We thank the U.S. Geological Survey Hammond Bay Biological Station for the use of their research facilities and the staff of U.S. Fish and Wildlife Service and Fisheries and Oceans Canada for providing sea lampreys. This research was supported by grants from the Great Lakes Fishery Commission to Weiming Li and Ke Li.

All methods described here have been approved by the Institutional Animal Care and Use Committee of Michigan State University (AUF# 03/14-054-00 and 02/17-031-00).
1. Collection and Extraction of Sea Lamprey Conditioned Water
<ol>
	<li>Place sexually mature male sea lampreys (15 - 30 animals) in a tank supplied with 250 L of aerated Lake Huron water maintained at 16 - 18&#160;&#176;C.</li>
	<li>Collect the male-conditioned water throughout each night from June to July. 
	Note: Sea lampreys naturally die after spawning. If a fish is nearing this point in its life, replace it with a fresh mature male.</li>
	<li>Extract the conditioned water by solid phase extraction.
	<ol>
		<li>Soak the resin in methanol for 4 h before loading it into a column. Load the resin into the column, then pump 10 L of water through the column to eliminate the organic solvent.</li>
		<li>Pass the conditioned water from the tank holding the fish via the pumping system to the top of the column. The water passes through a bed of 2 kg of moderately polar polymeric ion-exchange resin (e.g., Amberlite XAD-7 HP resin), contained in a series of four 2.5 L-capacity glass columns. Maintain load speeds between 400 and 600 mL/6 min. Elute the metabolites with 10 L of methanol followed immediately by 5 L of acetone. 
		CAUTION: This step uses methanol and acetone. Both are flammable and poisonous. 
		Note: The pooled residual can be stored at -80 &#176;C until further processed.</li>
	</ol>
	</li>
	<li>Reactivate the column after 1 week of enrichment by washing it with water (approximately 10 L).</li>
	<li>Remove the organic solvent (the mixture of methanol and water) and harvest the extract by rotatory evaporation for 5 h under reduced pressure (under 300 mbar) at 40 &#176;C. Concentrate the water residue by freeze dryer lyophilization for 48 h at -20 &#176;C until dry.</li>
</ol>
2. Isolation of Fraction Pools with Chromatography
<ol>
	<li>Soak the silica gel (230 - 400 mesh) in the initial mobile phase for 30 min. Transfer the gel with the solvent (suspension) to a glass column. Open the valve of the column to allow the mobile phase to go through. Allow the silica gel to precipitate and form a silica gel bed.</li>
	<li>Mix the extracts with silica gel (70 - 230 mesh) thoroughly and load them into the liquid chromatography column over the silica gel bed. Elute them with a gradient from 95% CHCl3 (chloroform)/MeOH (methanol) to 100% MeOH, 2.5 L in total volume. Collect the eluent in individual vials (each 10 ml). 
	CAUTION: The chloroform used in this step is a poisonous reagent.</li>
	<li>Guide the pooling of the eluents into 20 fractions by a thin layer chromatography (TLC) analysis.
	<ol>
		<li>Perform the TLC experiment on pre-coated silica gel plates by applying the sample on the starting line and immersing the starting line of the plate in the developing solvents (choose the polarity by the ratio of CHCl3 to methanol from 100 - 0%) in a sealed glass tank.</li>
		<li>Select the ratio of developing solvent to make all the TLC spots with a retention factor (Rf) ranging from 0.3 - 0.8.</li>
		<li>After the developing solvent reaches the end line, take the plate out of the tank, and wait 10 min for the solvent to evaporate.</li>
		<li>Visualize the spots first under UV light at 254 nm and then stain them by spraying them with an acidic methanol solution of 5% anisaldehyde (20 &#956;L) by a chromatography sprayer and heating them at 85 &#176;C for 3 min. 
		Note: The colorful spots on the TLC plate indicate the main component in the fraction.</li>
		<li>Combine the eluent to 20 fractions based on the spot color and the Rf similarity of the main component.</li>
	</ol>
	</li>
	<li>Concentrate the fraction to a residue by rotatory evaporation under reduced pressure (300 mbar according the property of solvents) at 40 &#176;C for approximately 30 min.</li>
</ol>
3. Electro-olfactogram (EOG) Recordings to Identify Odorous Fractions/Compounds
<ol>
	<li>Pull borosilicate glass capillaries (outer diameter: 1.5 mm; inner diameter: 0.86 mm; length: 100 mm) with a micropipette puller with the heater level set to 65.</li>
	<li>Score and cut the opening at the tip of the capillary with a diamond-tipped glass cutter and fill it with molten 0.4% agar in 0.9% saline. The opening at the tip of the capillary should be around 10 &#181;m in diameter.</li>
	<li>Fill the pulled capillary electrodes from steps 3.1 - 3.2 and solid-state electrodes holders pre-fabricated with Ag/AgCl pellets (see Table of Materials) with 3 M KCl using a micropipette. 
	Note: Dislodge any air bubbles in the capillary or the electrode holder.</li>
	<li>Insert the pulled electrodes in the electrode holders.</li>
	<li>Prepare 100 mL of 10-5 M L-arginine in charcoal-filtered water from a 10-2 M L-arginine stock solution in deionized water (stored at 4 &#176;C) in a volumetric flask. Transfer 20 mL of the 10-5 M L-arginine to a glass vial.</li>
	<li>For the concentration-response curve, prepare 10 mL of 10-fold dilutions of the fraction pools from step 2.3 in glass vials. Prepare fresh dilutions daily prior to the experiments and use them within a day. Put the glass vials of the working solutions of L-arginine and the fraction pool dilutions in a recirculating water bath to allow the temperature to equilibrate to 8 &#176;C.</li>
	<li>Anesthetize the lamprey with 3-aminobenzoic acid ethyl ester (MS222; 100 mg/L) and immobilize it with an intramuscular injection of gallamine triethiodide (3 mg/kg of body weight, in 0.9% saline) with a sterile syringe. 
	Note: A sufficient depth of anesthesia is determined by observing no gill motion, an inability to maintain an upright posture, and no suction to the sides of the tank using its oral disk. To minimize any microbial contamination before and during surgery, wear sterile gloves and soak the dissection tools in 70% ethanol (v:v) in deionized water for at least 10 min before use.</li>
	<li>Orient the anesthetized lamprey in a V-shaped stand and wrap it with a wet paper towel to prevent desiccation. 
	Note: Do not obstruct the gill openings.</li>
	<li>Insert a tube of recirculating aerated water containing 50 mg/L of MS222 into the buccal cavity, adjust the flow rate, and ensure that water is exiting via the gill openings to continuously irrigate the gills.</li>
	<li>Use a sterile scalpel and forceps [under the stereoscopic microscope at a 1.25X magnification (see Table of Materials)] to remove a 5 mm2 section of the skin on the surface of the olfactory capsule to expose the olfactory epithelium.</li>
	<li>Flush the odorant delivery tubing with filtered water and connect it to the valve mounted on a micromanipulator. Place the odorant delivery capillary tube into the olfactory epithelium cavity using the micromanipulator to deliver filtered water to the olfactory epithelium to prevent desiccation when not administering odorants.</li>
	<li>Mount the recording and reference electrodes on the micromanipulators. Lower the reference electrode onto the external skin near the naris. Using the stereoscopic microscope (1.25X), lower the recording electrode to barely touch the surface of the olfactory epithelium.</li>
	<li>Transfer the uptake of the odorant delivery tube from the background filtered water to the 10-5 M L-arginine solution.</li>
	<li>Turn on the computer, amplifier (set to DC mode), filter, and digitizer. Using the valve driver software, program the procedure to administer a 4 s single pulse of odorant by checking the box of T1, setting T to 4 s, and checking the box of T2.</li>
	<li>In the data acquisition software (see Table of Materials), set the acquisition mode to a high-speed oscilloscope, click the play icon, and then click Start in the valve driver to trigger the odorant pulse. 
	Note: The data acquisition software automatically records the differential EOG response amplitude for 20 s (3 s before, 4 s during the odorant pulse and 13 s afterward). Use the micromanipulator to maneuver the positions of the recording electrode, reference electrode, or odorant delivery tube to increase the signal-to-noise ratio with a maximum response to the L-arginine standard and a minimal response to the blank control (filtered water). Ground the amplifier by switching the AC-DC amplifier switch to GND while moving the electrodes.</li>
	<li>Start recording the blank control, L-arginine, and then the odorants prepared in step 3.6 from low to high concentrations with a 2 min flush of filtered water between applications.</li>
	<li>After recording responses to all the odorants to be tested, ground the amplifier and carefully retract the electrodes and odorant delivery capillary tube. Following the approved Institutional Animal Care and Use Committee (IACUC) methods, at the termination of the EOG experiment, euthanize the anesthetized lamprey with an overdose of MS222 (1 g/L). 
	Note: Verify successful euthanasia by noting a lack of gill movement and heartbeat for at least 5 min followed by pithing the brain.</li>
