Analysis of an Intact G-Protein Coupled Receptor by MALDI-TOF Mass

Sandrine Sagan†. Synthe`se, Structure et Fonction de Molécules Bioactives, and Plateforme de Protéomique et de Spectrométrie de Masse,. Universit...
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Anal. Chem. 2007, 79, 2189-2198

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Analysis of an Intact G-Protein Coupled Receptor by MALDI-TOF Mass Spectrometry: Molecular Heterogeneity of the Tachykinin NK-1 Receptor Isabel D. Alves,*,† Emmanuelle Sachon,†,‡ Gerard Bolbach,†,‡ Lynda Millstine,§ Solange Lavielle,† and Sandrine Sagan†

Synthe` se, Structure et Fonction de Mole´ cules Bioactives, and Plateforme de Prote´ omique et de Spectrome´ trie de Masse, Universite´ Pierre et Marie Curie-Paris 6, UMR 7613 CNRS, Paris, France, and Applied Biosystems, 25 Avenue de la Baltique, BP 96, 91943 Courtaboeuf, France

Integral membrane proteins are among the most challenging targets for biomedical research as most important cellular functions are tied to these proteins. To analyze intrinsically their structure/function, their transduction mechanism, or both, these proteins are commonly expressed in cultured cells as recombinant proteins. However, it is not possible to check whether these recombinant proteins are homogeneously or heterogeneously expressed. Owing to difficulties in their purification, very few mass spectrometry studies have been performed with those proteins and even less with G-protein coupled receptors. Here we have set up a procedure that is highly compatible with MALDI-TOF mass spectrometry to analyze an intact histidine-tagged G-protein coupled, namely, the tachykinin NK-1 receptor expressed in CHO cells, solubilized and purified using cobalt or nickel chelating magnetic beads. The metal-chelating magnetic beads containing the receptor were directly spotted on the MALDI plate for analysis. SDS-PAGE, combined with ingel digestion analyzed by mass spectrometry, Western blot ((His)6 and FLAG M2 tags), photoaffinity labeling with a radioactive agonist, and Edman sequencing, confirmed the identity of the purified protein as the human tachykinin NK-1 receptor. Mass spectrometry study of both the glycosylated and deglycosylated intact protein forms revealed the existence of several receptor species that is tempting to correlate with the unusual pharmacological behavior of the receptor.

10.1021/ac062415u CCC: $37.00 Published on Web 02/13/2007

© 2007 American Chemical Society

Membrane proteins encode for ∼30% of the human genome, catalyzing a multitude of essential functions, thus being important with regard to human disease. Regardless of their importance, membrane proteins and particularly seven-transmembrane (7TM) receptors still remain a challenge for analyzing the structure/ function relationships mainly because they are difficult to purify in sufficient quantities for molecular characterization by physical methods (such as NMR or CD). In addition, the primary structure of 7TM receptors is being more than often not predictable from gene sequencing, as they can be modified because of alternative splicing, RNA editing, or post-translational modifications (glycosylation, palmitoylation, phosphorylation, protein backbone cleavage) that are crucial for the receptor functions.1-3 For all these reasons, membrane proteomic developments are increasing with the mass spectrometry analysis of peptides from 1D or 2D in-gel digestions or with nongel shotgun methods.4 Using these strategies, a few membrane receptors have been identified such as the µ5,6 and the δ7 opioid, CB1 and CB2 cannabinoid,8,9 or estrogen10 * To whom correspondence should be addressed. E-mail: alves@ ccr.jussieu.fr. † Synthe ` se, Structure et Fonction de Mole´cules Bioactives, Universite´ Pierre et Marie Curie. ‡ Plateforme de Prote ´ omique et de Spectrome´trie de Masse, Universite´ Pierre et Marie Curie. § Applied Biosystems. (1) Krishna, R. G.; Wold, F. Adv. Enzymol. Relat. Areas Mol. Biol. 1993, 67, 265-298. (2) Yang, X. J. Oncogene 2005, 24, 1653-1662. (3) Jensen, O. Nature Rev. Mol. Cell Biol. 2006, 7, 391-403. (4) Wu, C. C.; MacCoss, M. J.; Howell, K. E.; Yates, J. R., III Nature Biotechnol. 2003, 21, 532-538. (5) Christoffers, K. H.; Li, H.; Keenan, S. M.; Howells, R. D. Brain Res. Mol. Brain. Res. 2003, 118, 119-31. (6) Sarramegna, V.; Muller, I.; Mousseau, G.; Froment, C.; Monsarrat, B.; Milon, A.; Talmont, F. Protein Expr. Purif. 2005, 43, 85-93.

