Comprehensive Proteomic Mass Spectrometric Characterization of

Comprehensive Proteomic Mass Spectrometric Characterization of Human Cannabinoid CB2 Receptor Nikolai Zvonok,# Suma Yaddanapudi,# John Williams,#,† Shujia Dai,† Keling Dong,‡ Tomas Rejtar,† Barry L. Karger,† and Alexandros Makriyannis*,# Center for Drug Discovery, Northeastern University, 116 Mugar Hall, 360 Huntington Avenue, Boston, Massachusetts 02115, Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, and Applied Biosystems, 500 Old Connecticut Path, Framingham, Massachusetts 01701 Received December 13, 2006

The CB1 and CB2 cannabinoid receptors belong to the GPCR superfamily and are associated with a variety of physiological and pathophysiological processes. Both receptors, with several lead compounds at different phases of development, are potentially useful targets for drug discovery. For this reason, fully elucidating the structural features of these membrane-associated proteins would be extremely valuable in designing more selective, novel therapeutic drug molecules. As a first step toward obtaining information on the structural features of the drug-receptor complex, we describe the full mass spectrometric (MS) analysis of the recombinant human cannabinoid CB2 receptor. This first complete proteomic characterization of a GPCR protein beyond rhodopsin was accomplished by a combination of several LC/MS approaches involving nanocapillary liquid chromatography, coupled with either a quadrupole-linear ion trap or linear ion trap-FTICR mass spectrometer. The CB2 receptor, with incorporated N-terminal FLAG and C-terminal HIS6 epitope tags, was functionally expressed in baculovirus cells and purified using a single step of anti-FLAG M2 affinity chromatography. To overcome the difficulties involved with in-gel digestion, due to the highly hydrophobic nature of this membraneassociated protein, we conducted in-solution trypsin and chymotrypsin digestions of purified and desalted samples in the presence of a low concentration of CYMAL5. This was followed by nanoLC peptide separation and analysis using a nanospray ESI source operated in the positive mode. The results can be reported confidently, based on the overlapping sequence data obtained using the highly mass accurate LTQ-FT and the 4000 Q-Trap mass spectrometers. Both instruments gave very similar patterns of identified peptides, with full coverage of all transmembrane helices, resulting in the complete characterization of the cannabinoid CB2 receptor. Mass spectrometric identification of all amino acid residues in the cannabinoid CB2 receptor is a key step toward the “Ligand Based Structural Biology” approach developed in our laboratory for characterizing ligand binding sites in GPCRs using a variety of covalent cannabinergic ligands. Keywords: Cannabinoid Receptor 2 • CB2 • G Protein-Coupled Receptor • GPCR • Protein Expression • FLAG Affinity Purification • Membrane Protein • In-Solution Protein Digestion • Mass Spectrometry • Peptide Mapping • Proteomic Analysis • LTQ-FT • Q-Trap

Introduction G-protein-coupled receptors (GPCRs) are involved in a wide variety of important cellular processes and functions, including cell-cell communication, mediating hormonal activity, and sensory transduction, and they are therefore attractive targets * To whom correspondence should be addressed. Alexandros Makriyannis, Northeastern University Center for Drug Discovery, 116 Mugar Hall, 360 Huntington Avenue, Boston, MA 02115. Tel.: 617-373-4200. Fax: 617-3737493. E-mail: [email protected]. # Center for Drug Discovery, Northeastern University. † Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University. ‡ Applied Biosystems.

2068

Journal of Proteome Research 2007, 6, 2068-2079

Published on Web 05/02/2007

for drug discovery. GPCRs are integral membrane proteins, known to be difficult to express, purify, and characterize structurally. To date, the prototypical GPCR rhodopsin, purified in large quantities from natural tissue with relative ease, is the only one to be fully characterized using high-resolution 3D crystallographic methods1,2 and by a proteomic approach.23-25,33 Functional heterologous overexpression and purification of large quantities of GPCRs is the key to the rapid progress in understanding their structure and function.3 Numerous members of the GPCR family, accordingly, have been expressed in different cell lines including Escherichia coli,4-9 yeast,10,11 baculovirus,12-15 and mammalian,16-18 to provide adequate quantities of receptor protein for biochemical studies. Cur10.1021/pr060671h CCC: $37.00

 2007 American Chemical Society

Proteomic Mass Spectrometric Characterization of hCB2 Receptor

rently, characterization of GPCR ligand binding domains as a major goal in the design of improved drug molecules is limited to computational prediction combined with mutagenesis experiments. In our “Ligand Based Structural Biology” approach19,20 we are using different classes of affinity-labeled cannabinergic ligands to obtain structural information by identifying a number of key amino acid residues associated with ligand binding sites. This goal, that requires the full proteomic characterization of the receptor, involves a number of technically demanding experimental steps, which include (a) functional overexpression of the receptors; (b) design and synthesis of high affinity and selective covalent ligands; (c) interaction of these ligands with membrane preparations in which the receptors are overexpressed; (d) purification of the ligand-receptor covalent complex; and (e) identification of the amino acid residue(s) through mass spectrometric (MS) analysis of the digested receptors. The precise knowledge of receptor-bound ligand structures would provide unique opportunities for drug discovery and likewise aid the development of tailor-made medicines substantially. However, as with other GPCRs, because of its highly hydrophobic nature, the complete MS analysis of the cannabinoid receptor 2 (CB2) receptor is fraught with substantial difficulties. Our initial attempts to characterize the CB2 receptor using a peptide fingerprinting approach by matrix-assisted laser desorbtion/ionization timeof-flight mass spectrometry (MALDI-TOF MS) yielded incomplete sequence coverage.15 Similar limitations were encountered with other GPCRs,14 overexpressed in yeast, CB1 (sequence coverage 35%) and CB2 (29%);10,11 E. coli, CB2 (11%);8 and mammalian cells, µ opioid (37%) and δ opioid (28%) receptors17,18 with significantly lower sequence coverage. The more accessible peptides are those predominantly derived from the hydrophilic N- and C-terminal ends, intracellular and extracellular loops, while fragments from the corresponding hydrophobic transmembrane helical domains are generally highly underrepresented. The limitations of our previously reported MS analysis include the size (3500-7000 Da) and poor solubility of some fragments produced by trypsin or double trypsin/CNBr digestion that were far beyond the dynamic range of detection and sensitivity of the TofSpec 2E MALDI-TOF mass spectrometer initially used to characterize the CB2 receptor. Additionally, many peptides from the digest were highly hydrophobic and tended to precipitate or aggregate during sample preparation and purification, and became exceedingly difficult to extract from a gel matrix. To overcome these limitations, we introduced a number of innovations in sample preparation, including a single-step procedure for protein purification followed by buffer-exchange desalting and in-solution proteolytic digestion. The digest sample subsequently was separated using nanocapillary liquid chromatography (nanoLC) and analyzed by the 4000 Q-Trap MS and the LTQ-FT MS. The peptide fragments, representing all cannabinoid CB2 receptor domains, were identified as singly or multiply charged species, and their sequence was confirmed with high confidence using electrospray tandem mass spectrometry. The work presented here demonstrates for the first time a complete peptide mapping of a GPCR beyond rhodopsin using nanoLC coupled with the 4000 Q-Trap MS and the high accuracy mass measurement LTQ-FT MS.

