Assay of Monoacylglycerol Lipase Activity.

Monoacylglycerol lipase (MGL) is a serine hydrolase involved in the biological deactivation of the endocannabinoid 2-arachidonoyl-sn -glycerol (2-AG). 2-AG is one of the main endogenous lipid agonists for cannabinoid receptors in the brain and elsewhere in the body. In the central nervous system (CNS), MGL is localized to presynaptic nerve terminals of both excitatory and inhibitory synapses, where it helps control the regulatory actions of 2-AG on synaptic transmission and plasticity. In this chapter, we describe an in vitro method to assess MGL activity by liquid chromatography/mass spectrometry (LC/MS)-based quantitation of the reaction product. This method may be used to determine the basal or altered MGL activity in various cells or animal tissues after pharmacological, genetic, or biological manipulations. In addition, this assay can be used for MGL inhibitor screening using puriﬁ ed recombinant enzyme or MGL-overexpressing cells.

MGL is highly expressed in the central nervous system (CNS) [ 1 , 2 , 7 ], where it is the primary enzyme responsible for the hydrolytic degradation of the endocannabinoid, 2-arachidonoyl-snglycerol (2-AG), into free arachidonic acid and glycerol [ 8 -11 ] (Fig. 1 ).Approximately 85 % of the 2-AG-hydrolyzing activity found in the rodent brain is attributable to this protein [ 1 , 3 , 12 , 13 ].MGL is also present in peripheral tissues where, in addition to degrading 2-AG, it completes the hydrolysis of triacylglycerols initiated by hormone-sensitive lipase and triacylglycerol lipase [ 14 ].
Earlier methods to measure MGL activity employed radioactive substrates such as 2-[ 3 H]arachidonoylglycerol and thin-layer chromatographic separation, to assess radioactivity in breakdown products [ 1 ].Advances in liquid chromatography/mass spectrometry (LC/MS) technology and greater availability of LC/MS instruments have contributed to the development of innovative methods for lipid analyses with greatly improved sensitivity, fi delity, and accuracy [ 15 , 16 ].Here, we describe an LC/MS-based MGL assay that is routinely used in our laboratory [ 11 , 13 , 17 ].The protocol includes details on enzyme preparation, lipid extraction, LC/MS analysis, and data processing.

Materials
Prepare all solutions using ultrapure water and analytical grade reagents.LC-grade solvents must be used for LC/MS analyses.

Methods
Comparative MGL assays can determine the basal or altered MGL activity in cells or in animal tissues under various conditions.Also, in vitro MGL activity assays can be performed to screen MGL inhibitors using MGL-overexpressing cells or purified recombinant enzyme ( see Note 4 ).The following is a general protocol for enzyme preparation, which can be adapted by individual laboratories.
1. Culture cells in appropriate tissue culture dishes, and treat them with test drug(s) if required.
2. On the day of harvesting cells, prepare an ice bucket, microcentrifuge tubes, glass tubes, and/or glass vials.Label tubes and vials appropriately.
3. Aspirate/discard the cultured media and wash cell cultures twice with a suffi cient volume of ice-cold phosphate-buffered saline (PBS).Remove PBS completely.
4. Add ice-cold MGL homogenization buffer (1 ml per a 100 mm culture dish).Scrape and collect cells in (micro)centrifuge tubes.
5. Homogenize the cells using a tissue homogenizer or sonicator on ice.Settings may vary depending on the system ( see Note 5 ).Be careful to prevent samples from heating up during homogenization.
6. Centrifuge samples for 10 min at 1000 × g at 4 °C.Carefully collect the supernatant into clean tubes.

