Ligand identification of the adenosine A2A receptor in self-assembled nanodiscs by affinity mass spectrometry

Jun Ma ae, Yan Lu ab, Dong Wu a, Yao Peng abc, Wendy Loa-Kum-Cheung d, Chao Peng e, Ronald J. Quinn *d, Wenqing Shui *ab and Zhi-Jie Liu *abc
aiHuman Institute, ShanghaiTech University, Shanghai 201210, China. E-mail: liuzhj@shanghaitech.edu.cn; shuiwq@shanghaitech.edu.cn; Tel: +86-20-20608182
bSchool of Life Science and Technology, ShanghaiTech University, 393 Middle Huaxia Road, Shanghai 201210, China
cInstitute of Molecular and Clinical Medicine, Kunming Medical University, Kunming 650500, China
dGriffith Institute for Drug Discovery, Griffith University, Brisbane, 4111, Australia. E-mail: r.quinn@griffith.edu.au
eNational Center for Protein Science (Shanghai), Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

Received 3rd August 2017 , Accepted 18th September 2017

First published on 12th October 2017


A nanodisc is designed as a vehicle to contain GPCRs in a native-like phospholipid environment. It maintains GPCRs in a better thermostability and homogeneity state in a detergent-free buffer compared to a detergent micelle environment. Here, we established an affinity mass spectrometry-based approach to assay the interaction between the adenosine A2A receptor in the nanodisc and eight known ligands. The affinity mass spectrometry results are consistent with the previously published data for binding affinities. The combination of nanodisc and affinity MS techniques allows the identification of the GPCR ligands in a relatively stable, unliganded/apo and native state.


Introduction

Membrane proteins are key players in processing foreign signals (such as taste, light, odour and chemicals) and maintaining cellular homoeostasis. They can trigger downstream signalling cascades and immune responses. A large number of membrane proteins belong to the superfamily of G-protein coupled receptors (GPCRs), which is predicted to have a number of 826 GPCRs in the human genome.1 The adenosine A2A receptor (A2AR) is one of the most important GPCRs in the human body.

There are four types of adenosine receptors discovered in mammalians, which are A1, A2A, A2B and A3 receptors.2 These receptors use adenosine as the endogenous ligand, which is a crucial neuromodulator regulating a broad range of physiological functions in the human central and peripheral nervous system.3,4 The A2AR plays a critical role in many physiological functions. For example, the knockout mice show a slower response to pain stimuli but increased platelet aggregation, heart rate and blood pressure.5 It is implicated that the A2AR can cause vasodilation of coronary arteries and increase the blood flow to the myocardium. The A2AR is also responsible for some pathophysiological conditions, such as inflammatory diseases, Parkinson's disease, epilepsies, sleep disorders, pain, and drug addictions.6–11 These properties indicate the therapeutic relevance of the A2AR in treating inflammation, psychiatric disorders and neurodegenerative diseases.

The GPCRs with solved structures usually contain bound ligands to stabilise the receptors in a certain state. The endogenous ligands include ions, organic compounds, odorants, amines, peptides, proteins, lipids, nucleotides, and photons.12,13 These ligands tend to bind at the extracellular domain, within the cavity of the GPCR binding pocket or in the outer half transmembrane region.14 These regulatory sites are readily available to circulating small ligands, which may be potential drug candidates. Approximately 33% of marketed small-molecule drugs are GPCR targeted.15 These ligands help to regulate cellular processes through GPCRs such as synaptic transmission, enzyme catalysis, signal transduction and immune response.

Ligand screening methods against protein targets include thermal shift assays, surface plasmon resonance (SPR), ligand-observed NMR spectroscopy, mass spectrometry (MS), radioactive ligand binding assay and X-ray crystallography. In the past decade, MS has emerged as a powerful and high-performance technique for ligand detection and screening in complex biological systems.16–20 For the direct capture of GPCR-ligand binding by MS, the major challenges are the sample preparation and instrument performance. The detergent signals can potentially overwhelm the GPCR signals significantly. Also, it needs well-tuned collisional energy to dissociate the GPCR from the local micelle environment. The most recent achievement by Robinson's lab is to directly identify the endogenous ligand of ADP binding to P2Y1R by nESI-MS.21 The mass spectrometer used for direct GPCR-ligand identification is internally modified22,23 and may not be feasible for some other MS labs.