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receptors. However, the homogeneity and molecular mass of these membrane proteins have only been inferred from their migration into polymer gels, although it has been shown that this method is not always reliable and can be biased.11 Therefore, mass spectrometry analysis is a grade one method to check for homogeneity through the precise measure of the molecular mass of these proteins. However, analysis by mass spectrometry of intact membrane proteins remains challenging. Indeed, there are many difficulties in solubilizing and purifying these proteins under conditions (buffers, salts, detergents) compatible with a suitable ionization during MALDI-TOF mass spectrometry analysis. The number of intact 7TM receptors that have been analyzed by mass spectrometry is restricted to a few ones. Among those, bacteriorhodopsin and rhodopsin, both of which are easily obtained from tissues in large quantities, have been analyzed by MALDI-TOF12,13 and ESI.14 In other examples, recombinant proteins had to be produced such as in the case of the thromboxane A2 receptor.15 In this study, we set up an efficient procedure that allowed the analysis of the intact His-tagged tachykinin NK-1 receptor by MALDI-TOF. The receptor, expressed in Chinese hamster ovarian (CHO) cells in high levels (10 pmol/mg og membrane proteins)16 was purified using cobalt or nickel magnetic beads. The magnetic beads containing the NK-1 receptor could be directly spotted on the plate for MALDI-TOF analysis. Both the intact glycosylated and deglycosylated protein species were analyzed by mass spectrometry. The identity of the proteins as the NK-1 receptor was confirmed by in-gel digestion combined with mass spectrometry, Western blot against the N-terminal FLAG M2 and the C-terminal (His)6 tags on the human recombinant NK-1 receptor, photolabeling with a radioactive and photoactivable analogue17 of SP, and Edman sequencing. EXPERIMENTAL METHODS Cell Culture. The FLAG M2 and His-tagged NK-1 receptor has been cloned in pTEJ8 vector and expressed in CHO-K1 cells with the SuperfectTM transfection reagent (Qiagen). CHO cells expressing human NK-1 receptors, with an expression level of 10 pmol/mg of total protein, were cultured in DMEM supplemented with 100 IU/mL penicillin, 100 IU/mL streptomycin, fungizone, and 10% fetal calf serum. Cultures were kept at 37 °C in a humidified atmosphere of 5% CO2. Stable transfectants were maintained by Geneticin (400 mg/L) periodic selection. (7) Christoffers, K. H.; Li, H.; Howells, R. D. Brain Res. Mol. Brain Res. 2005, 136, 54-64. (8) Filppula, S.; Yaddanapudi, S.; Mercier, R.; Xu, W.; Pavlopoulos, S.; Makriyannis, A. J. Pept. Res. 2004, 64, 225-236. (9) Kim, T.-K.; Zhang, R.; Feng, W.; Cai, J.; Pierce, W.; Song, Z.-H. Prot. Expr. Purif. 2005, 40, 60-70. (10) Yang, S. H.; Liu, R.; Perez, E. J.; Wen, Y.; Stevens, S. M., Jr.; Valencia, T.; Brun-Zinkernagel, A. M.; Prokai, L.; Will, Y.; Dykens, J.; Koulen, P.; Simpkins, J. W. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 4130-4135. (11) Murcia-Nicolas, A.; Bolbach, G.; Blais, J. C.; Beaud, G. Virus Res. 1999, 59, 1-12. (12) Cadene, M.; Chait, B. T. Anal. Chem. 2000, 72, 5655-5658. (13) Schey, K. L.; Papac, D. I.; Knapp, D. R.; Crouch, R. K. Biophys. J. 1992, 63, 1240-1243. (14) Whitelegge, J. P.; Gundersen, C. B.; Faull, K. F. Protein Sci. 1998, 7, 14231430. (15) Pawate, S.; Schey, K. L.; Meier, G. P.; Ullian, M. E.; Mais, D .E.; Halushka, P. V. J. Biol. Chem. 1998, 273, 22753-22760. (16) Alves, I. D.; Delaroche, D.; Mouillac, B.; Salamon, Z.; Tollin, G.; Hruby, V. J.; Lavielle, S.; Sagan, S. Biochemistry 2006, 45, 5309-5318. (17) Girault, S.; Sagan, S.; Bolbach, G.; Lavielle, S.; Chassaing, G. Eur. J. Biochem. 1996, 240, 215-222.

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Solubilization and Purification of the NK-1 Receptor. CHO cells were grown to confluence in 225-cm2 tissue culture flasks. Membranes were prepared as previously described.18 Membrane homogenates were resuspended in the solubilization buffers: 10 mM HEPES pH 7.6, 0.1 M KCl, 1% dodecyl maltoside, and protease inhibitors (1 mL/L) for His-tagged proteins (Sigma). The membranes were then homogenized by 15 strokes and kept for 1 h at 4 °C. The homogenate was centrifuged at 40000g. To the supernatant was added cobalt or nickel chelating magnetic beads (Invitrogen), prewashed with the same buffer system used for the protein solubilization and placed under gentle vortex for 1.5 h at 22 °C. The beads were kept in the solubilization buffer for MS analysis or incubated first (20 min) with the detergent buffer containing 30 mM imidazole to remove nonspecifically bound proteins and eluted with detergent buffer containing 200 mM imidazole. Alternatively, a lectin affinity purification of the crude protein was performed using wheat germ agglutinin (WGA) or concanavalin A (Con A). To the crude protein (∼0.5 mL in 10 mM HEPES pH 7.6, 0.1 M KCl, 1% dodecyl maltoside, and protease inhibitors (1 mL/L)) was added 1 mM CaCl2 and 1 mM MgCl2, and the resultant mixture was incubated with 1 mL of agarose-bound Con A or WGA for 2 h at 22 °C. Then, the beads were extensively washed with the above buffer and eluted from the Con A and WGA columns with the same buffer containing 0.5 M methyl R-D-mannopyranoside or 0.5 M N-acetylglucosamine, respectively. The purity and identity of the purified protein was monitored by gel electrophoresis and Western blot analysis targeted against the His-tag and FLAG M2 present in the receptor C- and N-terminal sides, respectively as described below. SDS-PAGE and Western Blot Analysis. The Western blot was performed on a PVDF membrane that was treated with an anti-(His)6 antibody (Qiagen) or a anti-FLAG M2 antibody (Sigma) followed by the Immun-Star chemiluminescencent protein detection system (Bio-Rad). Proteins were visualized on X-OMAT photographic films (Kodak). NK-1 Receptor Deglycosylation. Two different procedures were performed, either production of a deglycosylated receptor by tunicamycin treatment on cultured cells (performed as previously described17) or deglycosylation of the purified protein (Nglycosidase F). For N-glycosidase F treatment, the purified NK-1 receptor was simultaneously eluted from the cobalt beads and denatured by incubation with 20-40 µL of 50 mM Tris-HCl, pH 7.6, 3% SDS, and 17 mM DTT for 2 h at 37 °C. The solution was separated from the beads and the SDS diluted to a final concentration of 0.1% with NaH2PO4 50 mM, pH 8.5, to which Triton X-100 was added to a final concentration of 2%. This solution was incubated with 1 unit of N-glycosidase F (Roche) for 12 h at 37 °C. Additionally, the receptor was also incubated with a mixture of enzymes able to remove both N- and O-linked carbohydrates (Enzymatic Protein Deglycosylation Kit, Sigma). Alternatively, N-glycosidase F treatment was performed on the crude membrane preparation by the same method followed by protein purification with the magnetic Co2+ beads as described above. Photoaffinity Labeling and SDS-PAGE. Membranes from CHO cells (100 and 150 µg for the nontreated and N-glycosidase F-treated sample, respectively) expressing the NK-1 receptor were (18) Sagan, S.; Beaujouan, J. C.; Torrens, Y.; Saffroy, M.; Chassaing, G.; Glowinski, J.; Lavielle, S. Mol. Pharmacol. 1997, 52, 120-127.