	<li>Analyze and plot the data using analysis software. Determine the threshold of detection of the odorants22 to identify odorous fractions that elicit responses greater than the blank water control. The fraction pools that elicit concentration-dependent olfactory responses that are different than the blank water control are then tested in a behavioral assay (step 4).</li>
</ol>
4. Two-choice Maze Behavioral Bioassay to Identify Behaviorally Active Fractions/Compounds
<ol>
	<li>Acclimate the sexually mature female sea lamprey in the release cage in the maze (see Supplementary Figure 1) for 5 min. Maintain the flow rate of the river water in the maze 
	Note: The maze is built from varnished marine grade wood and measures 6.5 m in length and 1.2 m in width with a divider measuring 2.7 m in length to separate the maze into two channels at the upstream end. Water is temporarily diverted into the maze. The water depth should be maintained at 0.19 m and the velocity should be maintained at 0.07 m/s &#177; 0.01.</li>
	<li>Release the sea lamprey and record the cumulative amount of time the lamprey spends in the experimental and control channel each containing river water for 10 min. 
	Note: If the sea lamprey fails to enter the experimental and control channel for at least 10 s during this 10 min period, end the trial, as this is an indication of inactivity or strong side bias.</li>
	<li>Apply the test stimulus (i.e., a putative pheromone at 10-12 M dissolved in 50% methanol/deionized water) to the randomly assigned experimental channel and the vehicle (50% methanol/deionized water) to the control channel using peristaltic pumps at constant rates of 200 mL/min for 5 min. 
	Note: The detection threshold concentration of the test stimulus as determined with the electro-olfactogram recordings should be used as the initial concentration for the behavioral test.</li>
	<li>Apply the test stimulus and the vehicle for an additional 10 min and record the cumulative amount of time the lamprey spends in the experimental and control channel.</li>
	<li>Flush the maze with water for 10 min before the start of the next trial. Repeat steps 4.1 - 4.4 with at least 7 lampreys if sufficient test stimulus is available.</li>
	<li>Calculate an index of preference22 for each trial and evaluate the significance using a Wilcoxon signed-rank test. 
	Note: The index results in a single number that can be either positive or negative. A positive value of the index of preference indicates attraction, whereas a negative value of the index of preference indications repulsion. If the index of preference is significantly different from zero, the fraction is deemed active. 
	<img alt="Equation" src="/files/ftp_upload/58059/58059eq1v2.jpg" /> 
	Here, 
	Bc = the time spent by the test lamprey in the control channel before the odorant application, 
	Be = the time spent in the experimental channel before the odorant application, 
	Ac = the time spent in the control channel after the odorant application, and 
	Ae = the time spent in the experimental channel after the odorant application.</li>
</ol>
5. Chromatographic Isolation of Pure Compounds from Active Fractions
<ol>
	<li>Repeat steps 2.1 - 2.4 with the fraction pools that induce olfactory responses (step 3) and elicit behavioral responses (step 4).</li>
	<li>Further purify the active fractions into compounds with size-exclusion chromatography using a Sephadex LH-20 column.
	<ol>
		<li>Prepare the sample in 0.5 mL in the initial mobile phase [CHCl3-MeOH (1:1) or MeOH 100%], load into the corresponding column a CHCl3-MeOH (1:1) column and then a MeOH (100%) column, and elute them to yield the compounds.</li>
	</ol>
	</li>
</ol>
6. Structure Elucidation of a Pure Compound with Mass Spectrometry (MS) and Nuclear Magnetic Resonance (NMR)
<ol>
	<li>Dissolve and dilute the purified compound in the initial mobile phase (usually, methanol to water, 1:1, v:v) of high-performance liquid chromatography (HPLC) to form a 10 &#956;L solution of approximately 1 &#956;g/mL.
	<ol>
		<li>Transfer the sample solution to HPLC vials and set them into the autosampler of HPLC. Inject the sample (10 &#956;L) into the electrospray ionization mass spectrometry (ESIMS) and record the high-resolution electrospray ionization mass spectrometry (HRESIMS) spectra using a chromatography mass spectrometer23.</li>
	</ol>
	</li>
	<li>Predict the molecular formula according to the database in the mass spectrometer software (see Table of Materials)24.
	<ol>
		<li>Open the chromatogram of the injected sample and obtain its mass spectrum by selecting the chromatographic peak.</li>
		<li>Input the measured mass to ion (m/z) value (4 decimals) in Elemental composition under the Tool module. Set the tolerance parameter to PPM &#60; 5.</li>
		<li>Adjust the symbol parameters to fit the element composition of the measured molecule. The software produces a prediction of molecular formula based on the single mass analysis.</li>
	</ol>
	</li>
	<li>Select the deuterated solvents (600 &#956;L, CH3OH-d4 or DMSO-d6) according to the protocol25 to dissolve the samples.
	<ol>
		<li>Completely dissolve the sample in the selected deuterated solvents to form a solution with a concentration ranging approximately from 0.1 to 10 mg/mL. Transfer the sample solution into an NMR tube to record the 1D (1H, 13C) and 2D NMR [1H-1H correlation spectroscopy (COSY), heteronuclear single quantum coherence (HSQC), heteronuclear multiple bond correlation (HMBC)] spectra on a 900 MHz NMR spectrometer26.</li>
	</ol>
	</li>
	<li>Place the NMR tube with the sample inside the spinner turbine. Use the depth gauge to ensure the sample height is in the middle of the measuring window. Open the NMR software (see Table of Materials) and click the Lift button to change the sample in the magnet. 
	Note: Hold a hand over the top of the magnet to feel the gas coming from the top of the magnet. At the same time, a whistling sound should be audible.</li>
	<li>Gently place the sample on the air cushion on top of the magnet and push the Lift button again to descend the sample into the NMR magnet. 
	Note: Listen for a &#8220;click&#8221; noise to indicate the sample is in the right position.</li>
	<li>Create a new dataset and load the default parameters recommended by the NMR instrument (see Table of Materials).
	<ol>
		<li>Type &#8220;lock&#8221; to invoke the automatic locking procedure and select the solvent at the prompt page.</li>
		<li>For fine-tuning, in the Manipulate panel, adjust the button in the Unlock model, adjust the cursor on the manipulate panel, and lock the magnet again.</li>
		<li>On the prompt Atma page, adjust the parameter in the manipulate panel to the tuning procedure, which may take a few minutes. Wait until the tuning is completed to proceed.</li>
		<li>Start the automatic shimming routine by typing &#8220;topshim&#8221; in the prompt. Wait until the shimming is completed to proceed.</li>
		<li>Type &#8220;rga&#8221; to set automatic receiving gain adjustments, then type &#8220;d1&#8221; to set the delay between pulses, then type &#8220;zg&#8221; to start the acquisition, and wait until the acquisition finishes.</li>
		<li>Type &#8220;ef&#8221; or &#8220;efp&#8221; to process the data and then type &#8220;apk&#8221; for automatic phasing. Process the data on the NMR processing software.</li>
	</ol>
	</li>
	<li>Elucidate the chemical structure by an interpretation of the NMR data analysis.</li>
	<li>Count the carbon signals in the 13C NMR spectrum. Select the matched molecular formula from the predicted result in high-resolution mass spectrometry (HR-MS).</li>
	<li>Integrate the proton signals in the 1H NMR spectrum and assign the connectivity of the carbon- and proton-based on the signals in the HSQC spectrum.</li>
	<li>Assign the connectivity of the carbon skeleton based on the correlations in the 1H-1H COSY and HMBC spectrum.</li>
	<li>Tentatively assign the chemical structure based on the structure rationale27. Search the tentative structure in the chemical structure databases. Compare the tentative structure with the analogs in references.</li>
	<li>Assign the relative configuration with the Nuclear Overhauser Effect Spectroscopy (NOESY) spectrum. 
	Note: Since the identity properties of enantiomers displayed on spectrometry and spectroscopic methods are identical, the absolute configuration of some compounds, especially those with 2&#8217;-alcohol and 2&#8217;-NH2, has to be determined with a derivatization reaction. After the derivatization reaction, the differences indicated on the spectra facilitate the unequivocal assignment of the absolute configurations of most compounds28.</li>
</ol>
7. EOG and Bioassay to confirm pure compounds are odorous and behaviorally active
<ol>
	<li>Repeat steps 3.1 - 3.5.</li>
	<li>For the concentration-response curve, prepare 10 mL of 10-fold dilutions of the pure compounds from 10-6 M - 10-13 M of a 10-3 M pheromone stock solution in 50% methanol/water stored at -20 &#176;C based on the molecular weight determined in step 6.2. Put the glass vials with the working solutions of L-arginine and the pure compounds in a recirculating water bath to allow the temperature to equilibrate to 8 &#176;C.</li>
	<li>Repeat steps 3.7 - 3.18.</li>
	<li>Repeat steps 4.1 - 4.2.</li>
	<li>Apply the pure compound at the EOG detection threshold concentration to the randomly assigned experimental channel and the vehicle (50% methanol/deionized water) to the control channel using a peristaltic pump at constant rates of 200 mL/min for 5 min.</li>
	<li>Repeat steps 4.4 - 4.6.</li>
</ol>