In the past, affinity MS has been applied to ligand identification towards various soluble proteins24–27 and certain purified GPCRs such as human vasopressin (V2) and muscarinic type 1 (M1) receptors that are solubilised in detergent-containing buffer.28 Since the environment of detergent micelles tend to destabilise specific GPCRs during purification,29 an engineered nanodisc technology has been developed for GPCR studies.30,31 Nanodiscs constitute self-assembled disc-like phospholipid bilayers that are encircled by two amphipathic helical scaffold proteins, called membrane scaffold proteins (MSPs). The sizes of lipid bilayers normally range from 8.6–16 nm diameters, depending on MSP variants.32 From our dynamic light scattering (DLS) results, the average MSP1D1 diameter is estimated to be 9.8 nm. Nanodiscs can maintain GPCRs at a nearly native state with better thermostability and homogeneity in detergent-free buffer (such as phosphate-buffered saline and Tris/NaCl buffer). Our previous CPM thermostability assay (results not shown) indicates that the melting temperature of A2AR nanodiscs is 5 °C higher than it in DDM detergent and A2AR nanodisc is stable at 4 °C for at least one month. Moreover, nanodiscs will not influence the accessibility of ligands into the ligand binding pocket from extracellular and intracellular interfaces.33,34 Here we developed a method to apply the challenging GPCR nanodisc on affinity LC/MS with low interference and less false positives. In our study, we reconstitute the A2AR35 into a nanodisc and utilise the ultrafiltration-based LC/MS to measure the ligand binding affinity to the A2AR nanodisc.

Materials and methods

Constructs and GPCR expressions

The genetically engineered A2AR construct35 contained an HA signal sequence and a FLAG tag at the N-terminus and a 10× His-tag at the C-terminus. For the crystallisation purpose, the receptor also consisted of a fusion protein, apocytochrome b562RIL (BRIL), in the third intracellular loop and C-terminal truncation to residue A317 to further stabilise the receptor and minimise the flexibility. This A2AR construct was cloned into a pFastBac1 vector (Invitrogen). The recombinant baculoviruses were generated by the Bac-to-Bac system (Invitrogen) and infected into Spodoptera frugiperda (Sf9) cells. The passage 0 baculovirus was used to infect 5 mL of Sf9 cells at a density of 2–3 × 106 cells per mL. Cells were grown at 27 °C for 48 hours prior to harvest.

Purification and NuPAGE electrophoresis

For large scale purification of the A2AR, 1 L frozen Sf9 cells were thawed and dounce homogenised twice in the hypotonic lysis buffer containing 20 mM HEPES pH 7.5, 10 mM NaCl, 10 mM MgCl2, 20 mM KCl, and protease inhibitor cocktail tablets (Roche). The disrupted cells were collected by centrifuging at 40[thin space (1/6-em)]000 rpm for 35 minutes. To remove the unwanted soluble and membrane-associated proteins, dounce homogenisation was repeated another three times with the addition of 1 M NaCl in the hypotonic lysis buffer. The disrupted cell membrane pellet containing the A2AR was resuspended in the buffer containing 50 mM HEPES pH 7.5, 800 mM NaCl and 2 mg mL−1 iodoacetamide and rocked for 30 min, followed by solubilisation with 0.5% (w/v) DDM (Affymetrix) and 0.1% (w/v) cholesteryl hemisuccinate (CHS, Sigma) for 4 hours. The solubilised membranes were isolated by centrifuging at 35[thin space (1/6-em)]000 rpm for 40 minutes, and the supernatant containing the A2AR was incubated with the TALON® IMAC resin (Clontech) overnight in the presence of 20 mM imidazole. The resin was applied to a gravity column (Poly-Prep, Bio-Rad) and washed using 20 column volumes (CVs) buffer containing 50 mM HEPES pH 7.5, 800 mM NaCl, 0.05% (w/v) DDM, 0.01% (w/v) CHS and 20 mM imidazole. The A2AR was eluted with buffer containing 50 mM HEPES pH 7.5, 800 mM NaCl, 0.05% (w/v) DDM, 0.01% (w/v) CHS, and 220 mM imidazole.

