Anal. Chem. 2001, 73, 4774-4779
Mass Spectrometric Analysis of Integral Membrane Proteins at the Subnanomolar Level: Application to Recombinant Photopigments Zsolt Ablonczy,† Masahiro Kono,† Rosalie K. Crouch,† and Daniel R. Knapp*,‡
Departments of Ophthalmology and Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, 171 Ashley Avenue, Charleston, South Carolina, 29425
Integral membrane proteins produced by eukaryotic expression systems are a subject of much current interest in biomedical investigation. Due to the low efficiency of their expression and the limited quantity of the expressed to the total amount of the membrane proteins, they have evaded mass spectrometric analysis. The methodology previously developed for mass spectrometric analysis of integral membrane proteins required proteins that were obtained relatively pure from their native membranes. The previously developed methodology has been modified and applied to the analysis of subnanomolar samples of rhodopsin. Bovine rhodopsin purified by affinity chromatography, from native membranes and from a eukaryotic expression system, was successfully analyzed, obtaining complete sequence coverage for the detection and localization of posttranslational modifications. The methodology presented here will enable mass spectrometric analysis of subnanomolar levels of photopigments or other integral membrane proteins either from their native membranes or as products of expression systems.
Mass spectrometry (MS) has, over the last several years, become the method of choice for the analysis of the primary structure of proteins.1-5 The general approach for the analysis incorporates enzymatic or chemical fragmentation of the intact proteins and fractionation of the resulting digest mixture by chromatography prior to MS. These steps are relatively easy to perform for soluble proteins, but due to their amphiphilicity, integral membrane proteins are prone to irreversibly aggregate, adhere to the sample handling surfaces, and bind to the chromatographic column during this process. For purposes of protein * Corresponding author: (telephone) (843) 7925830; (fax) (843) 7922475; (email)
[email protected]. † Department of Ophthalmology. ‡ Department of Cell and Molecular Pharmacology and Experimental Therapeutics. (1) McCloskey, J. A. In Methods in Enzymology; Academic Press: San Diego, 1990; Vol. 193. (2) Shively, J. E. Methods 1994, 6, 207-212. (3) Karger, B. L.; Hancock, W. S. In Methods in Enzymology; Academic Press: San Diego, 1996; Vol. 270. (4) Burlingame, A. L.; Boyd, R. K.; Gaskell, S. J. Anal. Chem. 1998, 70, 647A716A. (5) Griffiths, W. J.; Jonsson, A. P.; Liu, S.; Rai, D. K.; Wang, Y. Biochem. J. 2001, 355, 545-561.
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identification, it is usually possible to observe at least one fragment from a soluble region of a membrane protein,6,7 but if the objective is to fully characterize the primary structure (including posttranslational and chemical modifications), the task is much more difficult. We have previously reported methodology for this type of analysis8 and present here improved methodology for MS analysis of integral membrane proteins where the objective is to observe fragments from the entire protein sequence. Integral membrane proteins are vital constituents of cells, since they are responsible for the material and signal transport of the cells. One of their groups, the large G protein-coupled receptor superfamily,9,10 is particularly interesting because they enable the primary reactions by which the cells gather information from their environment. Rhodopsin, the photopigment of the retinal rod cells, belongs to this superfamily and is in fact its best-characterized member.11 Due to its unique characteristics of reasonable stability and availability, it serves as a model for the G protein-coupled receptor superfamily. Since it constitutes 90% of the membrane protein content of the retinal outer segments, it can easily be extracted with high yields in the native membrane form,12-14 and its physical and biological properties can be examined by reliable assays and spectroscopy.15,16 Moreover, it has already been successfully expressed in recombinant form in several eukaryotic cell lines