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J., Pshenichnov, A., Tkachenko, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+&amp;#34;known+unknowns&amp;#34;+utilizing+accurate+mass+data+and+ChemSpider.">Identification of &amp;#34;known unknowns&amp;#34; utilizing accurate mass data and ChemSpider.</a> Journal of the American Society for Mass Spectrometry. 23 (1), 179-185 (2012).</li><li>Beckonert, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+profiling,+metabolomic+and+metabonomic+procedures+for+NMR+spectroscopy+of+urine,+plasma,+serum+and+tissue+extracts.">Metabolic profiling, metabolomic and metabonomic procedures for NMR spectroscopy of urine, plasma, serum and tissue extracts.</a> Nature Protocols. 2 (11), 2692 (2007).</li><li>Kaiser, B., Wright, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Draft Bruker XRF Spectroscopy User Guide: Spectral Interpretation and Sources of Interference. , (2008).</li><li>Breitmaier, E., Sinnema, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Structure Elucidation by NMR in Organic Chemistry: A Practical Guide. , (1993).</li><li>Seco, J. M., Quino&#225;, E., Riguera, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+assignment+of+absolute+configuration+by+NMR.">The assignment of absolute configuration by NMR.</a> Chemical Reviews. 104 (1), 17-118 (2004).</li><li>Li, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bile+Salt-like+Dienones+Having+a+Novel+Skeleton+or+a+Rare+Substitution+Pattern+Function+as+Chemical+Cues+in+Adult+Sea+Lamprey.">Bile Salt-like Dienones Having a Novel Skeleton or a Rare Substitution Pattern Function as Chemical Cues in Adult Sea Lamprey.</a> Organic Letters. , (2017).</li><li>Li, K., Buchinger, T. J., Li, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+and+characterization+of+natural+products+that+act+as+pheromones+in+fish.">Discovery and characterization of natural products that act as pheromones in fish.</a> Natural Product Reports. , (2018).</li><li>Yambe, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-Kynurenine,+an+amino+acid+identified+as+a+sex+pheromone+in+the+urine+of+ovulated+female+masu+salmon.">L-Kynurenine, an amino acid identified as a sex pheromone in the urine of ovulated female masu salmon.</a> Proceedings of the National Academy of Sciences. 103 (42), 15370-15374 (2006).</li><li>Rasmussen, L., Lee, T. D., Zhang, A., Roelofs, W. L., Daves, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification,+identification,+concentration+and+bioactivity+of+(Z)-7-dodecen-1-yl+acetate:+sex+pheromone+of+the+female+Asian+elephant,+Elephas+maximus.">Purification, identification, concentration and bioactivity of (Z)-7-dodecen-1-yl acetate: sex pheromone of the female Asian elephant, Elephas maximus.</a> Chemical Senses. 22 (4), 417-437 (1997).</li><li>Sorensen, P. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mixture+of+new+sulfated+steroids+functions+as+a+migratory+pheromone+in+the+sea+lamprey.">Mixture of new sulfated steroids functions as a migratory pheromone in the sea lamprey.</a> Nature Chemical Biology. 1 (6), 324-328 (2005).</li><li>Hoye, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Details+of+the+structure+determination+of+the+sulfated+steroids+PSDS+and+PADS:+New+components+of+the+sea+lamprey+(Petromyzon+marinus)+migratory+pheromone.">Details of the structure determination of the sulfated steroids PSDS and PADS: New components of the sea lamprey (Petromyzon marinus) migratory pheromone.</a> The Journal of organic chemistry. 72 (20), 7544-7550 (2007).</li><li>Fine, J. M., Sorensen, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+biological+activity+of+the+multi-component+sea+lamprey+migratory+pheromone.">Isolation and biological activity of the multi-component sea lamprey migratory pheromone.</a> Journal of Chemical Ecology. 34 (10), 1259-1267 (2008).</li><li>De Buchinger, T. J., Wang, H., Li, W., Johnson, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+a+receiver+bias+underlying+female+preference+for+a+male+mating+pheromone+in+sea+lamprey.">Evidence for a receiver bias underlying female preference for a male mating pheromone in sea lamprey.</a> Proceedings of the Royal Society of London B: Biological Sciences. 280, (2013).</li><li>De Bruyne, M., Baker, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odor+detection+in+insects:+volatile+codes.">Odor detection in insects: volatile codes.</a> Journal of Chemical Ecology. 34 (7), 882-897 (2008).</li><li>Bradshaw, J., Baker, R., Lisk, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separate+orientation+and+releaser+components+in+a+sex+pheromone.">Separate orientation and releaser components in a sex pheromone.</a> Nature. 304 (5923), 265-267 (1983).</li><li>Linn, C., Campbell, M., Roelofs, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pheromone+components+and+active+spaces:+what+do+moths+smell+and+where+do+they+smell+it.">Pheromone components and active spaces: what do moths smell and where do they smell it.</a> Science. 237 (4815), 650-652 (1987).</li></ol>