The protein samples were mixed with 4× LDS sample buffer (Novex) and separated on 10% NuPAGE bis–Tris gels. After Coomassie blue staining, the fractions containing the A2AR were pooled together and concentrated by using a 100 kDa cutoff concentrator (Sartorius). The final protein concentration was determined by colourimetric Bradford assay. All purification buffers and purification procedures were carried out at 4 °C.

Analytical size exclusion chromatography (aSEC)

Purified GPCRs were applied to a Sepax Nanofilm SEC-250 column using an Agilent model 2000 HPLC system at a flow rate of 0.5 mL min−1 and the signal detection set to 280 nm. The column was pre-equilibrated with 50 mM HEPES pH 7.5, 500 mM NaCl, 2% (v/v) glycerol, 0.05% (w/v) DDM (Affymetrix) and 0.01% (w/v) CHS (Sigma). Samples and columns were maintained at 4 °C throughout the analysis.

Membrane scaffold protein expression and purification

MSP1D1 was expressed by using a pET-28a vector system (Novagen) with the BL21-Gold (DE3) strain (Stratagene) as a host. A single colony was inoculated into 5 mL Luria–Bertani broth (LB) containing 50 μg mL−1 kanamycin as a starting culture and further transferred into 1 L sterilised LB. When OD600 reaches 0.8–1.2, the culture was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for another 4 hours. The bacteria were harvested by centrifugation at 4000g for 30 minutes. The pellet from the 1 L culture was resuspended in 40 mL 1× phosphate-buffered saline (PBS) and lysed by repeated sonication. The lysate was clarified by centrifugation at 20[thin space (1/6-em)]000g for 30 minutes.

The lysate was loaded onto the nickel column under gravity flow. The column was washed with buffer containing 1× PBS, pH 7.5 and 50 mM imidazole for 20 CVs, then the MSP1D1 was eluted with buffer containing 1× PBS pH 7.5 and 0.4 M imidazole. The purity and quantity of the fractions were analysed by electrophoresis. The fractions containing MSP1D1 were pooled together and buffer exchanged to 20 mM Tris/HCl, 0.5 M NaCl, 0.5 mM EDTA and pH 7.5 at 4 °C. The final concentration for MSP1D1 was 10 mg mL−1.

Reconstitution of the A2AR into the nanodisc

1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) were purchased from Sigma-Aldrich. The POPC and POPS stocks were solubilised by using sodium cholate. To optimise the ratio for nanodisc reconstitution, the A2AR was mixed with MSP1D1 and phospholipids (a mixture of POPC/POPS) with different ratios, i.e. 1[thin space (1/6-em)]:[thin space (1/6-em)]2[thin space (1/6-em)]:[thin space (1/6-em)]200, 1[thin space (1/6-em)]:[thin space (1/6-em)]5[thin space (1/6-em)]:[thin space (1/6-em)]100, 1[thin space (1/6-em)]:[thin space (1/6-em)]5[thin space (1/6-em)]:[thin space (1/6-em)]250. For the phospholipids, the ratio of POPC[thin space (1/6-em)]:[thin space (1/6-em)]POPS was maintained at 7[thin space (1/6-em)]:[thin space (1/6-em)]3. The mixture was incubated with pre-treated biobeads (Bio-Rad) overnight at 4 °C, which adsorbed the detergents to aid in nanodisc formation.

To isolate the homogeneous nanodisc, the product was run through a fast protein liquid chromatography (FPLC) system with the Superdex 200, 10/300 GL column. A reconstituted nanodisc peak appeared at around 12.5 mL, after an aggregation peak and preceding a MSP1D1 peak. The fractions were further purified by immobilised metal ion affinity chromatography (IMAC) to separate the empty nanodisc from the A2AR nanodisc.