incubated for 15 min at room temperature with 1 nM Bapa-[([2,33H]CH CH CO)Lys,3 (p-Bz)Phe,8 Pro9]SP in 50 mM Tris-Cl, pH 3 2 7.6, containing 1 mM EDTA, 10 mM MgCl2, 1 mM PMSF, 40 µg/mL bacitracin, 5 µg/mL leupeptin, 5 µg/mL STI, and 400 µg/ mL BSA. The membrane preparation was cooled at 4 °C and irradiated on ice for 40 min with ultraviolet light at 365 nm (HPR 125-W lamp) at a distance of 6 cm. After photolysis, the membranes were diluted twice with the buffer described above and incubated for 5 min with 10 µM [Pro9]SP to remove noncovalently attached photoaffinity ligand. The membranes were further washed with 50 mM Tris-Cl buffer, centrifuged for three cycles to remove unbound ligand, and finally denatured by incubation at 37 °C for 2-4 h with 20-40 µL of 50 mM Tris-HCl, pH 7.6, 3% SDS, and 17 mM DTT. Alternatively, the detergent-solubilized membranes obtained as described above were photolabeled as indicated, and the receptor was purified using Co2+ magnetic beads. Photolabeled membranes or purified photolabeled receptors were denatured by incubation for 15 min at room temperature with 60 mM Tris-HCl, pH 6.8, containing 2% SDS, 10% glycerol, 0.025% bromophenol blue, and 5% 2-mercaptoethanol. Samples were transferred into polyacrylamide gels according to the method of Laemmli.19 After electrophoresis, the gels were sliced every 2 mm or stained with coomassie blue/silver nitrate, after which the protein bands were sliced. The gel slices were heated at 50 °C for 20 h in H2O2 (35% in 0.5 mL of H2O). After cooling, 0.5 mL of 1% SDS in 4 M urea was added before radiation counting in 8 mL of scintillation liquid. Mass Spectrometry. MALDI-TOF experiments were carried out on different apparatus (Voyager Elite, DE-Pro, and 4700 Proteomic Analyzer, Applied Biosystems). For the analyses of the entire protein (positive ions), the linear mode was used (using an accelerated voltage of 20 kV) in combination with the delayed extraction. External calibration was performed using standard proteins (BSA, apomyoglobin, thioredoxin, and insulin; Applied Biosystems). The analysis of the fragments resulting from the protein digestion was essentially carried out on the MALDI-TOF 4700 Proteomics Analyzers in reflector mode (positive ion) using an external calibration (4700 mixture from Applied Biosystems). Entire Proteins. Several samples were analyzed to detect intact proteins purified by Ni2+ and Co2+ magnetic beads. Two procedures were examined, either direct analysis of the intact receptor still bound to the beads or after elution from the beads using the matrix. The best method was to deposit the beads (1 µL) on a first matrix deposit (1 µL), and after drying at room temperature, the beads were covered with a few microliters of matrix. Indeed, it has been noticed that beads must be surrounded by matrix microcrystals in order to detect protein signals. Ni2+ beads (Qiagen) gave a nonhomogeneous target because of aggregation (arising from the large size of beads), while Co2+ beads (Invitrogen) gave homogeneous targets (beads of smaller diameter). The elution of protein was found efficient only with Co2+ beads; this was performed by incubating the beads with 3 µL of the matrix solution for 10 min (mixed a few times by vortex). From this, 0.5-1 µL of the solution (no beads) was deposited on the target holder. (19) Laemmli, U. K. Nature 1970, 227, 680-685.

The matrix (R-cyano-4-hydroxycinnamic acid, CHCA, saturated solution in acetonitrile/water 1:1 with 0.1%TFA) was found to be the best for membrane protein ionization, producing multiple charged ions. Preparation of CHCA as a saturated solution in a 3:1:2 (v/v/v) mixture of formic acid/water/isopropyl alcohol12 did not improve either the sensibility or the signal over noise ratio for this membrane protein. The production of multiple charged ions was useful to identify such large proteins. Many experiments with this receptor were also carried out with sinapinic acid, but the results in terms of sensibility or signal over noise ratio were always better with CHCA. This observation is consistent with the previous study reported by Cadene and Chait.12 Digest Analysis. Aliquots of 0.5-1 µL were mixed with 1.5 µL of CHCA. Data from mass spectra were analyzed through Mascot, PAWS, and ExPASy research engines by considering the following parameters: digestion (trypsin or trypsin/chymotrypsin, CNBr), three maximum missed cleavages, possible oxidation of Met, and carbamidomethylation of Cys. The peptide mass tolerance was set to 30-50 ppm. To avoid any interference with matrix ions, only peptides with m/z >700 were considered in database search. Edman Sequencing. All samples were sequenced on the Procise 492 cLC protein sequencing system (Applied Biosystems) integrated with an online HPLC 140D dual-syringe pump, UV detector connected to a computer for data analysis using the Data Analysis Software Sequence Pro. Phenylthiohydantoin (PTH) amino acids were visualized with a UV wavelength (269 nm) using a 0.8 × 250 mm PTH column. Samples were applied to Biobrene precycled filters, dried under argon for 5 min, and then subjected to chemical Edman sequencing for 20 cycles. Edman sequencing was performed on the entire protein eluted from the cobalt beads (purified protein) with 0.1% TFA and 20% acetonitrile. In-Gel Tryptic Digestion Followed or Not by CNBr Cleavage. After visualization of the bands in the gel by coomassie staining, the bands containing the protein of interest were excised from the SDS-polyacrylamide gel and the SDS on the gel removed by ion pair extraction (adapted from ref 20) by incubating the gel pieces with 1 mL of a solution containing 85% acetone, 5% acetic acid, 5% triethylamine, and 5% H2O for 1 h at room temperature under agitation. After aspiration of the solution from the gel pieces, destaining, reduction and alkylation was performed by standard protocols. Tryptic digestion was started with the addition of 5 µL of 75 ng/µL trypsin in 25 mM ammonium bicarbonate, pH 8, and 0.1% of dodecyl maltoside to enhance peptide solubility and digestion efficiency and help extraction of more hydrophobic fragments from the gel (adapted from ref 21), performed at 4 °C to avoid autodegradation of trypsin before entering the gel. After reswelling, the gel pieces were covered with the above-mentioned buffer (without trypsin), and digestion was performed for 12-15 h at 37 °C. Enzymatic activity was stopped by the addition of 10 µL of PMSF (0.1 M) and 10 µL of STI (5 µg/mL). In the case where CNBr cleavage was performed after trypsin digestion, 2040 µL of CNBr in TFA (one small crystal was dissolved in 200300 µL of TFA) was added to make a 70% TFA solution that was incubated in the dark for 12-15 h under inert atmosphere. After collection of the supernatant, an extraction procedure was (20) Zischka, H.; Gloeckner, C. J.; Klein, C.; Willmann, S.; Swiatek-de Lange, M.; Ueffing, M. Proteomics 2004, 4, 3776-3782. (21) van Montfort, B. A.; Canas, B.; Duurkens, R.; Godovac-Zimmermann, J.; Robillard, G. T. J. Mass Spectrom. 2002, 37, 322-330.

