G-protein coupled receptors (GPCRs) are the most important class of membrane receptors with over 800 identified to date in humans, many of which are involved in diseases, such as oncologic, psychiatric, metabolic, neurodegenerative, cardiovascular, and infectious diseases [1, 2]. Approximately 40%–50% of drugs circulating in the market target GPCRs for the treatment of various diseases [2]. Ligand binding induces conformational changes of GPCRs, which in turn regulate interactions with downstream signaling molecules, such as heterotrimeric G-proteins or β-arrestins [3, 4]. Understanding the conformational changes that are induced in GPCRs upon activation or inactivation would greatly advance our understanding of the mechanism of activation and inactivation induced by endogenous or synthetic ligands, and might ultimately lead to the design of more effective and less toxic drugs.

Hence, enormous efforts have been invested in the characterization of the structure of GPCRs. X-ray crystallography and NMR spectroscopy are standard techniques for obtaining high-resolution structures of proteins. Recent breakthroughs in obtaining high-resolution X-ray crystal structures of GPCRs provide the most comprehensive insights into the unique functional properties of these receptors in both inactive and active states [5]. Although X-ray crystallography gives an important three-dimensional overview of structure, it has certain limitations. The X-ray crystal structure represents a single static conformational state, giving little information about conformational changes or dynamics. Another major limitation is the crystallization process for GPCR engineering, which requires a lot of effort and time, and selection of a ligand and detergent [5, 6]. Additionally, the introduction of nonfunctional insertions, truncations, or point mutations into native GPCRs might affect the endogenous conformation. More importantly, the conditions under which proteins function are generally not compatible with the conditions required for X-ray diffraction. NMR has restrictions associated with protein size and sample preparation, such as expression and isotope labeling of proteins [7], and the application of NMR to structural studies of GPCRs is currently very limited. Therefore, other techniques are needed in order to study the conformation of GPCRs.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) measures the exchange rates of peptide amide hydrogen with deuterium in the solvent. In folded proteins, the exchange rate varies depending on the conformation of the proteins [8, 9]; exposed or highly dynamic regions show rapid exchange rates whereas excluded and rigid regions show slow exchange rates [8, 9]. Thus, HDX-MS has been successfully used to study conformational changes [10, 11], the protein–protein interaction interface, protein–small molecule interaction sites, and protein folding [12, 13]. Previously, the Griffin group presented methodology for HDX-MS analysis of β2-adrenergic receptor (β2AR) [14] and analyzed ligand-dependent perturbation of the conformational ensemble of β2AR by HDX-MS [15]. Their study showed approximately 71% sequence coverage but the transmembrane (TM) regions were mostly not covered [14, 15]. Other studies analyzed the conformational changes of GPCR-interacting molecules (e.g., G proteins and β-arrestin) upon binding to GPCRs by HDX-MS, but conformational information on the GPCRs themselves is limited [1618]. The low sequence coverage in the TM regions in the study by the Griffin group and the even lower sequence coverage when analyzed with GPCR-interacting molecules might reflect the technical challenges associated with studying membrane proteins by mass spectrometry.

GPCRs are insoluble and unstable membrane proteins, and the use of detergents is obviously required for their extraction and purification from cell systems. The detergents are used not only to solubilize and stabilize GPCRs but also to keep the purified GPCRs in functionally folded states in the absence of a phospholipid membrane. Among many commercially available detergents, n-dodecyl-β-D-maltopyranoside (DDM) is the most widely used for GPCR studies (Figure 1b). A few studies have analyzed detergent-solubilized GPCRs by mass spectrometry-based approaches and achieved fairly good sequence coverage [1921]. However, the LC-MS conditions of these studies are different from those of HDX-MS; for HDX-MS, the separation by LC should be performed at low temperature (near 0°C) over a short period of time (less than 10 min). As discussed above, when the Griffin group used DDM for HDX-MS analysis of β2AR, they could not get good sequence coverage in the TM regions [14, 15].