Experimental Section Reagents and Materials. Standard laboratory chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) and Fisher

research articles Chemical (Pittsburgh, PA) if not otherwise specified. Coomassie G-250 stain, Laemmli electrophoresis sample buffer, PVDF membrane, molecular weight markers and 1D SDS-PAGE gels were from Bio-Rad (Hercules, CA). n-Dodecyl-β-D-maltoside (DM), 5-cyclohexyl-1-pentyl-β-D-maltoside (CYMAL5), cholesteryl hemisuccinate trissalt (CHS), 3-[(chlolamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), and n-dodecylphosphocholine (FOS12) were purchased from Anatrace (Maumee, OH). Nanospray LC-MS/MS Using the LTQ-FT Mass Spectrometer. Chromatographic separation of tryptic digests was conducted by an Ultimate nanoLC system (Dionex, Mountain View, CA) with an in-house-packed reverse-phased PicoFrit column [75 µm i.d. × 15 cm, tip ) 15 µm (New Objective, Woburn, MA); Vydac 208TP, C8, 5 µm particle size, 300 Å (Grace Vydac, Hesperia, CA)]. The peptides were analyzed by an LTQ-FTMS instrument (Thermo Electron, San Jose, CA) equipped with the PicoView ESI source (New Objective). The digests were loaded manually using a 3 µL PEEK loop. The flow rate was 250 nL/ min for sample loading and separation. Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile. After desalting with 2% B for 30 min, the peptides were eluted by a linear gradient to 40% B over 60 min, and then to 95% B over 15 min. Very hydrophobic peptides were eluted only with constant 95% B. The mass spectrometer was operated in a data-dependent mode similar as described earlier.21 Briefly, data was acquired with one full scan event (m/z 350-2000) by FTMS using up to 106 ions and nine subsequent data-dependent scans by the linear ion trap. The relatively high number of ions allowed in the FT cell resulted in improved sensitivity, while the mass accuracy decreased to only 15 ppm. It should be noted that masses reported in this paper were corrected for 4 ppm systematic shift with respect to the default calibration. Peptide Assignment. BioWorks software (Version 3.2 SR1, Thermo Electron) searched raw data files against the sequence of the human cannabinoid receptor 2. The representative parameters for the Sequest search included missed cleavage sites 3; peptide tolerance (2.5 Da; fragment ion tolerance (1.0 Da. Peptides with the charge state lower than 4 were automatically assigned by the Xcorr scores and peptide probability with the thresholds at >3.75 for 3+ charge; >2.50 for 2+ charge; >1.90 for 1+ charge; and P-value < 0.01. For the peptides with higher charge states (g4+ charge), the data were processed according to ref 21. In addition to the standard evaluation of identified peptides using Bioworks, all accurate masses of precursor ions (15 ppm) and tandem MS spectra were inspected manually to ensure correct identification. Nanospray LC-MS/MS Using the 4000 Q-Trap Mass Spectrometer. Following CB2 receptor purification and either trypsin or chymotrypsin digestion, samples were analyzed by nanoLC-MS using a hybrid quadrupole-linear ion trap mass spectrometer (4000 Q-Trap, Applied Biosystems/MDS Sciex) for identifying and sequencing peptides. Briefly, 1.5 µL injections were loaded at 10 µL/min onto a trap column (150 µm × 4 cm, Waters Oasis HLB 15 µm, made in-house) for 5 min in mobile phase A, using a Tempo nanoMDLC system (Applied Biosystems) for concentration and desalting. Mobile phase A consisted of 98% water, 2% acetonitrile, and 0.1% formic acid. Mobile phase B contained 98% acetonitrile, 2% water, and 0.1% formic acid. Gradient separation was carried out on a 75 µm × 100 mm reverse-phase column (YMC-Pack C4, 5 µm, 120 Å, made in-house) at 350 nL/min. A linear gradient from 20% B Journal of Proteome Research • Vol. 6, No. 6, 2007 2069

research articles

Figure 1. Saturation binding of [3H]CP 55 940 ligand to the N-FLAG-tagged CB2HIS6 receptor expressed in Sf21 cells. Conditions for membrane preparation and radioligand binding are described in the Experimental Section.

Figure 2. Western blot analysis of N-FLAG-tagged CB2HIS6 receptor preparations solubilized using DM (1) or FOS12 (2) surfactants. Detection was performed using an anti-5-HIS antibody according to the procedure described in the Experimental Section.

to 70% B over 50 min followed by a 10 min isocratic wash at 90% B was employed. Eluted peaks were introduced into the mass spectrometer via positive mode nanospray ionization. Instrument conditions were as follows: source temperature, 180 °C; curtain gas, 15; nebulizer gas, 8; source voltage, 2200 V; and declustering potential, 80 V. Peptides were analyzed using Information Dependent Acquisition (IDA), scanning over a mass range from 400 to 1600 m/z. Specifically, the charge states of the three most intense ions in an Enhanced Multiply Charged (EMC) survey scan were determined, and those ions that met the selection criteria were fragmented and analyzed in the MS/MS mode. Rolling collision energies were applied based upon the detected precursor’s m/z. Dynamic fill time of the linear trap was used to eliminate space-charging effects. Acquired spectra were searched by Protein Pilot software (Applied Biosystems) using a single entry database of the human CB2 receptor (accession no. P34972) to identify and sequence CB2 peptides. The Paragon search algorithm used by Protein Pilot software was set to perform a Thorough ID, including over 90 biological modifications at a Detected Protein Threshold of 1.3 (95%) for all samples. Expression of CB2 Receptor in Insect Cells. Sf-21 cells from the pupal ovaries of army fallworm Spodoptera frugiperda (Clontech) were grown as suspension cultures and maintained 2070

Journal of Proteome Research • Vol. 6, No. 6, 2007

Zvonok et al.

Figure 3. Coomassie stained 10% PAGE SDS gel of purified N-FLAG-tagged CB2HIS6 receptor using one step, anti-FLAG M2 affinity chromatography (A) or two steps, anti-FLAG M2 affinity chromatography, followed by IMAC purification (B). M, D, and O indicate monomer, dimer, and oligomer forms of the receptor.

in a shaker incubator at 27 °C in Sf900 II medium (Invitrogen) supplemented with 2% FBS (Invitrogen). Cultures were maintained at a density between 1 and 4 × 106 cells/mL. Cells were infected by adding 400 µL of a 1:10 dilution of viral stock to 50 mL of logarithmically growing cells at a density of 4 × 106 cells/ mL. After 4-6 days of incubation, the cells were harvested by centrifugation at 1000g for 10 min at 4 °C. Cell pellets were used immediately or stored at -80 °C. Transfection procedures, plaque isolations, and generation of monoclonal viral stocks were performed, as described in the BacPAK Baculovirus Expression System instruction manual (Clontech). Cell Membrane Preparations and Radioligand Binding Assays. Cell membranes were prepared by cavitation and ultracentrifugation sedimentation, as previous described.15 Saturation binding assays were performed in a 96-well format. Membrane pellets were resuspended in TME containing 0.1% BSA (TME-BSA). A total of 25 µg of protein was added to each assay well. Radioligands (CP 55940 or WIN 55212-2) were diluted in TME-BSA to yield ligand concentrations ranging from an order of magnitude below and above the predicted Kd. Nonspecific binding was assayed in the presence of 5 µM of the corresponding unlabeled ligand. The assay was performed at 30 °C for 1 h with gentle agitation. The resultant material was transferred to Unifilter GF/B filter plates and unbound ligand removed using a Packard Filtermate-96 Cell Harvester (Perkin-Elmer Packard, Shelton, CT). Filter plates were washed four times with ice-cold wash buffer (50 mM Tris-base and 5 mM MgCl2 containing 0.5% BSA, pH 7.4). Bound radioactivity was quantitated in a Packard TopCount Scintillation Counter. Nonspecific binding was subtracted from the total bound radioactivity to calculate specific binding of the radioligand (represented as pmol/mg protein). All assays were conducted in triplicate, and data points presented as the mean ( SEM. Bmax and Kd values were calculated by nonlinear regression using GraphPad Prism version 3.03 (one site binding analysis equation Y ) BmaxX/(Kd + X); GraphPad Software, San Diego, CA) on a Windows platform. Metal-Affinity Purification of CB2 HIS6-Tagged Receptor under Denaturing Conditions. Membrane pellets were resuspended in lysis/binding buffer [50 mM sodium phosphate, 300


research articles

Figure 4. Amino acid sequence of the N-terminal FLAG, C-terminal HIS6-tagged cannabinoid CB2 receptor. The transmembrane domains are marked in bold.

