascaroside is detected as [M−H]⁻ (see Table 1 for which ascarosides ionize in negative ion mode). In order to use LC-MS to identify the presence of a specific ascaroside and to determine the concentration of that ascaroside in the extract, a synthetic standard is needed for the ascaroside of interest. The crude pheromone extract can contain non-ascaroside compounds with masses and retention times that are similar to the ascarosides of interest. For this reason, LC-MS-based identification of ascarosides is usually confirmed with additional techniques, including LC-MS/MS or NMR spectroscopy. LC-MS can be used to analyze the ascarosides in (1) the crude pheromone extract from the culture medium of a non-synchronized culture (generated by method given in Subheading 3.2), (2) the crude pheromone extract from the culture medium of a synchronized culture [11], (3) the crude extract from the worms themselves [10], or (4) the secretions from worms produced over a short period of time (“worm water”) [4]. LC-MS is not useful for characterizing the chemical structures of novel ascarosides, as this must be done using techniques that provide additional structural information, such as LC-MS/MS and NMR spectroscopy.

LC-MS/MS analysis on a triple quadrupole mass spectrometer (Subheading 3.4) allows the simultaneous identification of many of the ascarosides present in the crude pheromone extract [7]. LC-MS/MS is similar to LC-MS in that the molecules in the extract are separated on a column based on their polarity and ionized. The m/z ratios of the ionized species are then detected. However, in LC-MS/MS, the molecules are not only ionized to detect the ionized species (called the precursor ion), but also broken apart, resulting in product ions that provide additional structural information about the molecules. When LC-MS/MS is performed on a triple quadrupole mass spectrometer, a precursor ion scan can be used to detect only molecules that give rise to a specific product ion and report back the precursor ions that produced the specific product ion. This technique is particularly useful for the detection of classes of molecules with similar structures, such as the ascarosides, that all produce a common product ion. Those ascarosides that ionize in negative ion mode can be fragmented in LC-MS/MS to produce a common product ion of m/z 73 [C₃H₅O₂]. Thus, this technique, which was developed by von Reuss and coworkers [7], can be used to detect all ascarosides in a crude pheromone extract that ionize as negative ions and report back the precursor ions (that is, [M−H]⁻) corresponding to those ascarosides (Subheading 3.4). Those ascarosides that ionize only in positive ion mode can be monitored by looking for a neutral loss of 130 amu [C₆H₁₀O₃] that corresponds to the loss of the ascarylose sugar [7]. However, fragmentation of the ascarosides in positive ion mode is not as efficient as that in negative ion mode, leading to lower and less reliable signal, and will not be described in this review.

NMR spectroscopy can be used to analyze the ascarosides in a crude pheromone extract or chromatography fractions derived from that extract (Subheading 3.5). One type of NMR experiment that is particularly informative is the double quantum-filtered correlation spectroscopy (dqf-COSY) experiment [5 , 10]. Like a standard COSY experiment, this experiment detects correlations between protons on neighboring carbons in a molecule, and these correlations appear as peaks off the diagonal of the spectrum. However, the dqf-COSY experiment is generally run in a phase-sensitive mode and gives a much cleaner spectrum near the diagonal than COSY, which is important for analysis of mixtures containing multiple components, such as crude pheromone extract, which may have many peaks near the diagonal. Each dqf-COSY correlation provides additional information about the protons involved in the correlation, including the coupling constant between those two protons (active coupling), as well as the coupling constants of those protons with other neighboring protons (passive coupling). The dqf-COSY spectrum of the crude pheromone extract contains peaks that are diagnostic for the ascarosides. Furthermore, dqf-COSY spectra have been acquired for many of the known ascarosides, and thus, the presence of a particular ascaroside in a crude pheromone extract or chromatography fraction can be substantiated by dqf-COSY. It should be noted, however, that the dqf-COSY spectrum of ascarosides from the same structural class, but with different side chain lengths, will be similar. One drawback of using NMR spectroscopy to analyze the ascarosides in the crude pheromone extract is that less abundant ascarosides cannot be detected because NMR spectroscopy is an inherently less sensitive technique than mass spectrometry. Further fractionation of the crude pheromone extract to give a less complex sample containing the specific ascaroside of interest is needed. However, chromatographic purification of individual ascarosides is dependent on the polarity of those ascarosides and done on an ad hoc basis, and thus, it is beyond the scope of this review.