retaining its main characteristics.17-20 This paved the (6) Papac, D. I.; Oatis, J. E., Jr.; Crouch, R. K.; Knapp, D. R. Biochemistry 1993, 32, 5930-5934. (7) Roos, M.; Soskic, V.; Poznanovic, S.; Godovac-Zimmermann, J. J. Biol. Chem. 1998, 273, 924-931. (8) 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. Protein Sci. 1998, 7, 758-764. (9) Marchese, A.; George, S. R.; Kolakowski, L. F., Jr.; Lynch, K. R.; O’Dowd, B. F. Trends Pharmacol. Sci. 1999, 20, 370-375. (10) Gershengorn, M. C.; Osman, R. Endocrinology 2001, 142, 2-10. (11) Okada, T.; Ernst, O. P.; Palczewski, K.; Hofmann, K. P. Trends Biochem. Sci. 2001, 26, 318-324. (12) Applebury, M. L.; Zuckerman, D. M.; Lamola, A. A.; Jovin, T. M. Biochemistry 1974, 13, 3448-3458. (13) Smith, H. G., Jr.; Stubbs, G. W.; Litman, B. J. Exp. Eye Res. 1975, 20, 211217. (14) McDowell, J. H.; Kuhn, H. Biochemistry 1977, 16, 4054-4060. (15) Oprian, D. D. Curr. Opin. Neurobiol. 1992, 2, 428-432. (16) Pepe, I. M. J. Photochem. Photobiol. B 1999, 48, 1-10. (17) Oprian, D. D.; Molday, R. S.; Kaufman, R. J.; Khorana, H. G. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 8874-8878. (18) Khorana, H. G.; Knox, B. E.; Nasi, E.; Swanson, R.; Thompson, D. A. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 7917-7921. 10.1021/ac015563n CCC: $20.00
© 2001 American Chemical Society Published on Web 09/19/2001
way for the study of site-directed mutagenesis21-23 and the examination of other photopigments whose pure extraction from the retina is difficult due to their low abundance.24,25 The key aspects of the approach previously developed for the mass spectrometric analysis of integral membrane proteins were the reduction and alkylation of cysteine residues prior to removal of the protein from the membrane, dissolution of the protein or fragments in neat trifluoroacetic acid (TFA), cyanogen bromide cleavage, subsequent dilution to achieve the required solvent compositions, and application of cleavage fragments to the reversed-phase HPLC column in a large volume of injection solvent. With this methodology, all of the CNBr fragments of bacteriorhodopsin8 bovine and rat rhodopsin,8,26,27 and bovine, rat, and human MIP28-30 could be detected and sequenced with electrospray ionization mass spectrometry. The greatest difficulty with the application of this method is that the sample can easily be lost during the processing due to the multiple washes performed. These washes are, however, necessary in order to remove the contaminants and the reagents present in the sample during the procedure. Therefore, the necessity of using 5-10 nmol of pure starting material limits the usefulness of the method. Moreover, the less abundant native membrane proteins are routinely purified and concentrated using affinity chromatography, and the necessary use of detergents for their solubilization is incompatible with the previously developed methodology. MALDI MS analysis of picomolar amounts of rhodopsin solubilized in detergent was recently reported.31 Although this methodology is fast and efficient, it used approximately the same amount and quality of starting material as our previously reported method (2 nmol of rhodopsin in the membrane form) and the MALDI approach reported gave mainly molecular weight information regarding the fragments. The objective of this work was to modify the methodology of our previously reported ESI-MS analysis of integral membrane proteins so that subnanomolar amounts of rhodopsin obtained by affinity chromatography either from native membrane preparations or as expressed recombinant pigment can both be processed and analyzed. An additional aim was to simplify the procedures and reduce the time and effort necessary for the sample preparation. Although native and recombinant bovine rhodopsin was studied as a model protein, the results should be applicable to other (19) Kaushal, S.; Ridge, K. D.; Khorana, H. G. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 4024-4028. (20) Mollaaghababa, R.; Davidson, F. F.; Kaiser, C.; Khorana, H. G. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 11482-11486. (21) Karnik, S. S.; Sakmar, T. P.; Chen, H. B.; Khorana, H. G. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 8459-8463. (22) Govardhan, C. P.; Oprian, D. D. J. Biol. Chem. 1994, 269, 6524-6527. (23) Kono, M.; Yu, H.; Oprian, D. D. Biochemistry 1998, 37, 1302-1305. (24) Oprian, D. D.; Asenjo, A. B.; Lee, N.; Pelletier, S. L. Biochemistry 1991, 30, 11367-11372. (25) Fasick, J. I.; Lee, N.; Oprian, D. D. Biochemistry 1999, 38, 11593-11596. (26) Ablonczy, Z.; Knapp, D. R.; Darrow, R.; Organisciak, D. T.; Crouch, R. K. Mol. Vision 2000, 6, 109-115. (27) Gelasco, A.; Crouch, R. K.; Knapp, D. R. Biochemistry 2000, 39, 49074914. (28) Schey, K. L.; Fowler, J. G.; Schwartz, J. C.; Busman, M.; Dillon, J.; Crouch, R. K. Invest. Ophthalmol. Visual Sci. 1997, 38, 2508-2515. (29) Schey, K. L.; Fowler, J. G.; Shearer, T. R.; David, L. Invest. Ophthalmol. Visual Sci. 1999, 40, 657-667. (30) Schey, K. L.; Little, M.; Fowler, J. G.; Crouch, R. K. Invest. Ophthalmol. Visual Sci. 2000, 41, 175-182. (31) Kraft, P.; Mills, J.; Dratz, E. Anal. Biochem. 2001, 292, 76-86.
photopigments or integral membrane proteins. This modified methodology removes a major obstacle to the study of lowabundance and recombinant membrane proteins by enabling the MS analysis of primary structure, including posttranslational and chemical modifications on subnanomolar samples. EXPERIMENTAL SECTION Expression, Generation, and Purification of Bovine Rhodopsin. Bovine rhodopsin was expressed and purified from COS cells essentially as described previously.15,17 Briefly, confluent COS cells in 10, 10-cm tissue culture plates were transiently transfected with a synthetic rhodopsin gene in a pMT3 expression plasmid using the DEAE-dextran method.17,32,33 Cells were harvested on the third day after transfection and incubated in the dark with 5 µmol/L 11-cis-retinal (MUSC, Charleston, SC, and NEI-NIH, Bethesda, MD) in 10 mL of 10 mmol/L sodium phosphate/150 mmol/L NaCl/pH 7.0 phosphate-buffered saline (PBS) at 4 °C. Bovine rhodopsin was also obtained as a rod outer segment (ROS) preparation isolated from bovine retinas in the dark.14 The cells and rod outer segment membranes were centrifuged at 3000g, and the pellets were solubilized in 1% CHAPS (Sigma Chemical Co., St. Louis, MO) in PBS and incubated at 4 °C for 1 h. This sample was then centrifuged at 3000g, and the solubilized fraction was incubated with anti-rho 1D4-conjugated Sepharose 4B resin for 2 h at 4 °C. The 1D4 antibody (Cell Culture Center, Minneapolis, MN) recognizes the last eight amino acids of bovine rhodopsin.34 It was coupled to Sepharose 4B by the method of Cuatrecasas.35 The 1D4-Sepharose was then transferred to a 1-mL syringe with a glass wool plug and washed with 0.1% dodecyl maltoside (DM, Calbiochem-Novabiochem, La Jolla, CA) in PBS. Rhodopsin was eluted from the column with a peptide corresponding to the carboxyl terminal 18-amino acid residues of bovine rhodopsin (DEASTTVSKTETSQVAPA, synthesized by the MUSC Biotechnology Resource Facility) at a concentration of 88 µM in the wash buffer. Care was taken to keep the rhodopsin in the dark. For control purposes, rhodopsin from rod outer segments was also examined without affinity purification. In this case, the membranes were centrifuged at 100000g and the pellets solubilized in 0.1% DM in water and incubated at room temperature for 15 min. This sample was then centrifuged and the solubilized fraction used for the experiments. The rhodopsin concentration of the samples was determined by spectrophotometric measurements at 495 nm. Generation of Peptides for Sequence Analysis. The purified rhodopsin samples (10-50 µg or 0.25-1.25 nmol, depending on the experiment, in 100 µL of 0.1% DM) were reduced with the addition of 5 µL of tributylphosphine (Aldrich Chemical Co., Milwaukee, WI; more than 1000 molar excess over the cysteine content) and alkylated with 5 µL of 4-vinylpyridine (Sigma; more than 2400 molar excess over the cysteine content). The reaction was performed at 4 °C for 1 h under argon with intermittent (32) Ferretti, L.; Karnik, S. S.; Khorana, H. G.; Nassal, M.; Oprian, D. D. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 599-603. (33) Franke, R. R.; Sakmar, T. P.; Oprian, D. D.; Khorana, H. G. J. Biol. Chem. 1988, 263, 2119-2122. (34) Molday, R. S.; MacKenzie, D. Biochemistry 1983, 22, 653-660. (35) Cuatrecasas, P. J. Biol. Chem. 1970, 245, 3059-3065.