The authors have nothing to disclose.

Fish live in a chemical world full of compounds yet to be identified. Bioassay-guided fractionation has proven essential to identify and characterize bioactive molecules that mediate many chemical interactions, such as those observed in masu salmon31, Asian elephants32, and sea lampreys33,34,35. Bioassay-guided fractionation is an effective approach to accurately trace and pinpoint...

Pheromones are specific chemical signals released by individuals that aid them in locating food sources, detecting predators, and mediating social interactions of conspecifics1. Pheromone communication in insects has been well studied2; however, the chemical identification and biological function of aquatic vertebrate pheromones have not been studied as extensively. Knowledge of the identity and function of the pheromones released can be applied to facilitate the recovery of threatened species3,4 or control pest species5,6. The application of these techniques necessitates the isolation and characterization of the bioactive pheromone components.
Pheromone identification is a branch of natural product chemistry. Progress in pheromone research has been partially limited due to the nature of the pheromone molecules themselves. Pheromones are often unstable and released in small quantities, and only a few sampling techniques exist to detect minute amounts of volatile7,8 or water-soluble compounds9. Approaches to identify pheromones include 1) a targeted screening of known compounds, 2) metabolomics, and 3) bioassay-guided fractionation. A targeted screening of known compounds tests commercially available metabolic by-products of physiological processes hypothesized to function as pheromones. This approach is limiting because researchers can only test known and available compounds. However, it has resulted in the successful identification of sex hormones in goldfish that function as pheromones10,11,12. Metabolomics is a second pheromone identification approach that distinguishes potential small molecule metabolic products within a biological system13. A comparison of the metabolic profiles of two groups (i.e., an active versus an inactive extract) enables the identification of a potential metabolic profile from which the metabolite is purified, the structure is elucidated, and the bioactivity is confirmed14. Additive or synergistic effects of complex formulations of specific mixtures are more likely to be detected with metabolomics because metabolites are considered together rather than as a series of fractions13. Yet, the implementation of metabolomics relies on the availability of synthetic references because the resulting data do not facilitate the elucidation of novel structures.
Bioassay-guided fractionation is an integrated, iterative approach that spans two fields: chemistry and biology. This approach uses the results of physiological and behavioral bioassays to guide the isolation and identification of an active pheromone compound. A crude extract is fractionated by a chemical property (i.e., molecular size, polarity, etc.) and tested with electro-olfactogram (EOG) recordings and/or in a bioassay. The bioactive components are screened out by repeating these steps of fractionation and EOGs and/or bioassays. The structures of pure active compounds are elucidated by spectrometric and spectroscopic methods, which provide the molecular weight and structural information to produce a template of the compound to be synthesized. Bioassay-guided fractionation can yield diverse metabolites and potentially novel pheromones with unique chemical skeletons that are unlikely to be predicted from the biosynthetic pathways.
Here, we describe the bioassay-guided fractionation protocol used to isolate and characterize the bioactivity of male sea lamprey sex pheromone compounds. The sea lamprey (Petromyzon marinus) is an ideal vertebrate model to study pheromone communication because these fish rely heavily on the olfactory detection of chemical cues to mediate their anadromous life history comprised of three distinct stages: larvae, juvenile, and adult. Sea lamprey larvae burrow into the sediment of freshwater streams, undergo a drastic metamorphosis, and transform into juveniles that migrate to a lake or ocean where they parasitize large host fish. After detaching from the host fish, the adults migrate back into spawning streams, guided by the migratory pheromones released by stream-resident larvae15,16,17,18,19. Mature males ascend to the spawning grounds, release a multi-component sex pheromone to attract mates, intermittently spawn for approximately a week, and then die15,20. The identification of sea lamprey pheromones is important because a modulation of the pheromone communication system is among the options considered to control the invasive sea lampreys in the Laurentian Great Lakes21.