2 Materials

2.1 25× Bacterial Stock

1. E. coli strain OP50 or HB101, grown on Luria-Bertani (LB)-agar.

2. LB broth.

3. Falcon tube (50 mL) for bacterial mini-culture.

4. Laminar flow hood.

5. 2.8 L baffled flask for large bacterial culture.

6. Shaker for shaking bacterial culture at 37 °C.

7. Refrigerated table-top centrifuge.

8. Potassium phosphate (1 M, pH 6): In 0.8 L water, dissolve 136.1 g KH₂PO₄ . Add potassium hydroxide until the pH reaches 6. Then add water to a final volume of 1 L. Autoclave.

9. S basal: 0.1 M NaCl, 0.05 M potassium phosphate (pH 6). Autoclave.

10. Microfuge and 1.5 mL Eppendorf tubes.

2.2 Worm Culturing

1. C. elegans, wild-type (N2) or mutant strain.

2. Stereomicroscope and worm pick for manipulating worms.

3. Cholesterol (5 mg/mL in ethanol): Dissolve 50 mg of cholesterol in 10 mL of ethanol (200 proof). Sonicate for approximately 10 min or shake until the cholesterol dissolves.

4. 25× bacterial stock from Subheading 3.1 for feeding worm cultures. 25 mL of 25× stock (corresponding to 625 mL-worth of bacterial culture) is needed for every 150 mL of worm culture that will be grown.

5. 10 cm NGM-agar plates with bacterial lawn: In 500 mL water, add 1.5 g NaCl, 8.5 g granulated agar, and 1.25 g peptone. Autoclave, and then add: 0.5 mL 1 M CaCl₂, 0.5 mL 1 M MgSO₄, 12.5 mL potassium phosphate (pH 6), and 0.5 mL 5 mg/mL cholesterol. Pour into 10 cm plates, and let them dry on the benchtop overnight. Pipet 0.75 mL 25× bacterial stock onto each plate, spread on plate (but do not allow lawn to touch plate edge), and dry in laminar flow hood or on benchtop. Approximately one plate is needed for every 150 mL of worm culture that will be grown.

6. Potassium citrate (1 M, pH 6): In 0.8 L water, dissolve 210.14 g citric acid monohydrate. Add potassium hydroxide until the pH reaches 6. Then add water to a final volume of 1 L. Autoclave.

7. Trace metals solution: In 1 L water, dissolve 1.86 g disodium EDTA (5 mM), 0.69 g FeSO₄ · 7H₂O (2.5 mM), 0.20 g MnCl₂ · 4H₂O (1 mM), 0.29 g ZnSO₄ · 7H₂O (1 mM), 0.025 g CuSO₄ · 5H₂O (0.1 mM). Autoclave. Wrap bottle in foil, and store in the dark.

8. S medium: Autoclave 150 mL of S basal in a 500 mL Erlenmeyer flask, covering the top with foil. Just before use, add 1.5 mL 1 M potassium citrate (pH 6), 1.5 mL trace metals solution, 0.45 mL 1 M CaCl₂ , 0.45 mL 1 M MgSO₄ , and 150 μL cholesterol (5 mg/mL).

9. Optional: To help prevent contamination of the cultures, add the antifungal nystatin (100 U/mL final concentration) and the antibiotics penicillin–streptomycin (50 U/mL final concentration for penicillin and 50 μg/mL final concentration for streptomycin).

10. Refrigerated shaker for culturing worms in shaker flasks at 22.5 °C.

11. Graduated cylinder (250 mL) and ice bath.

12. Refrigerated table-top centrifuge and polypropylene centrifuge bottles with screw caps.

13. Lyophilizer and lyophilization flask (600 mL).

14. Mortar and pestle.

15. Erlenmeyer flask (500 mL) and funnel.

16. Ethanol (190 proof).

17. Tabletop shaker for performing extractions.

18. Filter paper (Whatman, No. 41), Buchner funnel, neoprene adaptor, and Erlenmeyer filtering flask (500 mL).

19. Rotary evaporator and round bottom flask (250 mL).

20. Short (5.75 in.) and long (9 in.) Pasteur pipets and bulbs.

21. Clean ball of cotton.

22. Bendable spatula.

23. Speedvac, rotor for 8 mL glass vials, and 8 mL glass vials (e.g., Fisher, cat. 03-340-60B) (see Note 1).

2.3 LC-MS

1. 1. Methanol.