Cell Homogenate
Kwang-Mook Jung and Daniele Piomelli 7. Determine protein concentration of the supernatant using the bicinchoninic acid (BCA) assay or Bradford protein assay, with BSA as a standard ( see Note 6 ).
1. Collect the tissues of interest.If the tissues are going to be processed right away, keep them in cold MGL homogenization buffer on ice.Otherwise, snap-freeze them after sacrifi ce using liquid N 2 or powdered dry ice.
2. Place the (frozen) tissues into 10 tissue volumes of MGL homogenization buffer in an appropriate tube.
3. Homogenize the tissues using a tissue homogenizer or sonicator on ice.Be careful to prevent samples from heating up during homogenization.Settings may vary depending on the homogenization system and the amount and nature of tissue ( see Note 5 ).
4. Centrifuge the samples for 10 min at 1000 × g at 4 °C.Carefully collect the supernatant into clean tubes.
5. Determine protein concentration of the supernatant by using the BCA assay or Bradford protein assay with BSA as a standard ( see Note 6 ).
1.The day before transfection, detach HeLa cells from culture dishes by trypsin treatment and count them.Plate cells into 100 mm dishes using complete growth medium.The optimal number of cells to be plated varies depending on the transfection method.
2. Transfect the cells with plasmid DNA encoding MGL under a strong mammalian promoter.Our laboratory uses vectors encoding rat MGL under the human EF-1α promoter [ 17 ].
3. Incubate the cells at 37 °C in a CO 2 incubator for 24-72 h after transfection.

Confi rm MGL expression by Western blotting or other methods ( see Note 7 ).
The fi nal reaction consists of 50 mM Tris-HCl, pH 8.0, 0.05 % fatty acid-free BSA, an appropriate amount of enzyme, and 10 μM 2-OG, in a total reaction volume of 0.5 ml.If required, MGL can be pre-incubated with test drugs for 10 min, alongside an appropriate vehicle.MGL activity is unaltered by dimethyl sulfoxide (DMSO) at concentrations ≤1 %.
1. Design the experiment.Determine the volume of enzyme source ( χ μl) that will be used for the assay, referring to the protein quantitation results ( see Note 8 ).Run all reactions in triplicate.Include a blank triplicate assayed without any enzyme for blank subtraction.
4. If testing a drug, add it to the mixture, vortex gently, and incubate in a 37 °C water bath for 10 min.
1. Quench the reaction by adding 1 ml of Stop solution to each sample.
6. Centrifuge the samples at 4 °C, 2000 × g for 15 min.After centrifugation, the mixture should be separated into two phases with a whitish protein disk at the interface.The lower phase is mainly chloroform and contains most of the lipids; the upper phase is methanol and water containing more polar metabolites.
7. Prepare another set of 8 ml glass vials with the same labeling.
8. Collect the lower (organic) phase using a glass Pasteur pipette attached to a pipette aid (do not pipette by mouth!).Carefully transfer the organic phase to a new 8 ml glass vial ( see Note 9 ).Discard the protein disk and the upper (aqueous) phase ( see Note 10 ).
9. Evaporate the solvent to dryness in the vials with a gentle N 2 stream ( see Note 11 ).
11. Transfer them into 1.5 ml glass LC vials with 0.2 ml conical inserts and proceed to LC/MS analysis.
Fatty acids are identifi ed based on their retention times and MS properties.
1. Set up the column and the LC/MS parameters.We use a reverse-phase Zorbax XDB Eclipse C18 column (50 × 4.6 mm i.d., 1.8 μm, Agilent Technologies).Detection and analysis are performed using Agilent Chemistation and Bruker Daltonics software.
2. Lipids are eluted with a linear gradient from 90 to 100 % of A in B for 2.5 min at a fl ow rate of 1.5 ml/min with the column 3. Extract chromatograms for oleic acid and heptadecanoic acid from LC/MS runs: For oleic acid, m / z = 281, and for heptadecanoic acid, m / z = 269.Figure 2 shows a representative LC/MS chromatogram, where the integrated peak area can be obtained using the LC/MS software.
The fatty acid product of MGL activity in this assay, oleic acid (18:1Δ 9 FA), is quantifi ed using the internal standardization method.Briefl y, the method consists in adding known amounts of a structurally related internal standard to the sample under analysis.Unlike traditional analytical methods that rely on signal intensity, this method employs signal ratios.
In the current method, a fi xed amount of standard heptadecanoic acid (17:0 FA) is added to the sample immediately following the incubation.Extracted chromatograms for oleic acid and heptadecanoic acid from LC/MS analyses are used to obtain the integrated peak area and the ratio of oleic acid to heptadecanoic acid for each sample.Then, the amount of oleic acid is determined based on a standard curve that is generated from mixtures of oleic acid and heptadecanoic acid with known ratios.This protocol is

