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,  [ 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.

LC/MS Analysis
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. 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 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 ). 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 ].

Calculation of MGL Activity
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 0.2-5.0 μg 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. 10. At this step, the procedure can be stopped. Keep the organic phase at −20 °C. 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.