2. Sonicator.

3. Speedvac and rotor for 8 mL glass vials (see Note 1).

4. Microfuge and methanol-safe 1.5 mL Eppendorf tubes (e.g., Fisher, cat. 05-408-129).

5. Mass spectrometer: Agilent 6130 single quad mass spectrometer with API-ES source and operating in dual negative/positive scan mode.

6. LC: Agilent 1260 infinity binary pump with autosampler.

7. Column: Phenomenex 5 μm Luna C₁₈ 2, 100 Å (100 × 4.6 mm).

8. LCMS-grade water with 0.1 % formic acid (solvent A).

9. LCMS-grade acetonitrile with 0.1 % formic acid (solvent B).

10. Autosampler vials and glass inserts with springs.

2.4 LC-MS/MS

1. Methanol.

2. Sonicator.

3. Speedvac and rotor for 8 mL glass vials (see Note 1).

4. Microfuge and methanol-safe 1.5 mL Eppendorf tubes (e.g., Fisher, cat. 05-408-129).

5. Mass spectrometer: Thermo TSQ quantum ultra triple quadrupole mass spectrometer using argon collision gas, operating in negative ion, heated (H)-ESI, precursor scanning mode, selecting for a product ion of 73.0.

6. LC: Accela UHPLC (max pressure of 1,000 bar) with autosampler.

7. Column: Kinetex 2.6 μm C18 100 Å (100 × 2.10 mm).

8. LCMS-grade water (solvent A).

9. LCMS-grade acetonitrile with 0.1 % acetic acid (solvent B).

10. Autosampler vials and glass inserts with springs.

2.5 NMR Spectroscopy

1. Lyophilizer.

2. Methanol-d₄.

3. Sonicator.

4. Short (5.75 in.) and long (9 in.) Pasteur pipets and bulbs.

5. Clean ball of cotton.

6. 5 mm methanol-matched Shigemi NMR tube (Shigemi, cat. MMS-005v) (see Note 2).

7. Parafilm.

8. NMR spectrometer: Varian 500 MHz NMR spectrometer.

9. NMR probe: 5 mm Varian indirect-detection, triple resonance probe (¹H/¹³C/(³¹P-¹⁵N)PFG).

10. Pasteur pipets with extra long tips for removing samples from NMR tubes (Fisher, cat. 13-678-6).

11. Speedvac, rotor for 8 mL glass vials, and 8 mL glass vials (e.g., Fisher, cat. 03-340-60B).

3 Methods

3.1 Preparing 25× Bacterial Stock

This method prepares the bacterial food (25× bacterial stock) needed for growing the worm cultures in Subheading 3.2. Sterile technique is important in the preparation of the 25× bacterial stock. If the 25× bacterial stock is contaminated with bacteria or fungus, this contamination may overwhelm the worm cultures that are grown in Subheading 3.2 , regardless of whether antibiotics and antifungals are added to the worm culture medium.

1. Inoculate 5 mL LB with a colony of OP50 or HB101 bacteria. Let the mini-culture grow for 6–8 h in a shaker at 250 rpm at 37 °C (see Note 3).

2. Use 1 mL of this mini-culture to inoculate 1 L of LB broth in a 2.8 L baffled flask. Grow culture overnight in a shaker at 250 rpm at 37 °C.

3. Collect bacteria by centrifuging at 2,700 × g for 10 min. Discard the supernatant, and resuspend in enough S basal to make the final volume equal to 40 mL to give a 25× bacterial stock. Transfer to a 50 mL falcon tube, and store at 4 °C (see Notes 4-6).

3.2 Unsynchronized Worm Cultures and Generating Crude Pheromone Extract

This method describes how to grow unsynchronized worm cultures and how to extract the culture medium to generate crude pheromone extract. Production of many of the ascarosides is maximized by feeding the worm cultures regularly with sufficient bacterial food to allow reproduction and increase worm density. It is important not to feed the worms too much on any 1 day because they will suffocate, but it is also important not to deprive the worms of food. The exact culture conditions used will influence the amounts of the ascarosides produced.