Figure 5. Trypsin and chymotrypsin N-FLAG-tagged CB2HIS6 receptor coverage using LTQ-FT and Q-Trap. Amino acids are color coded to represent the enzyme used in their identification. Yellow ) trypsin only. Green ) chymotrypsin only. Blue ) both trypsin and chymotrypsin.

mM NaCl, 8 M urea, 7 mM imidazole, 1.5% DM or 0.5% SDS, and 0.5% protease inhibitor cocktail (P8849; Sigma), pH 8.0] and manually homogenized. Membrane solubilization was completed by gently mixing samples in a rotator at room temperature for 2 h, and the solubilized membrane preparation

was centrifuged at room temperature for 30 min at 27 000g. The supernatant was collected and incubated with preequilibrated BD Talon metal affinity resin (Clontech) for 1 h at room temperature in a rotator. The suspension was then centrifuged at room temperature for 4 min at 700g, and the Journal of Proteome Research • Vol. 6, No. 6, 2007 2071

research articles

Zvonok et al.

Table 1. Determination of the CB2 Receptor Binding Parameters on Membrane Preparations of Sf-21 Cells Expressing Untagged and Differently Tagged CB2 Receptor Using [3H]CP 55 940 Ligand construct

Kd (nM)

Bmax (pmol/mg)

CB2 C-terminal HIS6-tagged CB2 N-terminal HIS6-tagged CB2 N-terminal FLAG, C-terminal HIS6-tagged CB2

10.4 3.3 11.1 2.6

1.7 9.3 5.6 3.9

supernatant removed. The resin was washed six times in 5 vol of wash buffer (50 mM sodium phosphate, 300 mM NaCl, 8 M urea, 15 mM imidazole, 0.5% DM or 0.25% SDS, and 0.5% protease inhibitor cocktail, pH 8.0). After the final wash, the resin was transferred to a gravity-flow column with an endcap in place and allowed to settle. HIS-tagged receptor was eluted by adding 5 times the bed volume of elution buffer (50 mM sodium phosphate, 300 mM NaCl, 8 M urea, 200 mM imidazole, 0.5% DM, and 0.5% protease inhibitor cocktail, pH 8.0), and the eluate was collected in 500 µL fractions and analyzed by SDS-PAGE. Anti-FLAG M2 Affinity Purification of N-FLAG-Tagged CB2. Membrane pellets were resuspended in lysis/binding buffer (50 mM sodium phosphate, 300 mM NaCl, 1.0% DM, 20% glycerol, and 0.5% protease inhibitor cocktail, pH 7.4) and manually homogenized. Membrane solubilization was completed by gently mixing samples in a rotator at 4 °C for 2 h. The solubilized membrane preparation was centrifuged at 4 °C for 20 min at 14 000g, and the supernatant was collected, diluted twice with the same buffer without DM, and mixed with preequilibrated Anti-FLAG M2 Affinity resin (Sigma). After 1 h of rotation at 4 °C, the suspension was centrifuged at 4 °C for 4 min at 700g, and the supernatant was removed. The resin was washed six times in wash buffer (50 mM sodium phosphate, 300 mM NaCl, 0.1% DM, 20% glycerol, and 0.25% protease inhibitor cocktail, pH 7.4), and centrifuged as above. Following the final wash, the suspension was transferred to a gravity flow column. Fusion proteins were eluted with elution buffer (50 mM sodium phosphate, 300 mM NaCl, 100 µg/mL FLAG peptide, 0.1% DM, and 20% glycerol, pH 7.4). SDS-PAGE and Western Blot Analysis. Samples were preincubated at room temperature for 20 min in Laemmli sample buffer containing 5% β-mercaptoethanol. Protein samples were resolved on 10% Tris-HCl gels. Proteins were transferred to PVDF membranes, and the membranes were prepared for immunodetection following the procedures outlined in the QIAexpress Detection and Assay Handbook (QIAGEN, Valencia, CA). Membranes were incubated with a 1:10 000 dilution of anti-5HIS-HRP antibody (Qiagen) or a 1:1000 dilution of rabbit anti-CB1 or CB2 antibody (Sigma) followed by incubation in a 1:10 000 dilution of goat anti-rabbit IgG-HRP (Sigma). Protein bands were visualized using the ECL Western blotting analysis system (GE Healthcare, Piscataway, NJ). In-Solution Digestion. Anti-FLAG M2 affinity purified Nterminal FLAG-tagged CB2HIS6 receptor was reduced with dithiothreitol (DTT) and alkylated with iodoacetamide (IAM) using a standard procedure.22 Samples were desalted with a Zeba Desalt Spin Column (Pierce) or BioSpin Column (BioRad) and subjected to overnight digestion with trypsin (Trypsin Gold, mass spectrometry grade; Promega, Madison, WI) (37 °C) or R-chymotrypsin (C3142; Sigma) (30 °C) followed by nanoLC separation and MS analysis. 2072


Results and Discussion CB2 Receptor: Expression, Purification, and Sample Preparation. The full characterization of GPCRs is essential for understanding their role in fundamental biological processes, and for developing medications based on these protein targets. The majority of GPCRs are expressed naturally at very low levels. Although the CB2 cannabinoid receptor expression is elevated in immune cells, larger quantities are required for detailed structural studies. In this work, we have expressed the human CB2 receptor fused to various epitope tags in the baculovirus system to obtain adequate amounts of membrane material and facilitate its purification for MS characterization. In earlier work, a preliminary comparison of expression efficiency between different CB2 cassettes found the C-terminal HIS6-tagged CB2 construct to be optimal.15 Our previous effort, however, failed to provide us with the full MS characterization of the receptor protein. MS coverage represented 50% of the total protein sequence, while 70% of the hydrophobic transmembrane domains were not detected.15 This represented a serious limitation in our effort to characterize the binding domain of cannabinergic ligands that are known to be centered within the cannabinoid receptor transmembrane helix bundle. In this earlier work, the C-terminal HIS6-tagged protein was purified in a two-step procedure using metal chelating affinity chromatography and SDS-PAGE separation, followed by in-gel trypsin digestion and MALDI-TOF MS analysis. Because of the amphipathic nature of the transmembrane domains, GPCR proteins are highly hydrophobic, and during purification, they tend to aggregate irreversibly, adhere to surfaces, and are exceedingly difficult to extract from a gel matrix. For these reasons, we developed an in-solution digestion procedure that is better suited for preventing sample losses and increases recovering peptide fragments, as was demonstrated successfully for rhodopsin and bacteriorhodopsin.23-25 We found that neither the C- nor the N-terminal HIS6-tagged CB2 receptor, when solubilized under native conditions in the presence of different surfactants, allowed the protein to bind properly to the metal chelating affinity resin. The possible reason for this failure may be that under native conditions the hexa-histidine tag is prevented from binding to the IMAC resin because of competing nonspecific interactions with other proteins. On the other hand, when purified under denaturing conditions in the presence of 8 M urea, the receptor preparation contained significant amounts of other proteins and was susceptible to aggregation and precipitation during the reduction/alkylation and buffer exchange process. To improve receptor purification for MS analysis, we used as our key step an anti-FLAG M2 affinity chromatography, an approach generally considered to be superior to others.26,27 Initially, we sought to improve the quality of purified protein by performing a twostep purification of a doubly tagged cannabinoid CB2 receptor preparation, containing a FLAG epitope at the amino-terminus and a HIS6 tag at the carboxyl-terminus. Toward this goal, we introduced significant improvements in our earlier effort15 that had afforded low expression yield of the double-tagged CB2 receptor. We improved the yield by using a plaque-pick assay for screening and selection of recombinant virus, according to the BacPAK Baculovirus Expression System instruction manual (Clontech). The doubly tagged CB2 receptor was expressed as a functional protein (Figure 1) at a high level (Table 1) and was predominantly located in the membrane fraction, which was prepared, as described earlier.15 Because anti-FLAG M2 affinity chromatography requires the protein to be in its native state,