<table border="0" cellpadding="0" cellspacing="0" width="1135">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:340px;">Premium standard wall borosilicate capillaries with filament&#160;</td>
			<td style="width:199px;">Warner Instruments</td>
			<td style="width:160px;">G150F-4</td>
			<td style="width:436px;">recording and reference electrode (OD 1.5 mm, ID 0.86 mm)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pipette puller instrument&#160;</td>
			<td style="width:199px;">Narishige</td>
			<td style="width:160px;">PC-10</td>
			<td>pulls electrodes for EOGs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Diamond-tipped glass cutter</td>
			<td style="width:199px;">Generic</td>
			<td style="width:160px;"></td>
			<td>cut tip of electrodes for EOG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Borosilicate glass capillaries</td>
			<td style="width:199px;">World Precision Instruments</td>
			<td style="width:160px;">1B150-4</td>
			<td>odorant delivery tube for EOG</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:340px;">Recording electrode holder E Series straight body with Ag/AgCl pellet for glass capillary OD 1.5 mm</td>
			<td style="width:199px;">Warner Instruments</td>
			<td style="width:160px;">ESP-M15N</td>
			<td>recording electrode holder</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:340px;">Reference electrode holder E Series with handle with&#160; Ag/AgCl pellet&#160; for glass capillary OD 1.5 mm</td>
			<td style="width:199px;">Warner Instruments</td>
			<td style="width:160px;">E45P-F15NH</td>
			<td>reference electrode holder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">1 mm pin</td>
			<td style="width:199px;">Warner Instruments</td>
			<td style="width:160px;">WC1-10</td>
			<td>to bridge reference and recording electrode holders</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">2 mm pin</td>
			<td style="width:199px;">Warner Instruments</td>
			<td style="width:160px;">WC2-5</td>
			<td>to bridge reference and recording electrode holders</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Agar</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">A1296</td>
			<td>molten agar to fill electrodes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Potassium chloride (KCl)</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">P9333</td>
			<td>3M KCl to fill electrodes and electrode holders</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Micropipette microfil</td>
			<td style="width:199px;">World Precision Instruments</td>
			<td style="width:160px;">MF28G-5</td>
			<td>to fill electrodes and electrode holders&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">L-Arginine</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">A5006</td>
			<td>positive control odorant for EOG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Methanol</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">34860</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Water bath</td>
			<td style="width:199px;">Custom made</td>
			<td style="width:160px;">N/A</td>
			<td>holds odorants for EOG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-aminobenzoic acid ethyl ester (MS222)</td>
			<td style="width:199px;">Syndel USA</td>
			<td>Tricaine1G</td>
			<td>EOG anesthetic&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Gallamine triethiodide</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">G8134-5G</td>
			<td>EOG paralytic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">1 mL syringe</td>
			<td style="width:199px;">BD Biosciences</td>
			<td>301025</td>
			<td>to administer paralytic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Subcutaneous needle 26G 5/8</td>
			<td style="width:199px;">BD Biosciences</td>
			<td style="width:160px;">305115</td>
			<td>to administer paralytic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Roller clamp</td>
			<td style="width:199px;">World Precision Instruments</td>
			<td style="width:160px;">14043-20</td>
			<td>adjust flow rate of anesthic into lamprey&#39;s mouth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Sodium chloride (NaCl)</td>
			<td style="width:199px;">J.T. Baker</td>
			<td style="width:160px;">3624-05</td>
			<td>for preparation of 0.9% saline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">V-shaped plastic stand as specimen stage</td>
			<td style="width:199px;">Custom made</td>
			<td style="width:160px;">N/A</td>
			<td>holds lamprey during EOG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Plastic trough</td>
			<td style="width:199px;">Custom made</td>
			<td style="width:160px;">N/A</td>
			<td>holds V-shaped plastic stand during EOG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Scalpel Blades - #11</td>
			<td style="width:199px;">Fine Science Tools</td>
			<td style="width:160px;">10011-00</td>
			<td>for EOG dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Scalpel Handle - #3</td>
			<td style="width:199px;">Fine Science Tools</td>
			<td style="width:160px;">10003-12</td>
			<td>for EOG dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Straight ultra fine forceps</td>
			<td style="width:199px;">Fine Science Tools</td>
			<td style="width:160px;">11252-00</td>
			<td>for EOG dissection, Dumont #5SF Forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Curved ultra fine forceps</td>
			<td style="width:199px;">Fine Science Tools</td>
			<td>11370-42</td>
			<td>for EOG dissection, Moria MC40B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Straight pring Scissors</td>
			<td style="width:199px;">Fine Science Tools</td>
			<td>15003-08</td>
			<td>for EOG dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Stereomicroscope</td>
			<td style="width:199px;">Zeiss</td>
			<td style="width:160px;">Discovery V8</td>
			<td>for EOG dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Illuminator light</td>
			<td style="width:199px;">Zeiss</td>
			<td style="width:160px;">CL 1500 ECO</td>
			<td>for EOG dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Plastic tubing</td>
			<td>Generic</td>
			<td style="width:160px;"></td>
			<td>to connect re-circulating EOG setup and water baths</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Odorant delivery tubing&#160;</td>
			<td style="width:199px;">Custom made</td>
			<td style="width:160px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">In line filter and gasket set</td>
			<td style="width:199px;">Lee Company</td>
			<td style="width:160px;">TCFA1201035A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Micromanipulators</td>
			<td style="width:199px;">Narishige</td>
			<td style="width:160px;">MM-3</td>
			<td>to position electrodes and odorant delivery capillary tube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnetic holding devices</td>
			<td style="width:199px;">Kanetec</td>
			<td style="width:160px;">MB-K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Valve driver</td>
			<td style="width:199px;">Arduino</td>
			<td style="width:160px;">custom made</td>
			<td>to control the opening of the valve for odor stimulation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electromagnetic valve</td>
			<td style="width:199px;">Lee Company</td>
			<td style="width:160px;">LFAA1201618H</td>
			<td>valve for odor stimulation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">NeuroLog AC/DC amplifier</td>
			<td>Digitimer Ltd.</td>
			<td style="width:160px;">NL106</td>
			<td>to increase the amplitude of the elictrical signal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">NeuroLog DC pre-amplifier with headstage</td>
			<td>Digitimer Ltd.</td>
			<td>NL102G</td>
			<td>to increase the amplitude of the elictrical signal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Low-pass 60 Hz filter</td>
			<td>Digitimer Ltd.</td>
			<td>NL125</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:340px;">Digitizer</td>
			<td>Molecular Devices LLC</td>
			<td style="width:160px;">Axon Digidata 1440A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:340px;">Dell computer (OptiPlex 745) running Axoscope data acquistion software</td>
			<td style="width:199px;">Molecular Devices LLC</td>
			<td>AxoScope version 10.4&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Faraday cage</td>
			<td style="width:199px;">Custom made</td>
			<td style="width:160px;">N/A</td>
			<td>Electromagnetic noise shielding</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Two-choice maze</td>
			<td style="width:199px;">Custom made</td>
			<td style="width:160px;">N/A</td>
			<td>waterproofed marine grade plywood covered with plastic liner</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Trash pump</td>
			<td style="width:199px;">Honda</td>
			<td>WT30XK4A</td>
			<td>fills maze with water from nearby river</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:340px;">Peristaltic pump with tubing</td>
			<td style="width:199px;">Cole Parmer</td>
			<td style="width:160px;">Masterflex 07557-00</td>
			<td>to adminster odorants in maze</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Inverter Generator&#160;</td>
			<td style="width:199px;">Honda</td>
			<td style="width:160px;">EU1000i</td>
			<td>powers perstaltic pump</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Release cage</td>
			<td style="width:199px;">Custom made</td>
			<td style="width:160px;">N/A</td>
			<td>used to acclimate lamprey in the maze</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Mesh</td>
			<td>Generic</td>
			<td style="width:160px;"></td>
			<td>used to contain the dimensions of the maze and minimize water turbulance with mesh rollers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Buckets (5 gallon)</td>
			<td>Generic</td>
			<td style="width:160px;"></td>
			<td>to mix odorants</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Flow meter</td>
			<td style="width:199px;">Marsh-McBirney</td>
			<td>Flo-Mate 2000</td>
			<td>to measure discharge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">XAD 7 HP resin</td>
			<td style="width:199px;">Dow chemical</td>
			<td style="width:160px;">37380-43-1</td>
			<td>for extraction of conditioned water&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Methanol</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">34860</td>
			<td>for extraction of conditioned water&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Water bath</td>
			<td style="width:199px;">Yamato</td>
			<td style="width:160px;">BM 200</td>
			<td>for extraction of conditioned water&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:340px;">Freeze dryer</td>
			<td style="width:199px;">Labconco</td>
			<td style="width:160px;">CentriVap&#160; Concentrator</td>
			<td>for extraction of conditioned water&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">chloroform</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">CX1050</td>
			<td>for isolation of fraction pools</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Silica gel 70-230 mesh</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">112926-00-8</td>
			<td>for isolation of fraction pools</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Silica gel 230-400 mesh</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">112926-00-8</td>
			<td>for isolation of fraction pools</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:340px;">Pre-coated silica gel TLC plates</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">99571</td>
			<td>for isolation of fraction pools</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anisaldehyde</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">A88107</td>
			<td>for isolation of fraction pools</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sephadex LH-20</td>
			<td style="width:199px;">GE Healthcare</td>
			<td style="width:160px;">17-0090-01</td>
			<td>for isolation of fraction pools</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amberlite XAD 7 HP resin</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">XAD7HP</td>
			<td>for extraction of conditioned water&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4, 2.5L capacity glass columns</td>
			<td>Ace Glass Inc.</td>
			<td>5820</td>
			<td>for extraction of conditioned water&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetone</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:160px;">650501</td>
			<td>for extraction of conditioned water&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TQ-S TOF LC Mass spectrometer (or equivalent)</td>
			<td style="width:199px;">Waters Co.</td>
			<td style="width:160px;">N/A</td>
			<td>for structure elucidation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Binary HPLC pump</td>
			<td style="width:199px;">Waters Co.</td>
			<td style="width:160px;">1525</td>
			<td>for isolation of fraction pools/compounds</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agilent NMR spectrometer, 900MHz (or equivalent)</td>
			<td style="width:199px;">Agilent</td>
			<td style="width:160px;">N/A</td>
			<td>for structure elucidation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rotovap drying system</td>
			<td style="width:199px;">Buchi</td>
			<td style="width:160px;">RII</td>
			<td>for extraction of conditioned water&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UV lamp (254 nm)</td>
			<td style="width:199px;">Spectronics Co.</td>
			<td style="width:160px;">ENF-240C</td>
			<td>for thin layer chromatography&#160;</td>
		</tr>
	</tbody>
</table>