Ligand identification by affinity MS

The A2AR nanodisc was incubated with a compound mixture consisting of eight known ligands (SCH-442416, ZM-241385, UK-432097, SCH-58261, NECA, adenosine, theophylline, and caffeine) and seven unrelated compounds at a final concentration of 2 μM (A2AR nanodisc) and 0.4 μM (ligand) at 4 °C for two hours. The incubated sample was diluted with 150 mM ammonium acetate and filtered through a 100 kDa MW cutoff concentrator (Sartorius) by centrifugation at 5000g for 5 min at 4 °C. After washing with the dilution buffer twice, the A2AR nanodisc proteins retained on the ultrafiltration membrane were collected into a new centrifugal tube from which the protein–ligand complexes were isolated. The bound compounds were released from proteins by methanol dissociation, followed by centrifugation at 13[thin space (1/6-em)]000g for 20 min at 4 °C. The supernatant was passed through the Ostro™ 96-well plate (Waters) to remove protein and phospholipid interferents. The eluent was dried in a speed vacuum and re-dissolved in 50% methanol. The empty nanodisc was used as a negative control under the same procedures. Three experimental replicates were prepared for each pair of the target and the negative control. Samples were analysed on an Agilent 6530 TOF coupled to an Agilent 1260 HPLC system. The compounds were eluted from the Eclipse Plus C18 column (2.1 mm × 100 mm, 3.5 μm, Agilent, USA) at a flow rate of 0.4 mL min−1, with the mobile phases of water/0.1% formic acid (A) and acetonitrile/0.1% formic acid (B). The LC gradient was as follows: 0–2 min, 5% B; 2–2.1 min, 5–20% B; 2.1–10 min, 20–35% B; 10–13 min, 35–60% B; 13–13.5 min, 60–90% B; 13.5–16.5 min, 90% B and re-equilibration for 4 min. Full-scan mass spectra were acquired in the range of 100–1000 m/z on an Agilent 6530 TOF with ESI source settings: voltage 3000 V, gas temperature 350 °C and fragmentor 150 V. For each pair of target and control samples, all replicates were injected after the reference sample (compound alone). LC/MS chromatograms for specific ligands were extracted using MassHunter software (Agilent, USA) based on the accurate mass measurement with a deviation of 10 ppm and also an RT tolerance of 0.2 min compared with the reference compound. MS responses were represented by the integrated peak areas of the corresponding extracted ion chromatograms. The MS binding index (BI) referred to the ratio of MS response of a specific ligand detected in the A2AR nanodisc incubation sample relative to the control. From our past affinity MS screening results, the positive hits were selected based on an average BI value >2 and relative standard deviation (RSD) <30%.25

Results and discussion

Here we introduce an affinity MS screening method for the A2AR nanodisc. An overview of the affinity MS ligand screening workflow for the A2AR nanodisc is shown in Fig. 1.
image file: c7ay01891f-f1.tif
Fig. 1 Schematic diagram of affinity MS ligand identification for the A2AR nanodisc. The A2AR nanodisc preparation steps include: (1) A2AR purification; (2) A2AR purified proteins, phospholipids and MSP1D1 were mixed and incubated; (3) the A2AR nanodisc formed after detergent removal by adding Biobeads™. The affinity MS steps include: (1) the A2AR nanodisc incubated with the compound mixture; (2) unspecific binding washed off by ultrafiltration; (3) methanol dissociation; (4) lipids and protein removal by solid phase extraction; (5) binding index (BI) determination by LC/MS.

MSP1D1 and A2AR purifications

To validate the effectiveness of the MS-based screening technique to the GPCR nanodisc, we chose the human A2AR nanodisc as a target. The MSP1D1 with a measured diameter of 9.8 nm was used in nanodisc reconstitution. The A2AR and MSP1D1 were purified as previously described.32,35 The A2AR and MSP1D1 were examined by NuPAGE electrophoresis (Fig. 2A). The predicted molecular weights of A2AR and MSP1D1 were 50 kDa and 22.5 kDa, respectively. The elution fractions with the most proteins were concentrated and the final concentrations were determined by Bradford assay. Usually, 1.5–2 mg of A2AR were purified from 1 L biomass. Further analysis of A2AR homogeneity was done by aSEC (Fig. 2B). The aSEC result showed that 95% of A2ARs were present as monomeric peaks compared to 5% of aggregation peaks. There was no compound added during the purification procedures.
image file: c7ay01891f-f2.tif
Fig. 2 (A) NuPAGE electrophoresis analysis of A2AR and MSP1D1-ΔHis6 purified proteins. M, W1, W2, W3, E1, E2 and E3 represent marker, 1st wash fraction, 2nd wash fraction, 3rd wash fraction, 1st elution fraction, 2nd elution fraction, and 3rd elution fraction, respectively. Most A2AR proteins were eluted out in the 2nd elution fraction; (B) aSEC profile of the A2AR in DDM buffer shows a homogeneous sample preparation.