Fig. 1
figure 1

Sequence coverage of β2AR. Illustration of β2AR in bicelle (a) and DDM (b). Sequence coverage of β2AR prepared in bicelles (c) and DDM (d). Blue bars represent the peptic peptides identified in this study. The figures are representative of four independent experiments

In addition to conventional detergents, various other approaches to the solubilization and stabilization of GPCRs have been investigated [22]. Among those, nanodiscs have been tested for HDX-MS compatibility [23, 24]. Nanodiscs are composed of scaffold protein-assisted phospholipid bilayers, which provide more physiological conditions than detergent micelles [23]. γ-Glutamyl carboxylase, a membrane protein, was reconstituted in nanodiscs and analyzed by HDX-MS with only approximately 45% sequence coverage overall and no coverage of most TM regions [23, 24]. Therefore, it is necessary to develop a new methodology that can provide reliable sequence coverage for HDX-MS studies of membrane proteins, including GPCRs.

Bicelles (bilayered micelles) are lipid-detergent assemblies with discrete bilayer fragments that are edge-stabilized by certain detergents (Figure 1a) [25]. They provide a lipid-rich medium that mimics the natural phospholipid bilayer environment and is compatible with structural studies of membrane proteins [26]. Of note, bicelles express the attractive characteristics of both micellar and lipid bilayer systems in structural studies of membranous biomolecules [26, 27]. Because of these attractive features, a number of studies have used bicelles to maintain functional membrane proteins in EPR [28] and NMR [2830] studies, as well as X-ray crystallography [31]. More recently, there have been several reports of the use of bicelles to study membrane proteins by mass spectrometry [32, 33]. However, to our knowledge, there is no published HDX-MS study using bicelles. In the present study, we tested the feasibility of using bicelles for structural analysis of GPCRs by HDX-MS. Three GPCRs, β2AR, μ-opioid receptor (μOR), and protease-activated receptor 1 (PAR-1), were used as GPCR models and the HDX-MS profiles were compared between β2AR reconstituted in bicelles and in DDM.


Expression and Purification of β2AR

Recombinant baculovirus was prepared using the Bestbac expression system (Expression Systems Inc., Davis, CA, USA) with pVL1392 as a vector, and β2AR was extracted and purified as previously described [34]. Briefly, cell pellets were lysed by incubation in lysis buffer (20 mM HEPES pH 7.5, 5 mM EDTA, 1 μM alprenolol, 2.5 mg/mL leupeptin, 160 mg/mL benzamindine) with stirring for 20 min. The receptors were extracted from the cell membrane by dounce homogenization in solubilization buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 1% DDM, 1 μM alprenolol, 2.5 mg/mL leupeptin, 160 mg/mL benzamindine) for 1 h at room temperature. After clarification by high-speed centrifugation at 18,000 × g for 30 min, receptors bearing the N-terminal FLAG epitope were captured by M1 antibody affinity chromatography. The column was extensively washed with HMS-CHS buffer (20 mM HEPES pH 7.5, 350 mM NaCl, 0.1% DDM, 0.01% cholesterol hemisuccinate [CHS], 2 mM CaCl2), and the receptors were eluted with HMS-CHS buffer supplemented with 5 mM EDTA and 200 μg/mL free FLAG peptide. The eluted receptors were further purified by affinity chromatography using alprenolol-Sepharose as previously described [35] to select functional receptors. Size-exclusion chromatography (SEC) with Superdex-200 column (GE Healthcare, Pittsburgh, PA, USA) equilibrated in HMS-CHS buffer was finally used to clean up the receptor. The receptor was concentrated to 125 μM and mixed with different ligands if needed. The purity of the sample was higher than 95% as assessed by SDS-PAGE.