the identification of sea lamprey pheromones using bioassay-guided fractionation

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and identification of an active pheromone compound. This method has resulted in the successful characterization of the chemical signals that function as pheromones in a wide range of animal species. Sea lampreys rely on olfaction to detect pheromones that mediate behavioral or physiological responses. We use this knowledge of fish biology to posit functions of putative pheromones and to guide the isolation and identification of active pheromone components. Chromatography is used to extract, concentrate, and separate compounds from the conditioned water. Electro-olfactogram (EOG) recordings are conducted to determine which fractions elicit olfactory responses. Two-choice maze behavioral assays are then used to determine if any of the odorous fractions are also behaviorally active and induce a preference. Spectrometric and spectroscopic methods provide the molecular weight and structural information to assist with the structure elucidation. The bioactivity of the pure compounds is confirmed with EOG and behavioral assays. The behavioral responses observed in the maze should ultimately be validated in a field setting to confirm their function in a natural stream setting. These bioassays play a dual role to 1) guide the fractionation process and 2) confirm and further define the bioactivity of isolated components. Here, we report the representative results of a sea lamprey pheromone identification that exemplify the utility of the bioassay-guided fractionation approach. The identification of sea lamprey pheromones is particularly important because a modulation of its pheromone communication system is among the options considered to control the invasive sea lamprey in the Laurentian Great Lakes. This method can be readily adapted to characterize the chemical communication in a broad array of taxa and shed light on waterborne chemical ecology.

A diagram summarizing the steps described in the protocol of the bioassay-guided fractionation is shown in Figure 1. The protocol involves steps to isolate and characterize the structure, the olfactory potency, and the behavioral activity of 5 putative sea lamprey pheromones (Figure 2). Using the mass spectrometric and NMR data (Figure 3 and Figure 4), the structures of ...