1. Passage 20 N2 worms onto a 10 cm NGM-agar plate with a bacterial lawn.

2. Let worms grow for ~4 days at room temperature until just before the bacterial food supply runs out.

3. Wash the plate with 10 mL of S medium, and put in a 15 mL tube. Count the number of worms in 3 × 10 μL drops under the stereomicroscope to gauge the density of worms. Make sure that you invert the tube several times before taking out each 10 μL sample (see Note 7).

4. Add ~90,000 worms per 150 mL culture.

5. Feed the worms 3 mL of 25× bacterial stock (110–125 mg/mL). For our feeding schedule, this day is considered day 0.

6. Shake at 22.5 °C at 225 rpm for 9 days. Feed worms 1 mL 25× bacterial stock on day 1, and 3 mL 25× bacterial stock on days 2–8.

7. On day 9, harvest the culture by transferring the culture to a tall container, such as a 250 mL graduated cylinder, and place the container in an ice-bath for 30 min to 1 h in order to settle the worms. Transfer the supernatant to a centrifuge bottle until you cannot pipet any more of the supernatant without disturbing the settled worms. Transfer the remaining worms and culture medium from the bottle to a 50 mL falcon tube, centrifuge at 200 × g for 5 min. Pipet the remaining supernatant from the falcon tube into the centrifuge bottle together with the previously pipetted supernatant. Centrifuge the centrifuge bottle at 2,700 × g for 10 min to remove the bacteria from the supernatant. Then carefully transfer the supernatant to a 600 mL lyophilization flask, and freeze by placing it at −20 °C overnight.

8. Lyophilize the frozen culture medium to dryness (2–3 days) (see Note 8).

9. Scrape out solid material into a mortar, and grind with a pestle to a fine powder.

10. Place the powder in a 500 mL Erlenmeyer flask using a funnel, and extract by adding 150 mL ethanol (190 proof) and shaking in a table-top shaker at 250 rpm for 2 h.

11. Filter the extraction mixture through filter paper in a Buchner funnel.

12. Place the retentate back in the Erlenmeyer flask from step 10 , and extract a second time with 150 mL of ethanol (190 proof), shaking overnight (see Note 9).

13. Filter the extraction mixture through filter paper in a Buchner funnel, and combine the filtrate with the filtrate from step 11.

14. Rotovap the combined filtrates in a round bottom flask to a small volume (~3 mL).

15. Place a small amount of cotton in a short Pasteur pipet, and pack it down inside the Pasteur pipet using a long Pasteur pipet, such that the packed cotton plug is about 3 mm in length.

16. Wash the cotton in the Pasteur pipet with a small amount of ethanol to remove any impurities from the cotton. Use a bulb to push the ethanol through the Pasteur pipet and dry the cotton.

17. Filter the sample through the cotton plug in the Pasteur pipet into a 8 mL glass vial. Use a bulb to push the sample through the Pasteur pipet.

18. Wash the round bottom with ~3 mL of ethanol (190 proof). Scrape the sides of the round bottom with a metal spatula to make sure that as much of the solid is exposed to the ethanol as possible.

19. Filter this ethanol wash through the cotton plug in the Pasteur pipet into the 8 mL glass vial from step 17 . Use a small amount of ethanol to wash the plug, and add this last wash to the glass vial as well.

20. Speedvac the sample in the glass vial to dryness. Store the sample at −20 °C until use.

3.3 LC-MS Quantitation of Ascarosides

This method can be used to quantify the concentrations of the ascarosides in a particular sample, such as in the crude pheromone extract (Fig. 1) or in worm secretions produced over a short period of time (“worm water”) [4]. The mass spectrometer must be well maintained with low background signal. It should be noted that this method can underestimate the amount of an ascaroside in a sample, especially if the sample is complex, if ion suppression occurs.

Fig. 1.