research articles


Table 2. Theoretical Tryptic Digestion of N-FLAG C-6HIS-Tagged CB2 Receptor and Identified Peptides by LTQ-FTa monoisotopic [M + H]+ (Da)

start

end

peptide sequence

556.2441 607.2211 1522.6848 976.4410 1151.5771 3450.9762 4190.1319 690.4554 2253.2164 724.3816 754.3776 573.3724 232.1410 7735.0380

1 5 10 23 32 42 74 112 118 140 145 151 156 158

4 9 22 31 41 73 111 117 139 144 150 155 157 223

(-) MDYK (D) (K) DDDDK (E) (K) EECWVTEIANGSK (D) (K) DGLDSNPMK (D) (K) DYMILSGPQK (T) (K)TAVAVLCTLLGLLSALENVAVLYLILSSHQLR(R) *(R)RKPSYLFIGSLAGADFLASVVFACSFVNFHVFHGVDSK (A) (K) AVFLLK (I) (K) IGSVTMTFTASVGSLLLTAIDR (Y) (R) YLCLR (Y) (R) YPPSYK (A) (K) ALLTR (G) (R) GR (A) (R)ALVTLGIMWVLSALVSYLPLMGWTCCPRPCSELFPLIPNDYLLSWLLFIAFLFSGIIYTYGHVLWK(A)

1542.7526 758.3983 306.1600 502.2989 3736.1313 3594.0207 147.1128 2690.3331 561.2996 1333.6224 434.2186 661.3521 601.2946 1123.5119 971.4951 1659.6835

224 238 245 247 251 254 287 288 311 316 328 331 338 343 354 362

237 244 246 250 286 286 287 310 315 326 330 337 342 353 361 374

(K) AHQHVASLSGHQDR (Q) (R) QVPGMAR (M) (R) MR (L) (R) LDVR (L) *(R)LAKTLGLVLAVLLICWFPVLALMAHSLATTLSDQVK (K) (K) TLGLVLAVLLICWFPVLALMAHSLATTLSDQVK (K) (K) K (A) (K) AFAFCSMLCLINSMVNPVIYALR (S) (R) SGEIR (S) (R) SSAHHCLAHWK (K) (K) CVR (G) (R) GLGSEAK (E) (K) EEAPR (S) (R) SSVTETEADGK (I) (K) ITPWPDSR (D) (R) DLDLSDCHHHHHH (-)

a

charge states

NF 1+ 2+, 3+ 1+, 2+ 2+ 3+, 4+ 4+, 5+ 1+ 2+, 3+ 1+ 1+ 1+ NF 5+, 6+, 7+, 8+ 2+, 3+ 1+ NF 1+ 4+, 5+ 3+ NF 2+, 3+ 1+ 1+, 2+, 3+ NF 1+ 1+ 1+, 2+ 1+, 2+ 2+,3+

“*” indicates there is a missed cleavage site. NF ) not found.

we explored the use of DM along with several other less frequently utilized detergents, such as CHAPS, CHAPS/CHS, and FOS12.6,8-10 We found the solubilization efficiency of these surfactants to be very similar, as indicated from SDS-PAGE and Western blot analysis (Figure 2). The double-tagged receptor was initially subjected to two purification steps, starting with anti-FLAG M2 affinity resin, followed by metal chelating chromatography. However, anti-FLAG M2 affinity chromatography alone provided a high purity monomeric protein, while the double-purified receptor preparation contained more oligomeric forms and tended to aggregate and precipitate irreversibly (Figure 3). For this reason we chose a single step of anti-FLAG M2 affinity chromatography for the purification of the CB2 receptor. Prior to digestion, purified samples were reduced with DTT, alkylated by IAM, and desalted on Zeba or micro Bio-Spin columns. Both columns efficiently removed salts and detergent excess. However, elution from the Zeba column contained polymeric material that interfered with downstream MS detection. The surfactant CYMAL5 was added to the buffer at low concentration (0.05%) to prevent aggregation and sample loss. Although the presence of detergents generally results in suppressed MS sensitivity, CYMAL5 at this concentration was shown to be tolerated by MALDI-TOF and electrospray ionization mass spectrometry (ESI-MS).28 CB2 Receptor Sample Preparation. The initial incomplete cannabinoid CB2 receptor characterization using peptide fingerprinting with MALDI-TOF MS15 motivated us to revise the methodologies applied to protein analysis, especially for integral membrane proteins. Typical procedures of comprehensive MS protein characterization included (1) in-solution enzymatic or chemical cleavage of purified protein; (2) fractionation of peptides using liquid chromatography (LC); (3) MS analysis of separated peptides with conventional tandem mass

spectrometry (MS/MS). While all steps of this procedure work well with soluble proteins, it is difficult to achieve efficient LCMS/MS analysis of highly hydrophobic integral membrane proteins principally because of their poor solubility. Bacteriorhodopsin and rhodopsin are the only integral proteins from the approximately one thousand member GPCR superfamily that have been completely characterized using two different sample preparation approaches. In the first procedure, 70% formic or trifluoroacetic acids served as solvents for CNBr cleavage. Indeed, under acidic conditions, even very hydrophobic proteins become soluble and remain in solution. Peptide identification was performed either directly by MALDITOF MS,24 or the peptides were separated using HPLC and followed by ESI-MS analysis.23 Improved sensitivity using microcapillary liquid chromatography and advanced mass spectrometry led to the complete sequence coverage of rhodopsin at subpicomolar concentration.25 Both rhodopsin and bacteriorhodopsin have been completely characterized using this approach. However, the CB2 receptor contains long stretches of amino acid sequences without methionine (35123; 179-242; 302-374) (Figure 4), and thus would be difficult to characterize fully using CNBr alone. The second procedure used surfactants for solubilization of the integral membrane proteins followed by trypsin digestion. Since arginine and lysine are generally found outside the membrane region, many tryptic fragments contain long hydrophobic transmembrane regions that tend to aggregate and bind irreversibly to the LC column. Different surfactants, including OBG29,30 and CHAPS,31 were incorporated in the digestion buffer to prevent the loss of hydrophobic integral membrane proteins through aggregation or adsorption. Surfactants, however, interfere with chromatographic separation and suppress peptide ionization and downstream MS detection. Therefore, MS analysis of these proteins Journal of Proteome Research • Vol. 6, No. 6, 2007 2073

research articles

Zvonok et al.