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Figure 1. Metal chelating chromatographic purification of the detergent-solubilized NK-1 receptor followed by N-glycosidase F treatment. Protein-stained SDS gel (10%) (1, 2, 3, 7, 8) showing the crude membrane extract from CHO cells expressing the NK-1 receptor (3), after purification using cobalt magnetic beads with (2) or without imidazole elution (1) and upon N-glycosidase F treatment (7 and 8 correspond to the purified receptor before and after N-glycosidase F treatment). Western blot negative control corresponding to the NK-1 receptor lacking the His-tag (4). Western blot analysis of the purified glycosylated receptor directed against the anti-(His)6 (5) and anti-FLAG M2 (6) antibodies and upon N-glycosidase F treatment (9 and 10 correspond to the purified receptor before and after N-glycosidase F treatment revealed by antiFLAG M2). A protein band with aa Mr of ∼60 000 was observed in the SDS-PAGE and recognized after blotting into nitrocellulose membrane together with a protein band with a Mr of ∼50 000 using both the anti-His and anti-FLAG M2 antibodies. The higher Mr band shifted to lower molecular weight following N-glycosidase F treatment.

performed in certain cases (as it did no seem to lead to the extraction of significant amounts of material) by sonicating the gel pieces for 10 min with 20 µL of acetonitrile in the presence of 0.1% dodecyl maltoside (the process was repeated twice). The overlay and extracts were concentrated in a SpeedVac either separated (to test the capability of the extraction step) or combined. RESULTS AND DISCUSION Affinity Purification and Western Blot Analysis of the NK-1 Receptor. As with any membrane protein, the NK-1 receptor must undergo a solubilization step before its purification can be envisaged. For this receptor, the nonionic detergent dodecyl maltoside was found better than octyl glucoside, and the solubilization of the receptor could be accomplished in its active form, as determined by its ability to bind the photoactivable ligand (discussed below). Dodecyl maltoside is compatible with subsequent MALDI-TOF mass spectrometric analysis of the purified protein. The immobilized-metal affinity chromatography method was used for purification. Both Ni2+ and Co2+ magnetic beads were tested to bind the (His)6-tag present in the C-terminal end of the NK-1 receptor. A better purification was accomplished with cobalt, in terms of both quantity of receptor and reduced nonspecific binding (data not shown). These magnetic beads are extremely practical since they can be easily washed to remove unspecifically bound proteins using a magnetic particle concentrator that separates the beads from the solution; the beads can be directly spotted on the MALDI plate for mass spectrometry analysis. We have tested many protocols for the removal of nonspecific binding and to elute the receptor from the beads, such as an imidazole or pH gradient. The use of a pH gradient was not successful at all, likely because low pH is known to lead to precipitation of membrane proteins. Generally, the method that led to good protein purity with minimal protein loss was to extensively wash the beads after protein binding (see Experimental Methods) without eluting the protein from the beads, which were subsequentially analyzed by mass spectrometry. We found that efficiency of the purification may depend on several factors arising from the resin itself such as such as the binding capacity of the 2192 Analytical Chemistry, Vol. 79, No. 6, March 15, 2007

resin, the spacing between the functional groups, and the size of the beads, from the type of cells used and possible contaminant proteins, hence from the receptor properties such as the accessibility of the His-tag and its location (N- or C-terminal or internal), or from other parameters we could not control. SDS-PAGE analysis revealed a complex protein mixture in crude detergent extracts, but a major species for the purified sample migrating as a diffuse band with an apparent molecular weight (Mr) of ∼55 000-66 000 (Figure 1, lanes 2 and 3). An additional band with a lower Mr of ∼45 000 was also often observed. The theoretical molecular mass of the FLAG M2 and His-tagged NK-1 receptor is 48,228. The broad and diffuse appearance of the protein bands on the gel was observed before for membrane proteins22 and was attributed to glycosylation heterogeneity. The identity of the protein as the NK-1 was revealed by Western blot directed against the His-tag at the C-terminal end (Figure 1, lanes 4 and 5) and the FLAG M2 (Figure 1, lane 6) at the N-terminal end. In these Western blot experiments, the protein band with a Mr of ∼55 000-66 000 was detected together with a protein with lower Mr of ∼45 000-50 000. In order to get insight into the receptor glycosylation pattern, the purified protein was subjected to N-glycosidase F treatment as well as treatment with a mixture of enzymes able to remove both N- and O-linked carbohydrates. Independently of the enzymes used, this resulted in a shift of the protein migration to ∼50 000, which is close to the theoretical mass of the receptor (Figure 1, lanes 7-10). The precise mass of the different receptor species observed was obtained from detailed mass spectrometry analysis of the protein (discussed further below). Photoaffinity Labeling of the NK-1 Receptor. The pharmacological properties of the photoactive analogue Bapa-[([2,33H]CH CH CO)Lys,3 (p-Bz)Phe,8 Pro9]SP were previously deter3 2 mined,17 demonstrating that this analogue is a potent and full agonist with nanomolar affinities and EC50 values. The photolabeled membranes (with or without N-glycosidase F treatment) were subjected to SDS-PAGE. Subsequently, the gels were cut in 2-mm slices and the radioactivity was counted.17 A broad radioac(22) Grisshammer, R.; Tate, C. G. Q. Rev. Biophys. 1995, 28, 315-422.