Expression and Purification of μOR

Expression and purification of the construct was performed essentially as described by Manglik et al. [36]. Briefly, the construct used was the WT receptor from residue 6 fused to an N-terminal signal FLAG-tag and a C-terminal 6-histidine His tag. The construct was expressed in sf9 cells. Harvested cells were lysed in lysis buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 10 μM naloxone). The cell pellet was solubilized in solubilization buffer (20 mM HEPES pH 7.5, 0.5 M NaCl, 0.5% DDM, 0.3% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulphonate, 0.03% CHS, 30% v/v glycerol, 10 μM naloxone). Nickel-NTA agarose was added and the mixture was stirred for 1 h at 4°C. The beads were recovered by centrifugation and washed in batches with nickel wash buffer, and then the receptors were eluted in wash buffer supplemented with 250 mM imidazole. The receptors were further purified by loading onto a M1 antibody column. The column was washed with wash buffer and eluted with elution buffer supplemented with 5 mM EDTA and 0.2 mg/mL FLAG peptide. Finally, SEC with Superdex-200 column (GE Healthcare) equilibrated in HMS-CHS buffer was used to clean up the receptor.

Expression and Purification of PAR-1

The human PAR-1 construct was prepared as described previously with slight modification [37]. Briefly, the construct was expressed in Sf9 cells at 27°C for 48 h before harvest. To purify the receptor, infected cells were lysed by osmotic shock in low-salt buffer containing 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 100 nM vorapaxar derivative, and 100 μM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP). The vorapaxar derivative was generated by reduction of the non-aromatic carbon–carbon double bond of vorapaxar and showed a much faster dissociation rate than vorapaxar in cell-based assays. The protein was further extracted from cell membranes with a buffer containing 20 mM Hepes, pH 7.5, 500 mM NaCl, 1% DDM, 0.03% CHS, 0.2% sodium cholate, 15% glycerol, 100 nM vorapaxar derivative, and 100 μM TCEP. Cell debris was removed by high-speed centrifugation. From this point on, 1 μM vorapaxar derivative was added to all of the buffers used for purification except for the buffer used in size exclusion chromatography. Nickel-NTA agarose resin was added to the supernatant after homogenization and stirred for 1 h at 4°C. The resin was then washed three times in batches with buffer containing 20 mM HEPES, pH 7.5, 500 mM NaCl, 0.1% DDM, 0.02% CHS, and 1 μM vorapaxar derivative, and transferred to a glass column. The bound receptor was eluted with buffer containing 300 mM imidazole and loaded onto an anti-Flag M1 affinity column. After extensive washing with buffer containing 20 mM HEPES, pH 7.5, 500 mM NaCl, 0.1% DDM, 0.02% CHS, 1 μM vorapaxar derivative, and 2 mM Ca2+, the receptor was eluted from M1 resin using the same buffer without Ca2+ but containing 200 μg/mL FLAG peptide and 5 mM EDTA. Size exclusion chromatography was used to obtain the final monodisperse receptor preparation. The running buffer contained 20 mM HEPES, pH 7.5, 100 mM NaCl, 0.1% DDM, and 0.02% CHS. The flow rate was set at 0.2 mL/min to give enough time for the vorapaxar derivative to dissociate from the receptor.

Reconstitution of Receptors into Bicelles

A 10% bicelle stock was prepared with a 3:1 molar ratio of DMPC and CHAPSO. One aliquot of the bicelles was thawed at room temperature until the phase changed to a clear gel and then transferred to ice to liquefy. The aliquot was vortexed briefly to reestablish a homogenous phase and placed back on ice. One microliter of 10% bicelle stock was added to 9 μL receptor to give a final 1% working concentration of bicelles. The protein–bicelle mixture was gently pipetted up and down until the solution became clear and homogenous. The mixture was incubated on ice for approximately 30 min to allow complete reconstitution of the receptor into bicelles, and was kept on ice until preparation for HDX-MS studies.