Calculation of MGL Activity
Fig. 2 Representative LC/MS chromatograms of oleic acid and heptadecanoic acid.Signals from LC/MS are extracted for oleic acid ( m / z = 281) and heptadecanoic acid ( m / z = 269).Insets show the peak integration summary report very similar to the isotope dilution method that is commonly used to determine the quantity of endocannabinoids such as 2-AG and anandamide [ 15 ].
1. Prepare 1 mM heptadecanoic acid and 1 mM oleic acid in a chloroform:methanol (1:3, vol/vol) mixture.Mix equal parts of 1 mM heptadecanoic acid and 1 mM oleic acid, to make the standard for 5 nmol oleic acid.
Mix equal parts of 1 mM heptadecanoic acid and 0.5 mM oleic acid, to make standard for 2.5 nmol oleic acid.
Mix equal parts of 1 mM heptadecanoic acid and 0.25 mM oleic acid, to make standard for 1.25 nmol oleic acid.
5. Run each standard in duplicate by LC/MS.Generate a standard curve using the average peak area of the duplicates of each standard.The Χ -axis is the quantity (nmol) of oleic acid and the Υ -axis is the ratio of oleic acid to heptadecanoic acid (Fig. 3 ) ( see Note 12 ).Oleic Acid (nmol) r 2 =0.99 y=0.231x + 0.005 Ratio (18:1/17:0FA) Ratio Fig. 3 A representative standard curve.An example standard curve for oleic acid quantitation using 5 pmol heptadecanoic acid as an internal standard is shown here.The result from a linear regression analysis of the data is also displayed.Inset : Magnifi cation of the same graph in the range of 0-0.625 nmol oleic acid.18:1 FA, oleic acid; 17:0 FA, heptadecanoic acid 1.Using the peak area of samples obtained in Subheading 3.4 , step 3 , determine the ratio of oleic acid to heptadecanoic acid for each sample.Then, calculate the quantity of oleic acid by entering this ratio into the standard curve.
2. Calculate the quantity of oleic acid from the blank samples.Subtract the average of the blank samples from each sample run.
3. Normally, enzyme specifi c activity can be displayed as the amount of product formed per unit time and protein.In the current assay, MGL-specifi c activity is calculated as follows: specifi c activity (nmol/[min mg of protein]) = oleic acid (nmol) × 1000/μg protein added to reaction × 30.
2. First, prepare 10 mM heptadecanoic acid stock solution in chloroform in a glass vial with Tefl on liner cap.Protect from light and store at −20 °C.Just before use, warm the 10 mM heptadecanoic acid stock solution at room temperature for about 5 min, and vortex.Prepare the Stop solution by adding the appropriate amount of 10 mM heptadecanoic acid stock solution to methanol, according to the number of samples to be analyzed (e.g., for ten samples, add 5 μl of 10 mM heptadecanoic acid stock solution in 10 ml of cold methanol).Keep on ice until use.
3. All procedures involving the use of chloroform should be handled in a chemical fume hood.