Figure 2. Photoaffinity labeling of the NK-1 receptor. (A) Membranes of CHO cells expressing the human NK-1 receptor were photolabeled with 1 nM Bapa-[([2,3-3H]CH3CH2CO)Lys,3(p-Bz)Phe8-Pro9]SP prior (solid line, closed circles) and after treatment with N-glycosidase F (doted line, open squares). Gels were not stained but were cut every 2 mm, and radioactivity was counted in each slice; curves show the distribution of radioactivity. (B) The detergent-solubilized NK-1 receptor was photolabeled and purified with cobalt beads (lane 2) and treated with N-glycosidase F (lane 3). Lane 1 shows the molecular weight markers. Radioactivity of each band was counted and is shown in dpm units.

Figure 3. Lectin affinity chromatography purification of the detergentsolubilized NK-1 receptor. Western blot analysis (target against the His-tag) of the protein purified using Con A (lane 1) or WGA (lane 2). The protein with a Mr of ∼60 000 binds to Con A but not to WGA, showing that this protein contains high (oligo)mannosidic sugars and no complex N-glycans.

tive band (Mr of ∼60 000) was observed for the sample that was not treated with N-glycosidase F (Figure 2A, solid line with closed circles). Treatment of the membranes with N-glycosidase F produces a shift in the radioactive band to a lower mass, with a Mr of ∼45 000 (Figure 2A, dotted line with empty squares). Since visualization of the protein bands of the photolabeled membranes was not optimal (many protein bands are observed and they are diffuse; data not shown), photolabeling experiments were also performed on the detergent-solubilized receptor, which was then purified with cobalt beads. As determined by radioactivity counting of the sliced bands, the receptor bands correspond to a Mr of ∼60 000 (Figure 2B, lane 2), which upon N-glycosidase F treatment shifts to ∼45 000-50 000 (Figure 2B, lane 3). This indicates that the NK-1 receptor is extensively glycosylated with mostly N-linked oligosacharides making ∼10 000. It should be noted that the peaks of radioactivity (Figure 2A) have shoulders for both the glycosylated and nonglycosylated samples (the experiment has been repeated more than five times and always gave the same pattern), suggesting that the receptor is heterogeneous. Lectin Affinity Chromatography. Lectin affinity chromatography is used to identify the oligosaccharides added on proteins, based on the selective interaction of different lectins with glycosylated proteins.23 Con A binds with a very high affinity to N-glycosylated proteins with high (oligo)mannosidic carbohy(23) Cummings, R. D.; Kornfeld, S. J. Biol. Chem. 1982, 257, 11235-11240.

drates, but has low affinity for other glycoproteins. In contrast, WGA lectin selectively binds with a very high affinity to proteins containing complex N-glycans.23 The Western blot analysis of the NK-1 receptor purified with either the ConA or the WGA columns (no metal chelating chromatography was performed here) is presented in Figure 3. The NK-1 receptor binds to ConA resin, but does not interact with WGA resin, although several protein bands were visualized in the SDS-PAGE for both lectins, which may result from contaminating proteins (data not shown). From these data one can conclude that the receptor mainly contains high (oligo)mannosidic sugars and no complex N-glycans. Mass Spectrometry Analysis of the Intact NK-1 Receptor. In order to get precise mass information of the intact NK-1 receptor, the solubilized and purified receptor bound to the Co2+ or Ni2+ beads was directly spotted on the MALDI plate for MS analysis (see Experimental Methods). These experiments were performed on 40-50 samples prepared in different conditions. A major feature of all these studies is the large variability of the detected species in terms of molecular mass. Analysis of eventual contaminant proteins that bind to the metal chelating beads, obtained from CHO cells expressing no receptor, and photolabeling experiments were very useful to distinguish the receptor ion species from those resulting from nonspecific binding to the magnetic beads used for the purification. A typical mass spectrum is shown in Figure 4A for the glycosylated receptor, which shows the detection of different species. The most abundant species (G, glycosylated) has a molecular mass in the range 56 000-58 000 u and was detected as single, double, and triple charged ions. Those can be attributed to the glycosylated receptor as confirmed by photolabeling and Western blot analysis. The lower abundant single charged ions (D, deglycosylated) are at m/z of 48 468, which is very close to that of the mass predicted from the cDNA sequence without post-translational modification24 (see table inset in Figure 4A). The existence of the “nude” receptor as well as post-translationally modified forms, as observed by MS analysis, is also supported by the Western blot experiments (Figure 1, lanes 5 and 6). Interestingly, one species is detected in the m/z range (24) Ovchinnikov, Yu. A.; Abdulaev, N. G.; Bogachuk, A. S. FEBS Lett. 1998, 230, 1-5.

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Figure 4. MALDI-TOF mass spectra of the cobalt-purified glycosylated NK-1 receptor (100 pmol of starting material) (A); of the nickel-purified NK-1 receptor from cells subjected to tunicamycin treatment and purified with nickel (500 pmol of starting material) (B); and of the nickel-purified NK-1 receptor treated with N-glycosidase F (200 pmol of starting material) (C). The Co2+/Ni2+ magnetic beads containing the purified receptor were analyzed with a “sandwich” method using a saturated solution of R-cyano-4-hydroxycinnamic acid in 50% acetonitrile/0.1% trifluoroacetic acid as the matrix (see Experimental Methods). The observed ions are presented in the inset table.