Hydrogen/Deuterium Exchange and Mass Spectrometry

The purified proteins were prepared at a concentration of 125 pmol/μL. Hydrogen/deuterium exchange was initiated by mixing 2 μL of protein with 28 μL of D2O buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, and 0.1% DDM or bicelles in D2O), and the mixtures were incubated for various time periods (10, 100, 1000, and 10,000 s) on ice. At the indicated time points, the mixtures were quenched by addition of 30 μL of ice-cold quench buffer (100 mM KH2PO4, 20 mM TCEP, pH 2.01) and immediately placed on dry ice to freeze. For non-deuterated samples, 2 μL of purified protein was mixed with 28 μL of H2O buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, and 0.1% DDM or bicelles in H2O), and 30 μL of ice-cold quench buffer was added. The quenched samples were digested online by passing through an immobilized pepsin column (2.1 × 30 mm) (Life Technologies, Carlsbad, CA, USA) at a flow rate of 100 μL/min with 0.05% formic acid in H2O at 10°C. Peptide fragments were collected on a C18 VanGuard trap column (1.7 μm × 30 mm) (Waters, Milford, MA, USA) for desalting with 0.05% formic acid in H2O and then separated by ultra-pressure liquid chromatography using an Acquity UPLC C18 column (1.7 μm, 1.0 × 100 mm) (Waters) at a flow rate of 40 μL/min with an acetonitrile gradient using two pumps, starting with 8% B and increasing to 85% B over the next 8.5 min. The mobile phase A was 0.1% formic acid in H2O, and solvent B was acetonitrile containing 0.1% formic acid. To minimize back-exchange of deuterium to hydrogen, the sample, solvents, trap, and UPLC column were maintained at pH 2.5 and 0.5°C during analysis. Mass spectral analyses were performed with a Xevo G2 Qtof equipped with a standard ESI source in MSE mode (Waters). The mass spectra were acquired in the range of m/z 100–2000 for 10 min in the positive ion mode.

Peptide Identification and HDX-MS Data Processing

Peptic peptides in non-deuterated samples were identified with ProteinLynx Global Server 2.4 (Waters). Searches were run with variable methionine oxidation modification, and the peptides were filtered on a peptide score of 6. To process the HDX-MS data, the amount of deuterium in each peptide was determined by measuring the centroid of the isotopic distribution using DynamX software (Waters). EX1 kinetics were determined by visual inspection of the distribution of the two isotopes. All experiments were conducted in at least triplicate.

Statistical Analysis

Results were expressed as means ± S.E.M. Statistical analysis was performed using Graph Prism 5.0 software. Statistical significance of differences between bicelles and DDM was determined by Student's t-test. Differences between data were considered statistically significant at PP < 0.05.


Successful HDX-MS analysis is often determined by the sequence coverage derived from the identified peptic peptides of non-deuterated samples. It is, therefore, necessary to optimize buffers, digestion conditions, and LC-MS conditions to achieve the highest sequence coverage. We tested quench buffer (100 mM KH2PO4, pH 2.01) supplemented with various concentrations of denaturant (guanidine-HCl, 0.0–4.0 M) and reductant (TCEP, 0.01–0.2 M). Addition of 20 mM TCEP was optimal but addition of guanidine-HCl with 20 mM TCEP had a deleterious effect, resulting in an average 23% decrease in sequence coverage compared with 20 mM TCEP alone (data not shown). Based on this result, we selected quench buffer composed of 100 mM KH2PO4, pH 2.01, and 20 mM TCEP.

The feasibility of using bicelles for mass spectrometry was investigated and compared with the use of DDM. Sequence coverage was analyzed for the selected GPCRs (β2AR, μOR, and PAR-1) prepared in either bicelles or DDM. High redundancy is also an important parameter because we can use more peptides for the analysis of a single region, thus increasing our confidence in evaluation of the deuterium uptake level of this region. All three GPCRs showed improved sequence coverage with higher redundancy when prepared in bicelles than in DDM (average sequence coverage of 93.6% versus 79.5% and average redundancy of 4.87 versus 3.28; Table 1, Figure 1, Supplementary Figure 1, and Supplementary Figure 2). Interestingly, worse sequence coverage (64.43 ± 0.44% with redundancy of 2.02 ± 0.07) was obtained when β2AR was solubilized in maltose neopentyl glycol-3 (MNG-3), a detergent that was recently introduced for GPCR structural studies [38] (Supplementary Figure 3).