LC-MS-based determination of the concentrations of asc-ΔC9, asc-C6-MK, asc-ωC3, and IC-asc-C5 in crude pheromone extract. A calibration curve of synthetic ascarosides was generated by injecting 5 μL of several concentrations of the ascarosides and fi tting a quadratic curve to the data. 5 μL of crude pheromone extract was also injected, and the relevant ion for each ascaroside was extracted from the positive total ion count trace. The area under the peak for each ascaroside was used to estimate the number of pmol in 5 μL of crude pheromone extract. This number then was used to estimate the concentration of each ascaroside in the conditioned medium that was used to generate the crude pheromone extract. ( a) The concentration of asc-ΔC9 in the conditioned medium is ~273.7 nM. ( b) The concentration of asc-C6-MK in the conditioned medium is ~204.9 nM. ( c) The concentration of asc-ωC3 in the conditioned medium is ~144.5 nM. ( d) The concentration of IC-asc-C5 in the conditioned medium is ~77.4 nM

1. Resuspend the sample from Subheading 3.2 , step 20 , by adding 1 mL methanol. Sonicate for 20 min, vortex briefly to dislodge any remaining solids on the sides of the vial, and sonicate again for 20 min. Centrifuge the vial (with the cap closed) in the speedvac for 2 min to pellet most of the particulates.

2. Pipet 100 μL of the supernatant into a 1.5 mL Eppendorf tube, and centrifuge at 17,600 × g for 1 min to pellet any remaining particulates. Pipet 50 μL from the top of the sample to an autosampler vial. Pipet the remaining 50 μL of the sample from the tube back into vial (see Note 10).

3. Make 0.2, 0.4, 2, 4, 8, 20, 40, 60, 80, and 100 μM stocks of each synthetic standard in methanol. Transfer 20 μL of each stock to an autosampler vial (see Note 11).

4. For an Agilent 6130 single quadrupole mass spectrometer, use the following parameters for the spray chamber of the ion source: Drying gas flow of 12 L/min, nebulizer pressure of 35 psig, capillary voltage of 3,000 V, and drying gas temperature of 350 °C. Acquire signals in dual positive–negative scan mode with a 50 % cycle time between the two modes, peak width of 0.1 min, and cycle time of 1.08 s/cycle. Use a mass range of 100–1,000, fragmentor voltage of 125 V, gain of 1.00, threshold of 150, and stepsize of 0.10 (see Note 12).

5. Set the column temperature to 25 °C.

6. Set up the following chromatography method, where the solvent A is water with 0.1 % formic acid and solvent B is acetonitrile with 0.1 % formic acid: (1) ramp from 5 % B to 100 % B over 20 min, (2) hold at 100 % B for 2 min, (3) ramp from 100 % B to 5 % B over 1 min, and (4) hold at 5 % B for 2 min. Set the flow rate at 0.7 mL/min (see Note 13).

7. In order to clean the column, wash the column with 100 % acetonitrile for 10 min at 0.7 mL/min.

8. In order to equilibrate the column, wash the column with 5 % B for 10 min at 0.7 mL/min.

9. Use the autosampler to inject 5 μL of methanol into the LC-MS. This blank run will allow you to assess the background level of signal (see Note 14).

10. Use the autosampler to inject 5 μL of sample into the LC-MS.

11. Extract the relevant ion for the ascaroside of interest. That is, extract [M+Na]⁺ for the ascaroside of interest from the positive total ion count trace, or extract [M-H]⁻ for the ascaroside of interest from the negative total ion count trace. Obtain the area under the peak for the extracted ion.

12. Use the autosampler to inject 5 μL of each concentration of the synthetic standards in turn into the LC-MS.

13. For each concentration of synthetic standard injected into the LC-MS, extract the relevant ion from the total ion count trace, and use the area under the peak to generate a standard curve.

14. Use the standard curve generated to estimate the concentration of the ascaroside of interest in the sample (see Fig. 1).

15. In order to clean and store the column, wash the column with 100 % acetonitrile for 10 min.

3.4 LC-MS/MS Analysis of Ascarosides

This method can be used to analyze the ascarosides in the crude pheromone extract that ionize in negative ion mode (Fig. 2). This method assumes that the user has a basic knowledge of LC-MS/MS and is assisted by an experienced mass spectrometry facility manager. The mass spectrometer must be well maintained with low background signal.

Fig. 2.

LC-MS/MS trace in precursor ion scanning mode (selecting for m/z 73.0 product ion) for crude pheromone extract. Asterisks indicate signals from unidentified compounds or non-ascarosides

1. Resuspend the sample from Subheading 3.2 , step 20 , by adding 1 mL methanol. Sonicate for 20 min, vortex briefly to dislodge any remaining solids on the sides of the vial, and sonicate again for 20 min. Centrifuge the vial (with the cap closed) in the speedvac for 2 min to pellet most of the particulates.