Watch this Scientific Journal Video about The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation at JoVE.com

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

Here, we present a protocol to isolate and characterize the structure, olfactory potency, and behavioral response of putative pheromone compounds of sea lampreys.

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and ...

the-identification-sea-lamprey-pheromones-using-bioassay-guided

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

8.9K Views.  Michigan State University. Here, we present a protocol to isolate and characterize the structure, olfactory potency, and behavioral response of putative pheromone compounds of sea lampreys.

Video: The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

Identification of Fatty Acids in Bacillus cereus

The Bacillus species contain branched chain and unsaturated fatty acids (FAs) with diverse positions of the methyl branch (iso or anteiso) and of the double bond. Changes in FA composition play a crucial role in the adaptation of bacteria to their environment. These modifications entail a change in the ratio of iso versus anteiso branched FAs, and in the proportion of unsaturated FAs relative to saturated FAs, with double bonds created at specific positions. Precise identification of the FA profile is necessary to understand the adaptation mechanisms of Bacillus species.
Many of the FAs from Bacillus are not commercially available. The strategy proposed herein identifies FAs by combining information on the retention time (by calculation of the equivalent chain length (ECL)) with the mass spectra of three types of FA derivatives: fatty acid methyl esters (FAMEs), 4,4-dimethyl oxazoline derivatives (DMOX), and 3-pyridylcarbinyl ester (picolinyl). This method can identify the FAs without the need to purify the unknown FAs.
Comparing chromatographic profiles of FAME prepared from Bacillus cereus with a commercial mixture of standards allows for the identification of straight-chain saturated FAs, the calculation of the ECL, and hypotheses on the identity of the other FAs. FAMEs of branched saturated FAs, iso or anteiso, display a constant negative shift in the ECL, compared to linear saturated FAs with the same number of carbons. FAMEs of unsaturated FAs can be detected by the mass of their molecular ions, and result in a positive shift in the ECL compared to the corresponding saturated FAs.
The branching position of FAs and the double bond position of unsaturated FAs can be identified by the electron ionization mass spectra of picolinyl and DMOX derivatives, respectively. This approach identifies all the unknown saturated branched FAs, unsaturated straight-chain FAs and unsaturated branched FAs from the B. cereus extract.

Identification of Fatty Acids in Bacillus cereus

We propose a protocol to identify fatty acids without the need to purify them. It combines information on the retention times with the mass spectra of three types of fatty acid derivatives: fatty acid methyl esters (FAMEs), 4,4-dimethyl oxazoline derivatives (DMOX), and 3-pyridylcarbinyl esters (picolinyl).

The Bacillus species contain branched chain and unsaturated fatty acids (FAs) with diverse positions of the methyl branch (iso or anteiso) and of the ...

Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, abnormal phosphorylation of Tau. In the course of the molecular investigation of Tau functions and dysfunctions in the disease, nuclear magnetic resonance (NMR) spectroscopy is used to identify the multiple phosphorylations of Tau. We present here detailed protocols of recombinant production of Tau in bacteria, with isotopic enrichment for NMR studies. Purification steps that take advantage of Tau's heat stability and high isoelectric point are described. The protocol for in vitro phosphorylation of Tau by recombinant activated ERK2 allows for generating multiple phosphorylations. The protein sample is ready for data acquisition at the issue of these steps. The parameter setup to start recording on the spectrometer is considered next. Finally, the strategy to identify phosphorylation sites of modified Tau, based on NMR data, is explained. The benefit of this methodology compared to other techniques used to identify phosphorylation sites, such as immuno-detection or mass spectrometry (MS), is discussed.

We describe here a method to identify multiple phosphorylations of an intrinsically disordered protein by Nuclear Magnetic Resonance Spectroscopy (NMR), using Tau protein as a case study. Recombinant Tau is isotopically enriched and modified in vitro by a kinase prior to data acquisition and analysis.

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, ...

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in regulating transcription. These include the direct interaction with transcription factors, the covalent modification of histones and other proteins, and the occasional chromatin conformation alteration. Accordingly, establishing relatively quick methods for identifying proteins that interact within this network is crucial to enhancing our understanding of the underlying regulatory mechanisms. LC-MS/MS-mediated protein binding partner identification is a validated technique used to analyze protein-protein interactions. By immunoprecipitating a previously-identified member of a protein complex with an antibody (occasionally with an antibody for a tagged protein), it is possible to identify its unknown protein interactions via mass spectrometry analysis. Here, we present a method of protein preparation for the LC-MS/MS-mediated high-throughput identification of protein interactions involving nuclear cofactors and their binding partners. This method allows for a better understanding of the transcriptional regulatory mechanisms of the targeted nuclear factors.

We have established a method for the purification of coregulatory interaction proteins using the LC-MS/MS system.

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in ...

Fractionation for Resolution of Soluble and Insoluble Huntingtin Species

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a key pathological feature of HD is the aberrant accumulation of mutant HTT (mHTT) protein into high molecular weight complexes and intracellular inclusion bodies composed of fragments and other proteins. Conventional methods to measure and understand the contributions of various forms of mHTT-containing aggregates include fluorescence microscopy, western blot analysis, and filter trap assays. 
However, most of these methods are conformation specific, and therefore may not resolve the full state of mHTT protein flux due to the complex nature of aggregate solubility and resolution.
For the identification of aggregated mHTT and various modified forms and complexes, separation and solubilization of the cellular aggregates and fragments is mandatory. Here we describe a method to isolate and visualize soluble mHTT, monomers, oligomers, fragments, and an insoluble high molecular weight (HMW) accumulated mHTT species. HMW mHTT tracks with disease progression, corresponds with mouse behavior readouts, and has been beneficially modulated by certain therapeutic interventions1. This approach can be used with mouse brain, peripheral tissues, and cell culture but may be adapted to other model systems or disease contexts.

A method is described for fractionation of insoluble and soluble mutant huntingtin species from mouse brain and cell culture. The method described is useful for characterization and quantification of huntingtin protein flux and aids in analyzing protein homeostasis in disease pathogenesis and in the presence of perturbations

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a ...

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of diverse cellular functions, including cytoskeletal dynamics, cell motility, cell polarity, axonal guidance, vesicular trafficking, and cell cycle control. Changes in Rho GTPase signaling play an essential regulatory role in many pathological conditions, such as&#160;cancer, central nervous system diseases, and immune system-dependent diseases. The posttranslational modification of Rho GTPases (i.e., prenylation by mevalonate pathway intermediates) and GTP binding are key factors which affect the activation of this protein. In this paper, two essential and simple methods are provided to detect a broad range of Rho GTPase prenylation and GTP binding activities. Details of the technical procedures that have been used are explained step by step in this manuscript.