Table 1 Comparison of Sequence Coverage Between GPCRs Prepared in Bicelles or DDM

The sequence coverage maps of β2AR are shown in Figure 1c and d, and the sequence coverage maps of μOR and PAR-1 are provided in Supplementary Figure 1 and Supplementary Figure 2. Hydrophobic peptides are generally more difficult to be analyzed by mass spectrometry than hydrophilic peptides [39]. As expected, the intracellular or extracellular regions are well covered, whereas the transmembrane (TM) regions are poorly covered for both bicelle and DDM samples (Figure 1, Supplementary Figure 1, and Supplementary Figure 2). Interestingly, bicelle-prepared samples yielded increased identification for peptic peptides in both intracellular/extracellular regions and TM regions and, therefore, showed enhanced sequence coverage in both intracellular/extracellular and TM regions compared to DDM-prepared samples (Figure 1, Supplementary Figure 1, and Supplementary Figure 2).

To test the compatibility of bicelles and DDM for HDX-MS, we analyzed the HDX profile of all three GPCRs prepared in bicelles and in DDM (Figure 2). The average standard deviation of each deuterium exchange time point was 2.2% with a range of 0.1% to 5.3%. The thermodynamic stability of all GPCRs in bicelles was not significantly different from that in DDM, and the stability of GPCRs over time of D2O buffer incubation was tested and showed no significant change in GPCR stability (Supplementary Figure 4). As shown in Figure 2, Supplementary Figure 5, and Supplementary Figure 6, the TM regions showed lower deuterium uptake than intracellular or extracellular regions for both bicelle- and DDM-prepared GPCRs. This is expected because the TM regions are highly ordered and relatively protected by bicelles or DDM. These results suggest that both bicelles and DDM are compatible with HDX-MS. However, because bicelles gave better sequence coverage than DDM, we were able to obtain HDX-MS information for more regions from GPCRs prepared in bicelles than from those prepared in DDM (Figure 2). In particular, we could get new information from TM regions (e.g., TM1, TM3, and TM7 of β2AR) in only the bicelle samples. These results suggest that bicelles are a better solubilization method than DDM for HDX-MS analysis of GPCRs.

Fig. 2
figure 2

Deuterium uptake profile of β2AR. Heat map of β2AR prepared in bicelles (a) and DDM (b). Incorporation of deuterium at 10, 100, 1000, and 10,000 s is indicated by color-coded blocks underlining the amino acid sequence. The color legend shows deuterium uptake levels. Snake maps showing deuterium uptake at 1000 s for β2AR prepared in bicelles (c) and DDM (d). The figures are representative of four independent experiments

As bicelles appeared to be a good system for HDX-MS analysis of GPCRs, we next compared the conformation of β2AR prepared in bicelles with that in DDM. The HDX profile is dependent on the peptic peptides used for the analysis, and analyzing the same region with different peptides could result in slightly different HDX-MS profiles even though the samples were in the same conformation. Bicelle- and DDM-prepared samples produce dissimilar peptic peptide patterns (Figure 1, Supplementary Figure 1, and Supplementary Figure 2). For this reason, it is not possible to directly compare the HDX-MS profile of bicelle- and DDM-prepared samples. For example, in Figure 3, TM5 and TM6 of β2AR show different deuterium uptake levels in bicelles compared with DDM; this might reflect either the different conformation or the effect of using different peptides. Therefore, we chose peptides commonly found in both bicelle and DDM samples for side-by-side comparison. New heat maps were generated using these common peptides (Figure 3a, Supplementary Figure 5c, and Supplementary Figure 6c), and differences in deuterium uptake of β2AR between bicelle and DDM samples at the 1000 s time point are illustrated in the snake map (Figure 3b). The deuterium uptake levels were not statistically different between bicelle- and DDM-prepared samples for μOR and PAR-1 (Supplementary Figure 5c and Supplementary Figure 6c). For β2AR, most regions showed statistically similar deuterium uptake except for ICL3, which showed slightly, but statistically significant, lower deuterium uptake in bicelles than in DDM (Figure 3a and b red boxes, and 3c). These results indicate that the conformation of β2AR is similar between bicelle- and DDM-prepared samples in most regions, but ICL3 is more dynamic in DDM.