2. Pipet 100 μL of the supernatant into a 1.5 mL Eppendorf tube, and centrifuge at 13,700 rpm for 1 min to pellet any remaining particulates. Pipet 50 μL from the top of the sample to an autosampler vial with glass insert with spring. Pipet the remaining 50 μL of the sample from the tube back into vial (see Note 10).

3. Set the mass spectrometer to operate in negative ion, heated (H)-ESI, precursor scanning mode, selecting for a product ion of 73.0. If you are using a Thermo TSQ quantum ultra mass spectrometer, use the following parameters for the mass spectrometer: spray voltage of 3,000 V, vaporizer temperature of 300 °C, sheath gas pressure of 40 arb, auxillary gas pressure of 20 arb, capillary temperature of 350 °C, skimmer offset of 5 V, collision pressure of 0.8 mTorr, collision energy of 25 eV, and scan time 0.4 s. We usually set the mass range to m/v 200–500 (see Notes 15 and 16).

4. Set the column temperature to 30 °C.

5. Set up the following chromatography method, where solvent A is water and solvent B is acetonitrile with 0.1 % acetic acid: (1) hold at 2 % B for 1 min, (2) ramp from 2 % B to 50 % B over 34 min, (3) ramp from 50 % B to 100 % B over 1 min, (4) hold at 100 % B for 4 min, (5) ramp from 100 % B to 2 % B over 1 min, (6) hold at 2 % B for 4 min. Set the flow rate at 0.25 mL/min (see Note 17).

6. In order to clean the column before use, wash the column with 100 % acetonitrile for 10 min at 0.25 mL/min.

7. In order to equilibrate the column, wash the column with 2 % solvent B for 10 min at 0.25 mL/min.

8. Use the autosampler to inject 5 μL of methanol into the LC-MS/MS. This blank run will allow you to assess the background level of signal (see Note 14).

9. Use the autosampler to inject 5 μL of sample into the LC-MS/MS.

10. After the run is complete, smooth the trace using a Boxcar or Gaussian function, with 5–7 smoothing points (see Note 18).

11. Obtain the areas under the peaks for each ascaroside by extracting each specific precursor ion from the total ion count trace (see Note 19).

12. In order to validate that a given peak is a given ascaroside, you will need to acquire a synthetic standard. Dissolve the standard(s) in ethanol (200 proof) at 270 μM. Transfer some of the sample to an autosampler vial, and use the autosampler to inject 5 μL from the autosampler vial into the LC-MS/MS.

13. After the run is complete, verify that the synthetic standard has the same retention time as the tentatively assigned peak from step 11.

14. In order to clean and store the column, wash the column with 100 % acetonitrile for 10 min.

3.5 NMR Spectroscopy-Based Analysis of Ascarosides Using the dqf-COSY Experiment

This method can be used to analyze the ascarosides in the crude pheromone extract (Fig. 3) or chromatography fractions generated from the crude pheromone extract. The ascarosides must be present at sufficient concentrations in the crude pheromone extract for detection by NMR spectroscopy. Subheading 3.5 assumes that the user has a basic knowledge of NMR and is assisted by an experienced NMR facility manager. The NMR spectrometer must be well maintained with an updated shim set. Although we use a Varian NMR spectrometer in our own experiments, we employ terminology in this method that should be interpretable by users familiar with either Agilent (Varian) or Bruker NMR spectrometers. In order to have the necessary chemical shift separation in the complex spectrum obtained from crude pheromone extract, an NMR spectrometer with a magnetic field of 500 MHz or higher should be used. The number of points in both dimensions should provide for a resolution of 1–2 Hz per point. The concentrations of the ascarosides in the crude pheromone extract are relatively low compared to the concentrations of other more abundant metabolites. Therefore, it is critical to maximize the signal-to-noise ratio for the sample, and thus, an indirect detection probe or a cold probe is recommended.