Calculations
time for the Branson probe-type sonicator.Using the Polytron homogenizer, we homogenize tissues for 30 s to 1 min.All procedures should be done on ice and the samples should be kept cold at all times.
6.We determine protein concentration using a BCA assay kit (Life Technologies), and a spectrophotometer, following the manufacturer's manual.
7. We confi rm MGL overexpression by Western blot using either anti-MGL antibody [ 1 ] or antibody for the C-terminal tag sequence of recombinant MGL [ 11 ].Alternatively, quantitative PCR or MGL activity assay can be performed [ 11 ].
8. Determining the amount of protein being used for the assay is an important factor for success.As shown in Fig. 4 , the increment of MGL enzyme in the assay shall eventually result in depletion of substrate, which causes the concentration of product to reach a plateau and saturate the assay system.Therefore, a comparative study must use protein amounts within the linear range of the dose-response relationship (1-10 ng for the purifi ed MGL, inset of Fig. 4 ).Although the concentration of the 2-OG substrate vastly exceeds the concentration of enzyme under normal conditions, it is recommended to run an enzyme dose-response curve whenever a new type of protein source (cells, tissues, etc.) is used.In our laboratory, we use 0.2-5.0μg protein for HeLa MGL homogenate, 10-50 μg for cell or brain homogenate, or 1-10 ng for purifi ed recombinant MGL. 9. When the bottom organic phase is removed, it is important not to contaminate it with the upper aqueous phase.We suggest inserting the tip of the glass Pasteur pipette through the upper phase while giving a very gentle positive pressure (which will result in a gentle bubbling).When the tip has reached the bottom layer, carefully withdraw the organic phase from the very bottom of the glass tube.To prevent contamination, it is better not to try recovering the last drops from the bottom phase, instead leaving 5-10 % of the phase in the vial.Opt 9. Transfer them into 1.5 ml glass LC vials with 0.2 ml conical inserts, and proceed to LC/MS analysis.
12. The standard curve may be used to obtain oleic acid levels only when 5 nmol heptadecanoic acid is used as an internal standard.
It is recommended to renew the standard curve periodically.

Fig. 1
Fig.1MGL catalyzes the conversion of monoacylglycerols into free fatty acids and glycerol.In this fi gure, 2-AG (monoarachidonoylglycerol) is converted to free arachidonic acid and glycerol by MGL.The catalytic serine residue in the MGL active site is shown in red temperature at 40 °C.ESI is in the negative mode, capillary voltage is set at 4 kV, and the fragmentor voltage is 100 V. N 2 is used as a drying gas at a fl ow rate of 13 l/min and a temperature of 350 °C.Nebulizer pressure is set at 60 psi.Fatty acids are measured by monitoring the mass-to-charge ratio ( m / z ) of deprotonated molecular ions [M−H] − in selected ion monitoring mode.

Fig. 4
Fig.4 Dose-response curve for the MGL assay.MGL activity assay was performed as described in this chapter, using the indicated amounts of purifi ed recombinant MGL.Inset shows the linear range of the dose-response relationship (0-10 ng purifi ed MGL protein)

10 .Opt 4 .
At this step, the procedure can be stopped.Keep  the organic phase at −20 °C.11. ( Optional ) After step 9 , the sample can be further purifi ed by open-bed silica column purifi cation if better separation is required for LC/MS analysis.Follow the steps Opt 1 to Opt 9 below.Opt 1. Reconstitute the dried lipid pellet from step 9 in 2 ml of chloroform.Opt 2. Prepare a suspension of Silica Gel G (60-Å 230-400 Mesh ASTM; Whatman, Clifton, NJ) in chloroform (1:1, vol/vol).Opt 3. Add 1 ml of the Silica Gel G suspension to the glass columns.Wash the columns with 2 ml of chloroform.Opt 5. Load the samples onto the columns.Wait until all liquid drops down by gravity.Opt 6. Elute the lipids with 2 ml of chloroform/methanol (9:1, vol/vol), and collect the eluate in another set of 8 ml glass vials with the same labeling.Opt 7. Evaporate the eluate to dryness under a gentle N 2 stream.Opt 8. Resuspend the dried lipids in 0.1 ml of chloroform:methanol (1:3, vol/vol) mixture.