Table 3. Tryptic Digestion of N-FLAG C-6HIS-Tagged CB2 Receptor; Identified Peptides by 4000 Q-Trapa

a

theoretical MW (Da)

precursor MW (Da)

1185.450 2109.880 991.428 1150.569 3606.069 4033.024 689.448 2957.572 1364.745 572.365 3206.652 4545.389 2714.324 501.291 3906.213 2833.415 2689.325 2758.413 1332.615 660.344 600.287 1122.504 970.487 1658.678

1185.479 2109.810 991.333 1150.497 3605.868 4033.708 2957.328 1364.628 3206.820 4545.960 2714.004 3906.291 2833.139 2689.032 2758.070 1332.852 1122.308 970.383 1658.770

start

end

peptide sequence

charge states

1 5 23 32 42 75 112 118 145 156 158 186 224 247 251 287 288 308 316 331 338 343 354 362

9 22 31 41 74 111 117 144 155 157 185 223 247 250 286 310 310 330 326 337 342 353 361 374

*(-) MDYKDDDDK (E) *(K) DDDDKEECWVTEIANGSK (D) (K) DGLDSNPMK (D) (K) DYMILSGPQK (T) *(K) TAVAVLCTLLGLLSALENVAVLYLILSSHQLRR (K) *(R)KPSYLFIGSLAGADFLASVVFACSFVNFHVFHGVDSK(A) (K) AVFLLK (I) *(K) IGSVTMTFTASVGSLLLTAIDRYLCLR (Y) *(R) YPPSYKALLTR (G) (R) GR (A) (R) ALVTLGIMWVLSALVSYLPLMGWTCCPR (P) (R) PCSELFPLIPNDYLLSWLLFIAFLFSGIIYTYGHVLWK (A) *(K) AHQHVASLSGHQDRQVPGMARMRL (D) (R) LDVR (L) *(R)LAKTLGLVLAVLLICWFPVLALMAHSLATTLSDQVK(K) (K) KAFAFCSMLCLINSMVNPVIYALR (S) (K) AFAFCSMLCLINSMVNPVIYALR (S) *(Y) ALRSGEIR SSAHHCLAHWKKCVR (S) R) SSAHHCLAHWK (K) (R) GLGSEAK (E) (K) EEAPR (S) (R) SSVTETEADGK (I) (K) ITPWPDSR (D) (R) DLDLSDCHHHHHH (-)

+2, +3 +3 +2 +2 +3, +4 +4 NF +3 +3 NF +3 +4 +4 NF +4 +3 +3 +3 +3 NF NF +2 +2 +3

“*” indicates there is a missed cleavage site. NF ) not found.

Table 4. Chymotryptic Digestion of N-FLAG C-6HIS-Tagged CB2 Receptor; Identified Peptides by 4000 Q-Trap theoretical MW (Da)

precursor MW (Da)

start

end

peptide sequence

charge states

1789.645 2105.975 1426.822 3791.127 1065.608 1109.576 958.4873 1208.684 850.454 2741.603 3002.66 5092.540 1568.730 4621.626 3357.596 1428.594

1789.591 2105.784 1426.652 3790.997 1065.489 1109.389 958.3903 1208.528 850.286 2741.262 3002.484 5092.12 1568.579 4622.141 3357.467 1428.463

1 14 34 37 73 80 106 115 134 153 154 181 232 248 333 357

13 33 47 72 80 90 114 125 140 177 180 222 245 289 363 369

(-) MDYKDDDDKEECW (V) (W) VTEIANGSKDGLDSNPMKDY (M) (Y) MILSGPQKTAVAVL (C) (L) SGPQKTAVAVLCTLLGLLSALENVAVLYLILSSHQL (R) (L) RRKPSYLF (I) (L) FIGSLAGADFL (A) (F) HGVDSKAVF (L) (F) LLKIGSVTMTF (L) LTAIDRY (L) (L) LTRGRALVTLGIMWVLSALVSYLPL (M) (L) TRGRALVTLGIMWVLSALVSYLPLMGW (T) (W)TCCPRPCSELFPLIPNDYLLSWLLFIAFLFSGIIYTYGHVLW(K) (L) SGHQDRQVPGMARM (R) (L)DVRLAKTLGLVLAVLLICWFPVLALMAHSLATTLSDQVKKAF(A) (L) GSEAKEEAPRSSVTETEADGKITPWPDSRDL (D) (W) PDSRDLDLSDCH (H)

+2 +3, +4 +2 +4 +2 +2 +2 +2 +2 +3 +3 +4 +2 +5 +3 +3

requires either addition of those detergents whose presence is tolerated during sample handling or the removal of the detergents prior to mass spectrometric analysis to avoid protein aggregation. Transmembrane helices 1, 4, 5, 6, and part of TM7 were identified using surfactant (OBG)-assisted trypsin digestion of rhodopsin followed by HPLC for peptide separation and MALDI-TOF analysis.32 The combination of nanoLC coupled with ESI-MS allowed complete peptide mapping of bacteriorhodopsin, digested with excess of trypsin in the presence of CHAPS.33 In our analysis, we found that low concentrations (0.05%) of CYMAL5, when added to the digestion buffer, provided preparations that were compatible both with nanoLC separation and MS analysis.28 However, trypsin digestion of CB2 receptor also produces very short (1-4, 156157, 245-246, 247-250, 287, and 328-330) fragments that are below the lower scan range of the analysis, as well as long hydrophobic peptides with MW exceeding the confidence limit of MS detection, such as the hydrophobic peptide (158-223, GRAVY 1.118) covering the TM4 and TM5 helices. To overcome these limitations and to obtain full coverage of the receptor, 2074


we also performed digestion using chymotrypsin. Chymotrypsin predominantly cleaves peptide bonds at the carboxy side of aromatic amino acids and less efficiently of large hydrophobic amino acids, such as leucine, methionine, and isoleucine. Since the membrane-spanning regions are relatively abundant in hydrophobic amino acids, more fragments are generated through chymotryptic digestion, although often with several missed cleavages. Thus, although chymotrypsin digestion improves sequence coverage of the TM domains, a higher confidence in peptide identification, such as accurate mass determination and MS/MS analysis, is required to minimize false-positive identifications. MS Characterization Using Nanocapillary Liquid Chromatography (nanoLC) Coupled with Q-Trap MS or High Accuracy Mass Measurement LTQ-FT MS. This work demonstrates the first complete peptide mapping of the human CB2 receptor by LC-MS/MS. The results can be reported confidently based on the overlapping sequence data obtained from independent tryptic and chymotryptic digestions of purified receptor using the 4000 Q-Trap MS and the LTQ-FT MS. The


research articles

Figure 6. LC-MS analysis of a large CB2 tryptic peptide using LTQ-FT instrument. (A) Extracted ion chromatogram for peptide ALVTLGIMWVLSALVSYLPLMGWTCCPRPCSELFPLIPNDYLLSWLL FIAFLFSGIIYTYGHVLWK 158-223 for charge state 7+. High-resolution MS spectrum of the precursor ion is shown in the inset. (B) MS/MS spectrum of the same peptide acquired in the low-resolution ion trap mode. See text for more details on experimental conditions.