Fig. 3
figure 3

Comparison of deuterium uptake profile of β2AR prepared in bicelles and DDM.(a) Heat map of β2AR prepared in bicelles and DDM analyzed from common peptides. Incorporation of deuterium at 10, 100, 1000, and 10,000 s is indicated by color-coded blocks underlining the amino acid sequence. The color legend shows deuterium uptake levels. (b) Snake maps showing the difference in deuterium uptake between β2AR prepared in bicelles and DDM. The color legend shows the difference in deuterium uptake levels, where blue indicates less deuterium uptake and red indicates more deuterium uptake in DDM than in bicelles samples. Regions that show differences between bicelles and DDM samples are highlighted with red boxes. (c) Deuterium uptake plot. Error bars are standard error of four independent experiments. * P < 0.05

The kinetics (i.e., EX1 and EX2 kinetics) of the HDX profile provide information on the folding status of a protein [40]. EX2 kinetics occur when the rate of HDX is much slower than the rate of protein unfolding, and might represent local conformational fluctuations close to the surface of the protein [41]. In contrast, EX1 kinetics occur when the rate of protein unfolding reaction is slower than the rate of HDX, which might represent the global unfolding/refolding of proteins [41, 42]. In physiological conditions, EX1 kinetics are rare but very interesting because they help us understand the dynamics of a protein [42]. We did not find EX1 kinetics in μOR or PAR-1. Interestingly, we identified intermediate EX1 and EX2 kinetics (i.e., the existence of both bimodal isotopic distribution [EX1] and time-dependent mass shifts [EX2]) from a peptide in the C-terminus of TM6/ECL3 (IVNIVHVIQDNL) of β2AR prepared both in bicelles (Figure 4, left) and DDM (Figure 4, right). The ratio between folded and unfolded states was not statistically different in bicelle samples compared with DDM samples.

Fig. 4
figure 4

EX1 kinetics observed from a peptide in the C-terminus TM6/ECL3 region. EX1 kinetics observed from a peptide from β2AR prepared in bicelles (right panel) and DDM (left panel). The figures are representative of four independent experiments


In this study, we could achieve greater than 90% sequence coverage of GPCRs in a HDX-MS experimental setup by solubilizing GPCRs with bicelles. Importantly, this was observed for three unrelated GPCRs—PAR-1, μOR, and β2AR—which gave 93.7%, 94.4%, and 92.7% sequence coverage respectively. Of note, not only the sequence coverage but also the redundancy covering an amino acid improved significantly with bicelles. Redundancy is an important parameter because a greater number of peptides covering a single region will provide a higher quality and more reliable HDX-MS profile. Good sequence coverage and increased redundancy led to successful analysis of GPCRs by HDX-MS, indicating that bicelles are a good solubilization method for HDX-MS studies on GPCRs.