Here we describe a protocol to investigate the prenylation and guanosine-5'-triphosphate (GTP)-loading of Rho GTPase. This protocol consists of two detailed methods, namely membrane fractionation and a GTPase-linked immunosorbent assay. The protocol can be used for measuring the prenylation and GTP loading of different other small GTPases.

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. This method utilizes hypotonic buffers, digitonin, mechanical lysis and differential centrifugation to isolate the cytoplasm, mitochondria and plasma membrane. The process can be scaled to accommodate the needs of researchers, is inexpensive and straightforward. This method will allow researchers to determine protein localization in cells without specialized centrifuges and without the use of commercial kits, both of which can be prohibitively expensive. We have successfully used this method to separate cytosolic, plasma membrane and mitochondrial proteins in the human monocyte cell line U937.

Here, we present a protocol to isolate the plasma membrane, cytoplasm and mitochondria of U937 cells without the use of high-speed centrifugation. This technique can be used to purify subcellular fractions for subsequent examination of protein localization via immunoblotting.

In this protocol we detail a method to obtain subcellular fractions of U937 cells without the use of ultracentrifugation or indiscriminate detergents. ...

Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets in both physiological and pathological states. CK2 is an evolutionarily conserved serine/threonine kinase with a growing list of hundreds of substrates involved in multiple cellular processes. Due to its pleiotropic properties, identifying and characterizing a comprehensive set of CK2 substrates has been particularly challenging and remains a hurdle in the study of this important enzyme. To address this challenge, we have devised a versatile experimental strategy that enables the targeted enrichment and identification of putative CK2 substrates. This protocol takes advantage of the unique dual co-substrate specificity of CK2 allowing for specific thiophosphorylation of its substrates in a cell or tissue lysate. These substrate proteins are subsequently alkylated, immunoprecipitated, and identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We have previously used this approach to successfully identify CK2 substrates from Drosophila ovaries and here we extend the application of this protocol to human glioblastoma cells, illustrating the adaptability of this method to investigate the biological roles of this kinase in various model organisms and experimental systems.

The objective of this protocol is to label, enrich, and identify substrates of protein kinase CK2 from a complex biological sample such as a cell lysate or tissue homogenate. This method leverages unique aspects of CK2 biology for this purpose.

The study of kinase-substrate relationships is essential to gain a complete understanding of the functions of these enzymes and their downstream targets ...

Identification of Orexin and Endocannabinoid Receptors in Adult Zebrafish Using Immunoperoxidase and Immunofluorescence Methods

Immunohistochemistry (IHC) is a highly sensitive and specific technique involved in the detection of target antigens in tissue sections with labeled antibodies. It is a multistep process in which the optimization of each step is crucial to obtain the optimum specific signal. Through IHC, the distribution and localization of specific biomarkers can be detected, revealing information on evolutionary conservation. Moreover, IHC allows for the understanding of expression and distribution changes of biomarkers in pathological conditions, such as obesity. IHC, mainly the immunofluorescence technique, can be used in adult zebrafish to detect the organization and distribution of phylogenetically conserved molecules, but a standard IHC protocol is not estasblished. Orexin and endocannabinoid are two highly conserved systems involved in the control of food intake and obesity pathology. Reported here are protocols used to obtain information about orexin peptide (OXA), orexin receptor (OX-2R), and cannabinoid receptor (CB1R) localization and distribution in the gut and brain of normal and diet-induced obese (DIO) adult zebrafish models. Also described are methods for immunoperoxidase and double immunofluorescence, as well as preparation of reagents, fixation, paraffin-embedding, and cryoprotection of zebrafish tissue and preparation for an endogenous activity-blocking step and background counterstaining. The complete set of parameters is obtained from previous IHC experiments, through which we have shown how immunofluorescence can help with the understanding of OXs, OX-2R, and CB1R distribution, localization, and conservation of expression in adult zebrafish tissues. The resulting images with highly specific signal intensity led to the confirmation that zebrafish are suitable animal models for immunohistochemical studies of distribution, localization, and evolutionary conservation of specific biomarkers in physiological and pathological conditions. The protocols presented here are recommended for IHC experiments in adult zebrafish.

Presented here are protocols for immunohistochemical characterization and localization of orexin peptide, orexin receptors, and endocannabinoid receptors in the gut and brains of normal and diet-induced obesity (DIO) adult zebrafish models using immunoperixidase and double immunofluorescence methods.

Immunohistochemistry (IHC) is a highly sensitive and specific technique involved in the detection of target antigens in tissue sections with labeled ...

Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis process, which maintains enzymes far from their substrate. Characterization of these complex interactions is thus a challenge in complex substrates such as LB. The method here measures molecular interactions between fluorophore-tagged molecules and native autofluorescent lignin, to be revealed by F&#246;rster resonance energy transfer (FRET). Contrary to FRET measurements in living cells using two exogenous fluorophores, FRET measurements in plants using lignin is not trivial due to its complex autofluorescence. We have developed an original acquisition and analysis pipeline with correlated observation of two complementary properties of fluorescence: fluorescence emission and lifetime. sFLIM (spectral and fluorescent lifetime imaging microscopy) provides the quantification of these interactions with high sensitivity, revealing different interaction levels between biomolecules and lignin.

This protocol describes an original setup combining spectral and fluorescence lifetime measurements to evaluate F&#246;rster resonance energy transfer (FRET) between rhodamine-based fluorescent probes and lignin polymer directly in thick plant sections.

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis ...

Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and isopycnic density gradient centrifugation. The major advantage of this procedure is that it does not rely on the sole use of solubilizing detergents to obtain subcellular fractions. This makes it possible to separate the plasma membrane from other membrane-bound organelles of the cell. This procedure will facilitate the determination of protein localization in cells with a reproducible, scalable, and selective method. This method has been successfully used to isolate cytosolic, nuclear, mitochondrial, and plasma membrane proteins from the human monocyte cell line, U937. Although optimized for this cell line, this procedure may serve as a suitable starting point for the subcellular fractionation of other cell lines. Potential pitfalls of the procedure and how to avoid them are discussed as are alterations that may need to be considered for other cell lines.

This fractionation protocol will allow researchers to isolate cytoplasmic, nuclear, mitochondrial, and membrane proteins from mammalian cells. The latter two subcellular fractions are further purified via isopycnic density gradient.

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and ...