identification of peptides from two different enzymatic digests coupled with sequencing using tandem mass spectrometry allowed for the unambiguous characterization of all detected peptides and the full proteomic coverage of this GPCR. This represents an essential step in our quest to identify those amino acid residues that react covalently with the various ligands designed for this purpose. The LTQ-FT MS and 4000 Q-Trap MS analysis of CB2 tryptic digest resulted in sequence coverages of 97% and 90%, respectively. A 4000 Q-Trap MS analysis of a CB2 chymotrypsin digestion resulted in 76% coverage, further strengthening the sequence determination obtained using trypsin. Figure 5 represents a two-dimensional plot of the epitope-tagged human CB2 receptor, whose complete amino acid sequence has been confirmed using liquid chromatography coupled with nanospray tandem mass spectrometry. Each amino acid in the sequence is color coded to represent the enzymatic digest from which in it was identified. Yellow or green shaded amino acid sequences were uniquely identified in tryptic or chymotryptic digests, respectively, while the blue shaded amino acid sequences were identified in both enzymatic digests. The high sequence coverage obtained by using either the LTQ-FT MS or the 4000 Q-Trap MS suggests that either instrument is capable of analyzing future preparations of the CB2 receptor. However, by exploiting each instrument’s unique features, we can optimize the characterization of ligand binding motifs at the CB2 receptor.34,35 Table 2 lists the tryptic N-FLAG C-6HIS-tagged CB2 peptides identified by the LTQ-FT mass spectrometer. Of the 29 theoretical peptides produced by trypsin digestion of the receptor, only five were not detected, namely, peptides at 1-4 (MDYK),

156-157 (GR), 245-246 (MR), 287 (K), and 328-330 (CVR) positions. This is understandable since a majority of these fragments have molecular weights under 300 Da, below the lower scan range of the analysis. Thus, the tryptic peptides that were detected and identified by mass spectrometry constitute coverage of 97% of the epitope-tagged cannabinoid CB2 receptor sequence, corresponding to 362 out of 374 total amino acids. Table 3 list the peptides obtained from the tryptic N-FLAG C-6HIS-tagged CB2 that were identified using the 4000 Q-Trap and represent 90% of the CB2 receptor sequence. The peptides not identified by the Q-Trap corresponded to fragments at the 112-117, 156-157, 247-250, 331-337, and 338342 positions. All of these, with the exception of dipeptide GR 156-157, were detected by the LTQ-FT MS as singly charged species. The information dependent acquisition (IDA) method used by the Q-Trap excluded singly charged species in order to minimize acquisition of background and detergent ions generally detected as singly charged ions. While this approach enhances analysis of multiply charged ions, it can lead to misses of short singly charged CB2 peptides. The IDA approach could probably explain discrepancies between the number of missed cleavages when comparing the peptides identified in Tables 2 and 3. Indeed, the data from the LTQ-FT MS in Table 2 show only two peptides, 74-111 and 254-286, containing one missed cleavage site. One of these between residues 74 and 75 is expected due to the presence of two adjacent arginines. Conversely, the Q-Trap data in Table 4 show nine fragments with a missed cleavage site. The Q-Trap’s IDA method of discriminating singly charged ions may be the reason. Journal of Proteome Research • Vol. 6, No. 6, 2007 2075

research articles

Zvonok et al.

Figure 7. Tandem mass spectrometric sequence annotation of CB2 helix 7 tryptic peptide AFAFCSMLCLINSMVNPVIYALR 288-310 using Q-Trap.

A comparison of the CB2 tryptic peptides identified by the LTQ-FT MS and the Q-Trap MS leads to some interesting observations. For example, when trypsin is used to digest integral membrane proteins, large hydrophobic peptides that can encompass an entire transmembrane helix may be obtained. This often occurs due to the absence of lysine and arginine residues within the mostly hydrophobic transmembrane domains, and those residues located within a transmembrane domain are typically difficult to access by trypsin.36 As seen in Figures 4 and 5, the entire human CB2 receptor has only one lysine (K117) within a transmembrane domain (helix 3). The resulting tryptic peptides for TM helices 1-7 are over 30 amino acids long, except for TM 3 and TM 7, which contain 21 and 22 amino acids, respectively. The longest tryptic peptide (158-223), spanning TM 4 and 5, has 66 amino acids and a mass of 7734 Da. This peptide shown in Table 2 was identified by the LTQ-FT MS in charge states ranging from +5 to +8. Figure 6A shows an extracted ion chromatogram of this peptide for the 7+ charge state. As expected, the peptide eluted in the last segment of the chromatographic gradient at 95% solvent B. Importantly, as indicated by our ability to detect the hydrophobic peptide (GRAVY 1.118), it appears that the use of CYMAL detergent during the sample preparation can help to keep large hydrophobic peptides in solution, yet does not affect chromatographic resolution or sensitivity of the analysis. The annotated MS/MS spectrum of this peptide in Figure 6B shows that most of the major fragment ions can be assigned to multiply charged y-ions close to the N-terminus. Interestingly, 2076


the same long peptide (158-223) was not observed using Q-Trap analysis, and yet, two smaller fragments (158-185 and 186-223) were detected unexpectedly. The tryptic peptide 158223 contains an arginine at position 185; however, the arginine is flanked on both sides by two prolines. This PRP sequence is not a site for cleavage based upon trypsin’s specificity with a proline in the P1′ position.37 However, the analysis of CB2 on the Q-Trap revealed that this site was indeed cleaved by trypsin. Table 3 shows two separate peptides, 158-185 and 186-223, corresponding to TM domains 4 and 5. This cleavage at R185 does not follow the specificity rules for trypsin. A possible explanation for this anomaly could be that P184 may introduce a kink into the secondary structure of extracellular loop 2 that makes R185 accessible to trypsin for cleavage. Preliminary data38 from mutation experiments indicated that a cysteine at either position 292 or 296 of the CB2 receptor may be involved in a covalent interaction with electrophilic antagonists. Therefore, identifying and sequencing peptide AFAFCSMLCLINSMVNPVIYALR, containing helix 7 with both cysteines, shown in (Figure 7), would be crucial in further experiments aimed at determining the exact position for ligand attachment. In addition, the highly hydrophobic peptide TLGLVLAVLLICWFPVLALMAHSLATTLSDQVK (GRAVY 1.491) that includes the conserved CWFP motif, believed to be involved in the CB2 receptor activation and a site of attachment for our electrophilic covalent agonists, was identified by the LTQ-FT MS. Figure 8A shows a high-resolution MS spectrum with a reliably deter-

research articles


Figure 8. LC-MS analysis of CB2 helix 6 tryptic peptide TLGLVLAVLLICWFPVLALMAHSL ATTLSDQVK 254-286 using LTQ-FT instrument. High-resolution MS spectrum of the precursor ion for charge state 3+ and MS/MS spectrum acquired using linear ions trap are shown in panel A and B, respectively.