With recent developments in instrumentation and technology, HDX-MS has become a powerful technique for studying the dynamic structures of various proteins. However, analysis of membrane proteins such as GPCRs remains challenging, mainly because of the poor compatibility of membrane proteins with LC-MS [39]. Recent collaborative studies with the Woods group successfully analyzed the conformational changes of Gs protein and β-arrestin-1 upon binding to β2AR [16, 18]; however, conformational information on β2AR was limited because of the poor sequence coverage of β2AR. The proteins for these studies were prepared in the newly introduced detergents MNG-3 or LMNG [16, 18]. Extensive optimization of conditions by the Griffin group yielded approximately 89% sequence coverage of β2AR solubilized in DDM [14]. However, this result was achieved by extending the LC gradient to 120 min. Since back-exchange of deuterium to hydrogen occurs continuously in the quenched sample, even at low pH and temperature, the analysis process should be performed as quickly as possible (e.g., within 10 min) after quenching to preserve the maximum amount of deuterium [43], and a 120-min LC gradient is too long to prevent back-exchange. Hence, when the Griffin group tested a condition more suitable for HDX-MS (9.5 min LC gradient) the sequence coverage of β2AR decreased to 71% [15].

Previous reports showed that careful selection of the type and concentration of detergent, as well as optimization of variable conditions, can enhance the mass spectral analysis of proteins and peptides that are hard to solubilize, such as membrane proteins [14, 32]. In the present study, we tested bicelles for HDX-MS compatibility and achieved greater than 90% sequence coverage for GPCRs solubilized in bicelles. It is important that these results were obtained using quick on-line digestion (1 min) and a rapid peptide separation system (8.5 min LC gradient), which are suitable conditions for minimizing back-exchange. Interestingly, both DDM and MNG-3 gave worse sequence coverage than bicelles, which hints at the generalization that bicelles are more compatible with mass spectrometric analysis of GPCRs than conventional detergents.

This study was the first to adopt bicelles as a solubilization method for HDX-MS-based conformational analysis of GPCRs. It is well known that GPCR-G protein coupling is slower in detergent-solubilized GPCRs than in the phospholipid bilayer membrane [44]. Therefore, more physiological GPCR conformations will be obtained in phospholipid bilayer-mimicking systems than in detergents. Bicelles are the best multipurpose model mimicking the physiological phospholipid membrane system currently available [30] and enable the natural folding of membrane proteins better than detergent micelles [28]. The structural properties and formation of bicelles are understood in detail, and bicelles have been routinely used in several structural studies of membrane proteins by EPR [28] and NMR [2830], X-ray crystallography [31], and in drug formulations [45]. Reconstituted high-density lipoprotein (rHDL) particles or nanodiscs are other well-characterized GPCR solubilization methods that mimic the phospholipid bilayer [46]. However, certain properties of rHDL particles make them less attractive than bicelles. The reconstitution process of rHDL particles is more complex than that of bicelles, and rHDL particles use a dimer of ApoA-I as a scaffolding protein to surround the lipid bilayer, which might add complexity to the HDX-MS data analysis [46]. The properties of bicelles make them well suited for investigating the conformation of GPCRs by HDX-MS.

The HDX-MS profile of all three GPCRs tested showed higher deuterium uptake in intracellular and extracellular regions and lower deuterium uptake in TM regions, which correlates well with the overall seven-transmembrane structure of GPCRs [14]. Interestingly, we observed EX1 kinetics at the C-terminus of the TM6/ECL3 region of β2AR but not μOR or PAR-1, suggesting that this region in β2AR slowly exchanges the folded and unfolded conformations. To our knowledge, this is the first report of EX1 kinetics in GPCRs. TM6 undergoes outward movement upon GPCR activation that leads to G protein coupling and plays an important role in GPCR oligomerization [34, 4750]. The importance of EX1 kinetics for β2AR function will be an interesting field to explore.


The present study suggests that bicelles are a better GPCR solubilization tool for HDX-MS studies than conventional detergents such as DDM or MNG-3. It will be interesting to investigate why bicelles are more compatible with HDX-MS than detergents such as DDM or MNG-3. Elucidation of the underlying mechanism might lead to the development of HDX-MS-compatible detergents for GPCRs and other membrane proteins.