A2AR nanodisc reconstitution

Several models are used to contain the GPCRs and facilitate the structural and functional elucidations, such as detergent micelles, mixed detergent/lipids micelles, bicelles, liposomes and nanodiscs.36 Among these models, nanodiscs have many advantages and are a versatile tool to maintain the GPCR in a native-like phospholipid bilayer environment. It not only controls the physical size of the nanodisc (macro-environment) by varying the lipid stoichiometry and MSP variant but also allows the adjustment of nanodisc lipid types (micro-environment). We chose a nanodisc constituted by a lipid mixture of POPC/POPS.37–39 Since POPS is a negatively charged lipid and POPC is a neutral zwitterionic lipid, a mixture of POPC/POPS was used to mimic the environment of the plasma membrane. The molar ratio of POPC and POPS was maintained as 7[thin space (1/6-em)]:[thin space (1/6-em)]3.37 To incorporate the A2AR into the nanodisc, the ratio of A2AR, MSP1D1-ΔHis6 and lipids (POPC/POPS) was systematically optimised. The empirical ratio of MSP1D1[thin space (1/6-em)]:[thin space (1/6-em)]lipids for the empty nanodisc was 1[thin space (1/6-em)]:[thin space (1/6-em)]70 in previous experiments. However, the incorporation of the A2AR will lead to the replacement with some amount of lipids in the nanodisc. Therefore the experimentally determined ratio of A2AR[thin space (1/6-em)]:[thin space (1/6-em)]MSP1D1[thin space (1/6-em)]:[thin space (1/6-em)]lipids was 1[thin space (1/6-em)]:[thin space (1/6-em)]5[thin space (1/6-em)]:[thin space (1/6-em)]250 for generating a monodisperse and homogeneous nanodisc sample (Fig. 3A). The NuPAGE gel indicated the successful incorporation of A2AR into the nanodisc (Fig. 3B).
image file: c7ay01891f-f3.tif
Fig. 3 (A) The optimisation of reconstitution ratios of A2AR[thin space (1/6-em)]:[thin space (1/6-em)]MSP1D1-ΔHis6[thin space (1/6-em)]:[thin space (1/6-em)]phospholipids. FPLC analyses the homogeneity and monodispersity of the reconstituted A2AR nanodisc. The chromatographic peaks at 9 mL represent aggregation formed during nanodisc reconstitution, and the peaks at 12.5 mL represent the monomeric A2AR successfully reconstituted into the nanodisc; (B) the reconstituted A2AR nanodisc is examined by NuPAGE analysis. The two bands correspond to A2AR and MSP1D1-ΔHis6.

Ultrafiltration-based affinity mass spectrometry analysis for A2AR nanodisc ligand identification

Ultrafiltration-based affinity LC/MS has been employed to screen small molecule ligands against various soluble protein targets24–27 yet never reported to be applied to any GPCR nanodisc. We have found that the variation of the detergent concentration in the sample preparation of purified GPCR proteins would affect ligand recovery from the ultrafiltration device (data not shown). In contrast, nanodiscs under the detergent-free conditions can better suit the need for ligand detection by ultrafiltration-based affinity LC/MS analysis.

We prepared a 15-compound mixture containing ZM-241385, UK-432097, SCH-58261, SCH-442416, NECA, adenosine, theophylline, caffeine and other seven unrelated compounds in 50% methanol. A final concentration of 0.4 μM of each compound was mixed with 2 μM of the A2AR nanodisc to assay the ligand binding affinity to A2AR nanodisc by ultrafiltration-based affinity LC/MS. The inhibitory constants (Ki) and types of compounds were extracted from ChEMBL (https://www.ebi.ac.uk/chembl/compound) and BindingDB database (https://www.bindingdb.org/bind/index.jsp). The Ki values were used as an indicator for protein–ligand interaction activities. The binding index (BI) referring to the ratio of MS response of a given ligand detected in the A2AR nanodisc incubation sample versus the control (empty nanodisc) was used to assess the specific enrichment of the ligand associated with the A2AR (Fig. 4). Previous studies have shown that ligands with BI values >2 are positive bindings to the target protein.25 As expected, all eight known ligands of the A2AR in the 15-compound mixture were verified in this assay and the rest of seven compounds unrelated to the A2AR all had BI values below the threshold (Table 1). More importantly, BI values for SCH-442416 (15.9 ± 4.3), UK-432097 (47.8 ± 4.9), SCH-58261 (24.8 ± 4.8), NECA (30.5 ± 3.9) and adenosine (27.7 ± 4.9) with relatively strong affinity (Ki < 1 μM) were much higher than those of two weakly bound ligands theophylline (3.7 ± 1.0) and caffeine (3.25 ± 1.2) in the compound mixture (Ki > 1 μM). Although the ranking order of BI values is in general agreement with the binding affinity of different ligands, the exception does exist in some cases, such as for ZM-241385 (BI = 3.7 ± 0.1). Its low BI value might be related to the kinetics of the ligand association/dissociation with the receptor. Nevertheless, the current affinity LC/MS indicates that the nanodisc system coupled to the ultrafiltration-based affinity LC/MS technique is feasible and promising for ligand identification from compound mixtures towards specific GPCR targets in a high-throughput manner.