mined charge state of the ion, and panel B presents its MS/ MS spectrum acquired using the linear ion trap. The majority of high-intensity fragment ions can be assigned to b- and y-ions, suggesting that covalently attaching a ligand may determine the exact amino acid-ligand link. On the basis of single site mutation experiments, we have identified C6.42 cysteine as the site of attachment of AM841, a high affinity ligand carrying an electrophilic isothiocyanate group. We have now obtained preliminary MS evidence confirming the above finding. Additional work aimed at fully confirming the results are currently underway. The LTQ-FT MS was able to identify and sequence 97% of the human CB2 receptor independently with high confidence, by providing a combination of high mass accuracy in the MS spectrum and extensive fragmentation in the MS/MS mode. To further confirm the data obtained on the LTQ-FT, the Q-Trap MS also identified over 90% of the CB2 sequence. Between the two instruments, the only tryptic fragment not confirmed by MS/MS was the dipeptide GR at position 156157, whose molecular weight of 231 Da was below the scan range for both analyses. To obtain entire sequence coverage of the human CB2 receptor, a chymotryptic digest was performed and analyzed by the Q-Trap MS. Table 4 lists the chymotryptic CB2 peptides identified using the 4000 Q-Trap MS. The theoretical digestion of CB2 by chymotrypsin yields 66 peptides, 39 of which are 4 amino acids or fewer. The large number of peptides compared to trypsin (29) is due to the abundance of aromatic/hydrophobic amino acid residues in the CB2 receptor. Eighteen peptides covering 76% of CB2 were identified (Table 4). The chymotryptic coverage included the N-terminus; TM helices 1, 4, 5, and 6; extracellular loops 2 and

3; and cytoplasmic loop 1. All other regions were partially sequenced. The dipeptide GR, missing from the trypsin map of CB2, was sequenced in the chymotryptic peptides 153-177 and 154-180.

Conclusion Sample quality is critical for achieving high sensitivity, resolution, and mass accuracy in the mass spectrometric characterization of integral membrane proteins. To improve the purification of the CB2 cannabinoid receptor and obtain material suitable for in-solution digestion, we have expressed CB2 in the baculovirus system as a functionally active protein with a FLAG epitope at the amino-terminus and a HIS6 tag at the carboxyl-terminus. Several purification schemes were employed, including IMAC and FLAG affinity chromatography, alone and combined. We established that a single step of antiFLAG M2 affinity chromatography resulted in the best preparations, while introducing the second IMAC step significantly decreases the quality and quantity of the purified protein. The purified receptor was prepared for in-solution proteolytic digestion using a spin desalting-buffer exchange column. The surfactant CYMAL5 was added to the buffer in amounts tolerated by ESI-MS, to keep the receptor in solution and prevent protein aggregation. In-solution trypsin and chymotrypsin digestion minimized sample losses, as compared to the in-gel procedure, and produced overlapping peptides representing all parts of the cannabinoid CB2 receptor. Following proteolytic digestion, CB2 peptides were separated on reversedphase microcapillary columns and analyzed using nanoESI source instruments. The combination of nanoLC coupled with the Q-Trap MS or the high accuracy mass measurement LTQJournal of Proteome Research • Vol. 6, No. 6, 2007 2077

research articles FT MS allowed the identification of the peptide fragments representing the whole protein sequence, resulting in complete MS characterization of the cannabinoid CB2 receptor. The results can be reported confidently, based on the overlapping sequence data obtained using electrospray tandem mass spectrometry (MS/MS). Abbreviations: BSA, bovine serine albumin; C-, carboxy; N-, amino; CB1, cannabinoid receptor 1; CB2, cannabinoid receptor 2; CHAPS, 3-[(chlolamidopropyl)dimethylammonio]-1propane sulfonate; CHS, cholesteryl hemisuccinate trissalt; CYMAL5, 5-cyclohexyl-1-pentyl-β-D-maltoside; DM, n-dodecylβ-D-maltoside; DTT, dithiothreitol; ECL, enhanced chemiluminescence; EDTA, ethylene diamine tetraacetic acid; ESI-MS, electrospray ionization mass spectrometry; FLAG, MDYKDDDK peptide tag; FOS12, n-dodecylphosphocholine; GPCR, G proteincoupled receptor; GRAVY, grand average of hydropathicity; HIS6, hexa-histidine peptide tag; HRP, horseradish peroxidase; LC, liquid chromatography; IAM, iodoacetamide; IDA, information dependent acquisition; IMAC, immobilized metal affinity chromatography; LTQ-FT MS, linear ion trap-Fourier transform mass spectrometer; MALDI-TOF MS, matrix-assisted laser desorbtion/ionization time-of-flight mass spectrometry; m/z, mass-to-charge ratio; P2 membrane, pellet from the second centrifugation step; Q-Trap MS, hybrid triple quadrupole/linear ion trap mass spectrometer; TM, transmembrane; TME, 25 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 2 mM EDTA.

Acknowledgment. This work has been supported by grants from the National Institutes of Drug Abuse: DA09158, DA00493, DA03801, DA07215, and DA07312 (A.M.), and GM15847 (B.L.K.). This work is contribution #893 from the Barnett Institute. References (1) Palczewski, K.; Kumasaka, T.; Hori, T.; Behnke, C. A.; Motoshima, H.; Fox, B. A.; Le, Trong, I.; Teller, D. C.; Okada, T.; Stenkamp, R. E.; Yamamoto, M.; Miyano, M. Crystal structure of rhodopsin: a G protein-coupled receptor. Science 2000, 289, 739-745. (2) Okada, T.; Fujiyoshi, Y.; Silow, M.; Navarro, J.; Landau, E. M.; Shichida, Y. Functional role of internal water molecules in rhodopsin revealed by x-ray crystallography. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5982-5987. (3) Functional heterologous expression and purification of large quantities of GPCRs, has proved to be very difficult [Tate, C. G.; Grisshammer, R. Heterologous expression of G-protein-coupled receptors. Trends Biotechnol. 1996, 14, 426-430. (4) Marullo, S.; Delavier-Klutchko, C.; Guillet, J.; Charbit, A.; Stotsberg, A. D.; Emorine, L. J. Expression of human beta 1 and beta 2 adrenergic receptors in E. coli as a new tool for ligand screening. BioTechnology 1989, 7, 923-927. (5) Bertin, B.; Freissmuth, M.; Breyer, R. M.; Schutz, W.; Strosberg, A. D.; Marullo, S. Functional expression of the human serotonin 5-HT1A receptor in Escherichia coli. Ligand binding properties and interaction with recombinant G protein alpha-subunits. J. Biol. Chem. 1992, 267, 8200-8206. (6) Weiss, H. M.; Grisshammer, R. Purification and characterization of the human adenosine A2a receptor functionally expressed in Escherichia coli. Eur. J. Biochem. 2002, 269, 82-92. (7) Calandra, B.; Tucker, J.; Shire, D.; Grisshammer, R. Expression in Escherichia coli and characterization of the human central CB1 and peripheral CB2 cannabinoid receptors. Biotechnol. Lett. 1997, 19 (5), 425-428. (8) Yeliseev, A. A.; Wong, K. K.; Soubias, O.; Gawrisch, K. Expression of human peripheral cannabinoid receptor for structural studies. Protein Sci. 2005, 14, 2638-2653. (9) Tucker, J.; Grisshammer, R. Purification of a rat neurotensin receptor expressed in Escherichia coli. Biochem. J. 1996, 317, 891899. (10) Kim, T.-K.; Zhang, R.; Feng, W.; Cai, J.; Pierce, W.; Song, Z.-H. Expression and characterization of human CB1 cannabinoid receptor in methylotrophic yeast Pichia pastoris. Protein Expression Purif. 2005, 40, 60-70.