Fig. 3.

dqf-COSY spectrum of crude pheromone extract, highlighting specific correlations for bolded protons for specifi c ascarosides (asc-ΔC9, asc-C6-MK, and asc-ωC3) that are visible in the spectrum. ( a and b) Highlight the below diagonal and above diagonal correlations between the bolded proton at 5.81 ppm and the bolded proton at 6.92 ppm in asc-ΔC9. The active coupling can be measured in ( a) and ( b) to give the coupling constant between the two bolded protons (15.6 Hz). The passive coupling can be measured in ( b) to give the coupling constant between the bolded proton at 6.92 ppm and the other protons to which it is coupled (6.8 Hz). ( c) Highlights the above diagonal correlations between the bolded protons at 2.61 ppm and the two nonequivalent bolded protons at 1.71 ppm and 1.77 ppm in asc-C6-MK. The active coupling between the bolded protons can be measured in ( c). The measured value of 14.8 Hz must be divided in two (to give coupling constants of approximately 7.4 Hz) because the bolded proton at 2.61 ppm is a triplet and oppositely phased peaks in the center of the triplet cancel each other. The below diagonal correlation is not highlighted as it is too faint. ( d) Highlights the below diagonal correlation between the two bolded protons at 2.56 ppm and the bolded proton at 3.94 ppm in asc-ωC3. The above diagonal correlation is not highlighted as it is obscured by a vertical stripe

1. Resuspend the sample from Subheading 3.2, step 20 , by adding 1 mL methanol-d₄ . Sonicate for 20 min, vortex briefly to dislodge any remaining solids on the sides of the vial, and sonicate again for 20 min. Centrifuge in the speedvac (with the cap closed) for 2 min to pellet any particulates that do not dissolve.

2. Transfer the supernatant to a new vial, and speedvac or rotovap the sample to dryness (see Note 20).

3. Place the vial of dry sample, a short Pasteur pipet, and an empty 5 mm methanol-matched Shigemi tube on the lyophilizer for 1–2 h. This drying step will remove any trace amounts of methanol and water in the sample that would otherwise interfere with the signal of the sample in the NMR spectrum.

4. Resuspend your sample by adding 300 μL of methanol-d₄ and sonicating for 5 min.

5. Place a small amount of cotton in the short Pasteur pipet, and pack it down inside the Pasteur pipet using a long Pasteur pipet, such that the packed cotton plug is about 3 mm in length.

6. Wash the cotton in the Pasteur pipet with a small amount of methanol-d₄ to remove any impurities from the cotton. Use a bulb to push the methanol-d₄ through the Pasteur pipet and dry the cotton.

7. Filter the sample through the cotton plug in the Pasteur pipet, directly into the Shigemi tube. Use a bulb to push the sample through the Pasteur pipet. Use a few drops of methanol-d₄ to wash the cotton plug, allowing the wash to drip directly into the Shigemi tube (see Note 21).

8. Insert the solvent-matched Shigemi tube insert into the Shigemi tube, taking care to avoid any bubbles between the sample and the insert. Use a small strip of parafilm (about 0.5 × 2 cm in length) to wrap around the top of the tube to prevent any evaporation of the sample while it is in the NMR spectrometer (see Notes 22 and 23).

9. Insert your sample into the spectrometer. Setup parameters for a ¹H spectrum in methanol (see Note 24).

10. Lock the sample, and optimize the lock phase.

11. Gradient shim the sample.

12. Tune the ¹H channel of the probe.

13. Turn off the spinner (see Note 25).

14. Acquire a ¹H spectrum with 16 scans (and with the spinner off) (see Notes 26 and 27).

15. Set the sweep width to the region of the ¹H spectrum that you are interested in for the 2D experiment. Usually you will select the region where there are peaks, plus 0.5 ppm on either side. In general, for the crude pheromone extract, the sweep width will extend from 0.5 to 9.5 ppm.

16. Determine the 90° pulse width. Make sure that the pulse width is equal to this new optimized value (see Note 28).

17. Acquire another ¹H spectrum (see Note 29).

18. Move the ¹H spectrum into a new experiment in order to move all optimized parameters, including sweep width, 90° pulse width, and gain, into the new experiment. Setup up a dqf-COSY experiment with the default parameters.

19. Change the default parameters in the following manner: the number of scans to 16, the number of iterations to 1,024, the number of points to 4,096, and the relaxation delay to 1 (see Notes 30 and 31).

20. Start the experiment, and once finished, process the data (see Note 32).