image file: c7ay01891f-f4.tif
Fig. 4 (A) Total ion chromatograms (TIC) acquired through compounds from the mixture alone (green), A2AR nanodisc (red) and negative control (blue). (B) Extracted ion chromatograms (EIC) of the compounds from the A2AR nanodisc (solid line) and the negative control (dashed line), each chromatogram (from left to right) represents a compound with strong, weak, or nonspecific binding to the A2AR. The MS spectra for individual compounds detected in the A2AR nanodisc samples and the standards are provided in Fig. S1.
Table 1 Identification and binding index determination for each compound in a 15-compound mixture by ultrafiltration-based affinity LC/MS analysis
Compounds BIa Rep-1 BIa Rep-2 BIa Rep-3 Retention time (min) Mass accuracy (ppm) A2AR ligandb K i (nM)
a BI determined in the affinity MS assay from three independent replicates. b Data from ChEMBL (https://www.ebi.ac.uk/chembl/compound) and BindingDB databases (https://www.bindingdb.org/bind/index.jsp).
SCH-442416 15.1 12.1 20.6 14.9 0.64 Antagonist 0.05
ZM-241385 3.70 3.84 3.65 8.62 0.12 Antagonist 1.6
UK-432097 50.7 50.5 42.2 10.6 −1.19 Agonist 4.0
SCH-58261 19.4 26.3 28.6 14.3 −1.61 Antagonist 5.0
NECA 34.8 27.4 29.2 3.62 −0.99 Agonist 20
Adenosine 26.1 23.7 33.2 1.05 −0.88 Agonist 700
Theophylline 3.27 2.95 4.78 4.74 −1.82 Antagonist 1584
Caffeine 3.39 2.02 4.34 5.51 −0.90 Antagonist 2000
Negative-1 1.14 1.24 1.20 11.3 −0.64 No N.A.
Negative-2 0.86 1.11 0.87 11.5 1.76 No N.A.
Negative-3 0.83 1.26 1.39 13.0 1.03 No N.A.
Negative-4 1.00 0.86 1.08 16.2 −0.50 No N.A.
Negative-5 0.00 0.00 0.00 11.5 −0.32 No N.A.
Negative-6 0.95 0.91 1.15 16.0 −0.17 No N.A.
Negative-7 1.42 1.30 1.18 7.23 0.21 No N.A.


Conclusions

The affinity LC/MS has been successfully applied to ligand screening against both soluble proteins and purified GPCR dissolved in detergents.24–28 This is the first report of combining nanodisc and affinity LC/MS techniques to identify ligands against unstable GPCRs. We provide a protocol for a robust, quantitative and reliable ligand identification method by combining GPCR nanodisc and affinity LC/MS techniques. The reconstitution of GPCRs into nanodiscs would potentiate ligand screening efficiency in an automated affinity LC/MS platform due to better thermostability of the protein target. The affinity LC/MS technique allows for sensitive and convenient detection of ligands in a wide range of affinities to GPCR targets, and can be readily set up without much instrumental modification for different GPCR target screening.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

We thank the Cloning, Cell Expression, and Protein Purification Core Facilities of iHuman Institute, ShanghaiTech University for their support. We specially thank the MS facility of National Center for Protein Science Shanghai (Shanghai, China) for assistance in LC-MS usage.

Notes and references

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Footnotes

Electronic supplementary information (ESI) available. See DOI: 10.1039/c7ay01891f
These authors contributed equally to this work.

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