112 300-114 000, which could correspond to the dimer of the most abundant ion (2G). Taking into account the expected low concentration of the receptor in the MALDI plate, the observation of the dimer can be interpreted as an effect of receptor aggregation during the different purification steps, which is highly probable considering the strong hydrophobic nature of this protein. However, this “dimer” ion species was not always observed, either in the SDS-PAGE or in the MS spectra. Other ions with low m/z (5 000-20 000) were also observed, suggesting a partial degradation of the receptor. Typical MALDI mass spectra resulting from purification of the receptor from cells treated with tunicamycin to inhibit glycosylation and that of the deglycosylated receptor using N-glycosidase F are shown (Figure 4, panels B and C, 2194 Analytical Chemistry, Vol. 79, No. 6, March 15, 2007

respectively). With the N-glycosidase F-treated sample (Figure 4B), the most abundant ions were at m/z 48 856 and 47 571. These ions were attributed to the intact deglycosylated receptor with a palmitoylation (theoretical mass of 48 466 u) and to the intact deglycosylated receptor lacking the N-terminal FLAG M2 and the adjacent methionine (as confirmed by Edman sequencing, see following section), respectively. Indeed, the N-terminal FLAG peptide extended with the following methionine has a mass of 1303.4 u, and the difference observed between the two protein species corresponds to 1284.8 u plus 18 (one water molecule), i.e., 1302.8 u (see information below the columns of Table 1). The two proteins have a mass difference with the theoretical truncated and full-length proteins (46 943 and 48 228, respectively) of 628 u

Table 1. Identification by MALDI/TOF-MS of the Peptides Derived from Trypsin Digestion of the Human Neurokinin 1 Receptor Containing the His-tag and FLAG M2 Expressed in CHO Cellsa,c measured mass (amu)b

theoretical mass (amu)

606.24 2752.52 1136.66 1039.54 1843.97 1692.76 1080.42 1560.72 876.47 729.35 941.37 1197.68 996.44 2281.28 1337.69 2279.28 1466.77 1616.66 1008.45 2734.28 1148.54 1059.59 1120.66 2399.18 1570.85 848.45 772.44 2362.31 2490.40 1366.73 1102.66 1165.72 1780.02 2260.18

606.21 2752.57 1136.70 1039.52 1843.98 1692.79 1080.47 1560.70 876.42 729.35 941.42 1197.64 996.47 2281.24 1337.73 2279.28 1466.81 1616.71 1008.46 2734.26 1148.56 1059.58 1120.65 2399.16 1570.89 848.43 772.43 2362.31 2490.41 1366.72 1102.64 1165.69 1780.01 2260.15

2295.16 722.31 1107.54 1074.37 2261.98 1212.62 1401.67 2237.11 1537.70 948.40

2295.11 722.28 1107.51 1074.42 2262.02 1212.58 1401.69 2237.07 1537.70 948.39

chymotrypsin, activity

CNBr cleavage

missed cleavages

δm (ppm)

location, position

no yes yes yes yes yes yes yes yes yes yes yes no yes no yes yes yes yes yes no yes yes yes no yes yes yes yes yes yes yes yes no

no no yes no no no no no no no no no no no no no no no no no yes no no no no no no no no no no no no yes

49 18 35 19 5 18 46 13 57 0 53 33 30 17 30 0 27 30 10 7 17 9 9 8 25 23 12 0 4 7 18 25 5 13

D[5-9]K, N-term (FLAG M2) A[45-70]K, TM1 I[65-73]R, ICL1 R[71-78]Y, ICL1 R[71-85]F, ICL1 + TM2 N[94-107]W, ICL1 A[102-109]Y, ECL1 A[102-113]Y, ECL1 G[110-116]F, ECL1 G[110-115]K, ECL1 C[114-120]F, ECL1 F[120-130]Y, TM3 S[131-139]R, TM3 + ICL2 D[138-157]K, ICL2 Y[140-150]R, ICL2 V[158-177]Y, TM4 V[165-177]Y, TM4 P[173-186]R, TM4 +ECL2 S[178-186]R, ECL2 Y[177-199]K, ECL2 I[191-199]K, ECL2 H[206-214]Y, TM5 L[216-225]Y, TM5 A[224-245]Y, ICL3 Y[245-257]K, ICL3 A[267-273]F, TM6 L[271-276]F, TM6 L[271-289]K, TM6 + ECL3 L[271-290]K, TM6 + ECL3 F[277-287]Y, ECL3 I[282-290]K, ECL3 L[288-296]Y, ECL3 L[288-301]W, ECL3 K[290-308]M, ECL3 + TM7

yes yes yes yes yes yes yes no no yes

no no no no no no no no no no

0 0 2 2 2 0 0 0 1 0 1 0 0 2 0 0 0 0 0 1 0 0 0 0 3 0 0 0 1 0 1 2 2 1 (tryp) + 2 (CNBr) 0 0 2 0 1 2 1 0 0 0

21 41 27 46 17 32 14 17 0 10

F[291-309]Y, ECL3 + TM7 C[315-320]R, ICL4 K[326-334]F, C-term C[331-340]Y, C-term R[330-349]R, C-term E[341-350]Y, C-term S[347-358]Y, C-term L[363-383]K, C-term A[384-398]R, C-term T[403-410]F, C-term

a This table represents the combined results of five independent experiments. CNBr cleavage was performed in a few experiments after trypsin enzymatic activity. b amu corresponds to arbitrary mass units. c Primary sequence of the FLAG M2 and His-tagged human tachykinin NK-1 receptor. Tags: Flag M2 at the N-terminus and His at the C-terminus are shown in boldface italics; potential extracellular N-glycosylation sites are in boldface; transmembrane domains are underlined and potential palmitoylation site in boldface underline. Above the sequence, the masses of the full length (48 228) and truncated D[11 422] receptor are indicated.

that may include one palmitoylation (+238 u) and additional posttranslational modifications. In tunicamycin-treated cells, only the N-terminal truncated receptor species was observed together with fragments that may correspond to protein degradation, as seen also in photolabeling experiments followed by SDS-PAGE (data

not shown). The protein in the range m/z (53 750-55 650) as single charged ion likely corresponds to the O-glycosylated form of the receptor since this sample was only treated with Nglycosidase F. It should be mentioned that a rather large variability was obtained in terms of molecular masses of the glycosylated Analytical Chemistry, Vol. 79, No. 6, March 15, 2007