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vortexing. The mixture was then precipitated with the addition of 1 mL of acetone at -20 °C for 15 min. The precipitant was centrifuged at 100000g for 5 min at 4 °C and the supernatant removed. The pellet was washed two more times with the repeated addition of 1 mL of acetone (Sigma), centrifugation with 100000g for 5 min at 4 °C, and removal of the supernatant. The remaining CNBr cleavage and HPLC separation were performed as previously reported.8 In short, the pellet was dissolved in 400 µL of TFA (Acros Organics, Fair Lawn, NJ), and 180 µL water was added to adjust the concentration to 70% TFA. The CNBr cleavage was performed by adding 10 µL of 5 mol/L CNBr solution in acetonitrile (Aldrich; more than 500 molar excess over the methionine content). The cleavage was carried out with stirring in the dark under argon overnight at room temperature. The reaction was quenched by the addition of 1 mL of water, and the solvents were evaporated under vacuum. The dried fragment mixture was redissolved in 5 mL of solution equivalent to the initial HPLC gradient mobile phase (2.5% organic; the aqueous phase was 0.05% TFA in water, the organic phase was 0.05% TFA in 2:1 isopropyl alcohol (J. T. Baker, Phillipsburg, NJ)/acetonitrile (Fisher Scientific, Fair Lawn, NJ)). It is important that the dried peptide mixture be dissolved first in neat TFA and the other components added subsequently with the water added last. The sample was loaded onto a 2.1 mm × 100 mm, C4 Aquapore column (Perkin-Elmer Brownlee column, Bodman Industries, Aston, PA), and the peptides were eluted at a flow rate of 400 µL/min with a gradient from 2.5 to 97.5% organic phase. The HPLC fraction containing the glycosylated rhodopsin CNBr fragment was dried under vacuum, dissolved in glycanase incubation buffer (Glyko Inc., Novato, CA), treated for overnight at 37 °C with N-glycanase-PLUS (Glyko Inc., Novato, CA), and analyzed by MALDI-MS as described previously.26 Mass Spectrometry. The column effluent was split, and 10% of the flow was directed into the ESI source of a Finnigan LCQ ion trap mass spectrometer (Thermo-Finnigan Instrument Systems Inc., San Jose, CA). MS and MS/MS data were automatically acquired using Xcalibur software (version 1.2). The remaining 90% of the effluent was collected in 4-min fractions, dried under vacuum, and later analyzed with a Perspective Voyager DE MALDI-TOF instrument (Applied Biosystems, Framingham, MA). Further details have been previously published.8 RESULTS AND DISCUSSION Two MS techniques were applied in this work for the analysis of integral membrane proteins. ESI-MS made it possible to sample the HPLC effluent during the chromatographic separation with a high frequency and determine both the mass composition (MS spectrum) and the collision-induced dissociation (CID) spectrum (MS/MS spectrum). MALDI provided a fast and efficient method for checking intact molecular weight or the progress of the cleavage of the protein or peptide. The practical application of mass spectrometry for analysis of the primary structure of integral membrane proteins requires that proteins present in lower abundance can also be analyzed in their entirety. The applicable protocol has to be sufficiently simple and efficient for generating digest peptides that provide sequence information and a complete map of the protein for the determination of the posttranslational modifications. The previously developed methodology8 has to be modified, because it requires 4776