2078


Zvonok et al. (11) Feng, W.; Cai, J.; Pierce, W. M., Jr.; Song, Z.-H. Expression of CB2 cannabinoid receptor in Pichia pastoris. Protein Expression Purif. 2002, 26, 496-505. (12) Akermoun, M.; Koglin, M.; Zvalova-Iooss, D.; Folschweiller, N.; Dowell, S. J.; Gearing, K. L. Characterization of 16 human G protein-coupled receptors expressed in baculovirus-infected insect cells. Protein Expression Purif. 2005, 44 (1), 65-74. (13) Glass, M.; Northup, J. K. Agonist selective regulation of G proteins by cannabinoid CB1 and CB2 receptors. Mol. Pharmacol. 1999, 56, 1362-1369. (14) Xu, W.; Filppula, S.; Mercier, R.; Yaddanapudi, S.; Pavlopoulos, S.; Cai, J.; Pierce, W. M.; Makriyannis, A. Purification and mass spectroscopic analysis of human CB1 cannabinoid receptor functionally expressed using the baculovirus system. J. Peptide Res. 2005, 66, 138-150. (15) Filppula, S.; Yaddanapudi, S.; Mercier, R.; Xu, W.; Pavlopoulos, S.; Cai, J.; Pierce, W. M.; Makriyannis, A. Purification and mass spectroscopic analysis of human CB2 cannabinoid receptor expressed in the baculovirus system. J. Peptide Res. 2004, 64, 225236. (16) Farrens, D. L.; Dunham, T. D.; Fay, J. F.; Dews, I. C.; Caldwell, J.; Nauert, B. Design, expression, and characterization of a synthetic human cannabinoid receptor and cannabinoid receptor/ Gprotein fusion protein. J. Peptide Res. 2002, 60, 336-347. (17) Christoffers, K. H.; Li, H.; Keenan, S. M.; Howells, R. D. Purification and mass spectrometric analysis of the µ opioid receptor. Mol. Brain Res. 2003, 118, 119-131. (18) Christoffers, K. H.; Li, H.; Howells, R. D. Purification and mass spectrometric analysis of the δ opioid receptor. Mol. Brain Res. 2005, 136, 54-64. (19) Picone, R. P.; Fournier, D. J.; Makriyannis, A. Ligand based structural studies of the CB1 cannabinoid receptor. J. Peptide Res. 2002, 60, 348-356. (20) Picone, R. P.; Khanolkar, A. D.; Xu, W.; Ayotte, L. A.; Thakur, G. A.; Hurst, D. P.; Abood, M. E.; Reggio, P. H.; Fournier, D. J.; Makriyannis, A. (-)-7′-Isothiocyanato-11-hydroxy-1′,1′-dimethylheptylhexahydrocannabinol (AM841), a high-affinity electrophilic ligand, interacts covalently with a cysteine in helix six and activates the CB1 cannabinoid receptor. Mol. Pharmacol. 2005, 68, 1623-1635. (21) Wu, S. L.; Kim, J.; Hancock, W. S.; Karger, B. Extended Range Proteomic Analysis (ERPA): a new and sensitive LC-MS platform for high sequence coverage of complex proteins with extensive post-translational modifications-comprehensive analysis of betacasein and epidermal growth factor receptor (EGFR). J. Proteome Res. 2005, 4 (4), 1155-1170. (22) Jensen, O. N.; Wilm, M.; Shevchenko, A.; Mann, M. Sample preparation methods for mass spectrometric peptide mapping directly from 2-DE gels. Methods Mol. Biol. 1999, 112, 513-530. (23) Ball, L. E.; Oatis, J. E., Jr.; Dharmasiri, K.; Busman, M.; Wang, J.; Cowden, L. B.; Galijatovic, A.; Chen, N.; Crouch, R. K.; Knapp, D. R. Mass spectrometric analysis of integral membrane proteins: application to complete mapping of bacteriorhodopsins and rhodopsin. Prot. Sci. 1998, 7, 758-764. (24) Kraft, P.; Mills, J.; Dratz, E. Mass spectrometric analysis of cyanogen bromide fragments of integral membrane proteins at the picomole level: application to rhodopsin. Anal. Biochem. 2001, 292, 76-86. (25) Ablonczy, Z.; Crouch, R. K.; Knapp, D. R. Mass spectrometric analysis of integral membrane proteins at the subpicomolar level: application to rhodopsin. J. Chromatogr., B. 2005, 825, 169-175. (26) Lichty, J. J.; Malecki, J. L.; Agnew, H. D.; Michelson-Horowitz, D. J.; Tan, S. Comparison of affinity tags for protein purification. Protein Expression Purif. 2005, 41, 98-105. (27) Arnau, J.; Lauritzen, C.; Petersen, G. E. Pedersen, John. Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins. Protein Expression Purif. 2006, 48, 1-13. (28) Katayama, H.; Tabata, T.; Ishihama, Y. Sato, T.; Oda, Y.; Nagasu, T. Efficient in-gel digestion procedure using 5-cyclohexyl-1pentyl-β-D-maltoside as an additive for gel-based membrane proteomics. Rapid Commun. Mass Spectrom. 2004, 18, 23882394. (29) Katayama, H.; Nagasu, T.; Oda, Y. Improvement of in-gel digestion protocol for peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 2001, 15, 1416-1421.

research articles

Proteomic Mass Spectrometric Characterization of hCB2 Receptor (30) Zhang, N.; Li, L. Effects of common surfactants on protein digestion and matrix-assisted laser desorption/ionization mass spectrometric analysis of the digested peptides using two-layer sample preparation. Rapid Commun. Mass Spectrom. 2004, 18, 889-896. (31) Zhang, N.; Li, L. Effects of common surfactants on protein digestion and matrix-assisted laser desorption/ionization mass spectrometric analysis of the digested peptides using two-layer sample preparation. Rapid Commun. Mass Spectrom. 2004, 18, 889-896. (32) Barnidge, D. R.; Dratz, E. A.; Sunner, J. J.; Jesaitis, A. Identification of transmembrane tryptic peptides of rhodopsin using matrixassisted laser desorption/ionization time-of-flight mass spectrometry. Protein Sci. 1997, 6, 816-824. (33) Hixson, K. K.; Rodriguez, N.; Camp, D. G. II; Strittmatter, E. F.; Lipton, M. S.; Smith, R. D. Evaluation of enzymatic digestion and liquid chromatography-mass spectrometry peptide mapping of the integral membrane protein bacteriorhodopsin. Electrophoresis 2002, 23, 3224-3232.

(34) Clauser, K. R.; Baker, P.; Burlingame, A. L. Role of accurate mass measurement ((10 ppm) in protein identification strategies employing MS or MS/MS database searching. Anal. Chem. 1999, 71, 2871-2882. (35) Le Blanc, J. C.; Hager, J. W.; Ilisiu, A. M.; Hunter, C.; Zhong, F.; Chiu. I. Unique scanning capabilities of a new hybrid linear ion trap mass spectrometer (Q TRAP) used for high sensitivity proteomics applications. Proteomics 2003, 3, 859-869. (36) Ablonczy, Z.; Kono, M.; Crouch, R. K.; Knapp, D. R. Mass spectrometric analysis of integral membrane proteins at the subnanomolar level: application to recombinant photopigments. Anal. Chem. 2001, 73, 4774-4779. (37) Keil, B. Specificity of Proteolysis; Springer-Verlag: Berlin-Heidelberg-NewYork, 1992; p 335. (38) Makriyannis, A. Unpublished results.

PR060671H

Journal of Proteome Research • Vol. 6, No. 6, 2007 2079

Comprehensive Proteomic Mass Spectrometric Characterization of

Recommend Documents