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proteins, even for experiments performed under identical conditions. This variability extent (typically ∼2000 u) is much larger than the uncertainty of m/z measurements (typically (100 u at m/z 40 000). Such results could arise from the heterogeneity in receptor post-translational modifications and some plausible receptor degradation since very often proteins with molecular masses around 35 000 and 23 000 u were also detected. However, for the deglycosylated samples, a rather high reproducibility in terms of the measured masses was observed. For example, four independent experiments lead to an average mass of 47 488 ( 56 u for the N-truncated receptor species D[11-422]H. Mass Spectrometry Analysis of the Trypsin and CNBr Digested NK-1 Receptor. The purified NK-1 receptor was analyzed by SDS-PAGE stained with coomassie blue. The band at 55 000-60 000 was excised and subjected to in-gel digestion with trypsin followed or not by CNBr cleavage. MALDI-TOF analysis of this digested sample resulted in peptide maps with a high variability as evidenced by five independent experiments with identical protocols. Among the observed ions, most of the less abundant ones could be attributed to peptides coming from the NK-1 sequence (peptide mass tolerance 50 ppm, due to the high mass of these fragments. Edman Sequencing. Automated Edman sequencing on the entire deglycosylated sample produced sequence data, which identify the N-terminus of the NK-1 receptor. The sequences were MDXXXXDXXM and DNVLPXXXXX that correspond to the fulllength NK-1 receptor and the NK-1 receptor lacking the FLAG M2 tag and the adjacent methionine, respectively. Another sequence was also inferred from the Edman sequencing, which produced a high signal, YKIKP/KF that did not correspond to the NK-1 receptor but could be attributed to a contaminant protein, e.g., keratin. From this Edman analysis, it can be deduced that the signal intensity specific to the NK-1 sequence was 1 pmol of aspartic acid in the first cycle and 150 fmol of proline in the fifth one. The existence of the full-length and the FLAG M2 truncated receptors is consistent with the data analysis obtained from MS and the primary amino acid sequence of the tagged NK-1 receptor (see below the columns of Table 1). CONCLUSIONS The identification of proteins isoforms and characterization of post-translational modifications are crucial steps to understand the pharmacology of membrane proteins. Due to a series of processes 2196

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ranging from post-translational modifications to alternative splicing and protein-regulated enzymatic cleavage, the mass of a protein is often far from that predicted from the primary amino acid sequence. Additionally, the accuracy of molecular weight determination of intact proteins by SDS-PAGE is low, with errors between 1 and 5% in the best cases but often much greater depending on protein structure.25 In this regard, MALDI-TOF mass spectrometry is a particularly attractive method, providing measurements with high sensitivity and a wide mass range and allowing accurate molecular weight determination of intact proteins and peptides in mixtures with a relatively high tolerance to most buffer components.26,27 However, the isolation of intact proteins with high purity, low losses of material, and under conditions compatible with MS analysis is rather challenging. Several strategies have been proposed to remove proteins from a gel for analysis, such as electroelution,28 chemical extraction,29 or passive diffusion.30 However, these methods are usually timeconsuming and lead to low protein recovery, especially when working with small (low-picomole) amounts of proteins, which is often the case with G-protein coupled receptors (GPCRs). In addition, SDS, which is usually extracted from the gel along with the protein, interferes with subsequent MS analysis, although its removal with organic solvents has been demonstrated but only for small proteins and working at high-picomole levels.31 Here, we have presented a novel and simple method for the characterization by MALDI-TOF MS of a GPCR (the tachykinin NK-1 receptor) purified on Co2+ magnetic beads. MALDI was preferred to ESI because we initially only analyzed beads (surely containing the NK-1 receptor) that were directly spotted on the MALDI plate. Indeed, releasing the protein from the beads with the acid matrix might have led to protein aggregation and loss of MS signal. The protocol used for receptor solubilization is highly compatible with its biological activity as the receptor is still able to bind photoactivatable ligand after being isolated from the cell membrane by detergent extraction. The analysis by MS of the intact receptor reveals a certain level of heterogeneity, which may result from various plausible post-translational modifications (palmitoylation, glycosylation, phosphorylation, backbone cleavage). Another possible source of recombinant protein heterogeneity arises from the existence of two initiation sites, Met1 and Met10, that are adjacent to the N-terminus FLAG M2 coming from the plasmid construct. The use of distinct initiation sites has already been observed in the rat neurotensin receptor as a consequence of two initiation sites of translation present in the mRNA.31 From the present data, at least three distinct proteins with masses ranging from about 56 000 to 58 000 can be identified. Such receptor heterogeneity was observed in most analyzed samples and may result from different stages of co- or posttranslational modification of the receptor as it is constantly being synthesized. However, a certain degree of reproducibility in (25) Creighton, T. E. in Proteins: Structures and Molecular Principles, eds Freeman WH (New York), 1984, pp 33-34. (26) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299-2301. (27) Beavis, R. C.; Chait, B. T. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 68736877. (28) Haebel, S.; Jensen, C.; Andersen, S. O.; Roepstorff, P. Protein Sci. 1995, 4, 394-404. (29) Claverol, S.; Burlet-Schiltz, O.; Gairin, J. E.; Monsarrat, B. Mol. Cell. Proteomics 2003, 2, 483-493. (30) Cohen, S. L.; Chait, B. T. Anal. Biochem. 1997, 247, 257-267. (31) Botto, J. M.; Vincent, J. P.; Mazella, J. Biochem. J. 1997, 324, 389-393.

MALDI-TOF MS experiments was observed for the deglycosylated species. The use of lectin affinity chromatography permitted the determination of the nature of the glycosylation. The selective interaction of the receptor with Con A but not with WGA demonstrates that the receptor contains high (oligo)mannosidic sugars and no complex N-glycans. Similar results have been obtained with the β2 adrenergic receptor;32 in contrast, the rat M3 receptor expressed in the same cell line was able to bind both on WGA and ConA resins, demonstrating microheterogeneity in glycosylation.33 N-Glycosylation is not the only source of receptor heterogeneity in this study since treatment of the receptor with N-glycosidase F still resulted in the appearance of more than one protein. The presence of O-glycosylation has been observed in other GPCRs such as V2 vasopressin34 and the human δ-opioid receptor.35 It has also been shown that differential N-linked glycosylation may function to maintain membrane receptors in different agonist binding states in vivo.36-37 The existence of several receptor species may also arise from differential palmitoylation yielding several forms of the receptor that may activate different intracellular pathways. Such behavior has been observed with the mutated endothelin receptor A,38 where the nonpalmitoylated receptor activates cAMP production but is unable to stimulate PLC activation while the wild type is able to activate both pathways. Whatever the reasons, it is tempting to conclude that this heterogeneity in NK-1 receptor population may correlate with the unusual pharmacological behavior of this receptor, namely, the existence of two binding sites in this protein.16,39-41 Studies from our16,18,40 and another laboratory39 have shown that these binding sites would be independent and non-interconvertible, suggesting that they could result from the existence of different receptor isoforms. In our studies, we have tried to enhance the production of one of the putative receptor isoforms by treating the cells expressing the NK-1 receptor with a ligand that binds with greater affinity to one of the binding sites. In the photolabeling experiments (data not shown), one of the receptor species seemed to vanish, although more than one species was still observed. Photolabeling experiments demonstrate that the protein with a Mr of ∼55 000 purified with the cobalt beads and observed by both SDS-PAGE and mass spectrometry corresponds to the NK-1 receptor. After treatment of the receptor with N-glycosidase F, the deglycosylated receptor migrates as a discrete band with a Mr of ∼50 000, which correlates well with the mass calculated (32) Reilander, H.; Boege, F.; Vasudevan, S.; Maul, G.; Hekman, M.; Dees, C.; Hampe, W.; Helmreich, E. J.; Michel, H. FEBS Lett. 1991, 282, 441-444. (33) Vasudevanm, S.; Hulme, E. C.; Bach, M.; Haase, W.; Pavia, J.; Reilander, H. Eur. J. Biochem. 1995, 227, 466-475. (34) Sadeghi, H. M.; Birnbaumer, M. Glycobiology 1999, 9, 731-737. (35) Petaja-Repo, U. E.; Hoque, M.; Laperriere, A.; Walker, P.; Bouvier, M. J. Biol. Chem. 2000, 275, 13727-13736. (36) Rens-Domiano, S., Reisine, T. J. Biol. Chem. 1991, 266, 20094-20102. (37) Benya, R. V.; Kusui, T.; Katsuno, T.; Tsuda, T.; Mantey, S. A.; Battey, J. F.; Jensen, R. T. Mol. Pharmacol. 2000, 58, 1490-1501. (38) Horstmeyer, A.; Cramer, H.; Sauer, T.; Muller-Esterl, W.; Schroeder, C. J. Biol. Chem. 1996, 271, 20811-20819. (39) Hastrup, H.; Schwartz, T W. FEBS Lett. 1996, 399, 264-266. (40) Sagan, S.; Karoyan, P.; Chassaing, G.; Lavielle, S. J. Biol. Chem. 1999, 274, 23770-23776. (41) Kim, H. R.; Lavielle, S.; Sagan, S. Biochem. Biophys. Res. Com. 2003, 306, 725-729. (42) Trester-Zedlitz, M.; Burlingame, A.; Kobilka, B.; Zastrow, M. Biochem. 2005, 44, 6133-6143.

from the primary sequence (NK-1 receptor with FLAG M2 and His-tag has a mass of ∼48 000). Additionally, the capability of the detergent-solubilized receptor to specifically bind the photoactivatable ligand demonstrates that the receptor retains its binding properties after the solubilization procedure. We have already observed this binding capacity in a previous study, where the binding between the detergent-solubilized and purified receptor and its ligands was monitored by plasmon waveguide resonance spectroscopy.16 Very few studies have been presented on mass spectrometry analysis of intact membrane proteins and even less with GPCRs with rhodopsin and bacteriorhodopsin constituting unique examples of those studies12-14 due to the facility in obtaining those proteins in high quantities and good purity. The His-tagged thromboxane A2 receptor constitutes another example of a GPCR for which the intact mass has been determined by MALDI.15 However, this study was restricted to the glycosylated receptor, which was transferred from the SDS gel to a nitrocellulose membrane, subsequently dissolved in acetone, TFA, and sinapinic acid for analysis. In this study, we show that the “one-pot” purification of proteins with magnetic beads and their direct MS analysis is advantageous when studying low-level expressed proteins such as membrane proteins. Starting from 100 to 500 pmol, we assess that 1 pmol or less of receptor was finally analyzed by mass spectrometry. This quantity was estimated from comparison of the receptor MS signal intensity with those of proteins with similar molecular weight but soluble in water. Thus, large hydrophobic intrinsic membrane proteins are completely amenable to mass spectrometric analysis. But, even though 1 pmol of this purified GPCR can be obtained, MS/MS analysis could not be achieved. The absence of MS/MS data was also the case in previous studies on the µ-opioid,5-6 the CB1 and CB2 cannabinoid,8,9 and the β2-adrenergic receptor,42 while for the δ-opioid7 receptor, the MS/MS mass spectra of one peptide from the C-terminus was reported. This latter point underlines the urgent need to improve membrane proteomics (in-gel digestion, shot-gun methods, peptide recovery from gels) that will allow further MS/MS analysis. However, for a better understanding of the structure/function relationships and signaling mechanisms of membrane proteins, MS should play a crucial role in proteomics to study protein isoforms through the analysis of intact proteins and not only via their identification and characterization from peptide fragments. ACKNOWLEDGMENT Dr. T. W. Schwartz is acknowledged for the kind gift of the pTEJ8 vector containing the FLAG M2 tagged human NK-1 receptor and Dr. B. Mouillac for adding the His-tag at the C-terminus and subcloning of the receptor in pRK5 vectors. The CNRS is acknowledged for funding meetings of the research group: GDR 2611 “Mass spectrometry and membrane proteins”. I.D.A. was the recipient of a postdoctoral fellowship from the city of Paris and from EMBO. SUPPORTING INFORMATION AVAILABLE A figure showing the sequence coverage of the FLAG-M2 and His-tagged NK-1 receptor obtained from the enzymatic (trypsin) Analytical Chemistry, Vol. 79, No. 6, March 15, 2007

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and CNBr cleavage followed by MALDI-TOF mass spectrometry analysis. This material is available free of charge via the Internet

Received for review December 21, 2006. Accepted January 30, 2007.

at http://pubs.acs.org.

AC062415U

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