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that the membrane proteins be purified in their native membranes in high yields. The main difference between the previous and the new, simplified methodology is that previously the proteins were removed from the membranes after reduction/alkylation since it was found that performing the reduction/alkylation procedure after the protein was removed from the membranes and completely denatured causes a serious decrease in the signal quality. This order of steps is not possible if the protein is already in detergent. However, the removal of the proteins from the membranes with detergents does not cause complete denaturation and therefore reduction/alkylation can be successfully performed in detergent as well. Since the detergent and lipids that are present in these samples can interfere with the mass spectrometric analysis, the protein has to be precipitated from the detergent. This was performed with three acetone washes, which also eliminate the need for delipidation and, thus, simplify the procedure. Figure 1 indicates the primary structure of bovine rhodopsin in the form of a two-dimensional model with the previously observed posttranslational modifications and the predicted CNBr cleavage sites. We have completely mapped rhodopsin from three different types of samples: 20 µg (500 pmol) of ROS solubilized in 0.1% DM, 20 µg of rhodopsin from immunopurified ROS in 0.1% DM, and 10 µg (250 pmol) of recombinant rhodopsin immunopurified from COS cells in 0.1% DM. With the new methodology, the mass spectrometric analysis was possible for all three types of the samples, and it showed no difference between the sequences. Recently, it has been demonstrated that 99% of a rhodopsin CNBr digest mixture was detected by MALDI mass spectrometry at the picomolar level.31 However, the initial sample (the amount of rhodopsin before reduction/alkylation) was approximately 3 nmol (125 µg) and comparable to our previous experiments. This amount was reduced in two later steps; first, after reduction/alkylation only 30 pmol (1 µg) was precipitated and cleaved with CNBr (this amount was minimally necessary for the digest for complete mapping), and later, only a thirtieth of the digest mixture was spotted on the MALDI plate. Therefore, 1 pM rhodopsin was detected but the processing for the mass spectrometric analysis started with 3 nmol of protein. With the method presented here, using the new methodology, low initial amounts can be both processed and analyzed. Table 1 shows the predicted and observed CNBr fragments of bovine rhodopsin and summarizes the data shown in Figures 2 and 3. Figure 2 is a base peak chromatogram of bovine rhodopsin expressed in COS cells, affinity purified, and analyzed by mass spectrometry. The base peak chromatogram was obtained by the mass spectrometer by repetitive scanning of the HPLC effluent and indicating at each time point the signal intensity of the most abundant mass. As can be seen, 14 of the total 17 CNBr fragments were observed in a single experiment, providing 90% sequence coverage. Fragments 1 and 15 are single methionines, and their masses were below the lower mass limit of the instrument. Fragment 2 is doubly glycosylated (at asparagines 2 and 15) and is known to evade analysis with the previous methodology8 because of its large size, heterogeneity, unfavorable chromatography, and fragmentation. Due to their unique characteristics, some of the fragments gave more intense peaks than the others. Fragments 4, 5, 6, 7, 8, 9, 10, 11, 14, and 17 gave distinct
Figure 1. Two-dimensional model and amino acid sequence of bovine rhodopsin. The detected posttranslational modifications and the first amino acids of the CNB fragments are numbered. Fragment 15 was not detected; dashed circles indicate fragments 1 and 2 that were detected by MALDI-MS.
peaks while fragments 3, 12, 13, and 16 were in the baseline and only observed as minor species in the mass spectra. In some cases, the CNBr fragments that contain tryptophan (fragments 6, 8, 9, and 13) were observed to contain oxidized tryptophans. This phenomenon has also been observed previously and may be attributed to contaminants in the CNBr.26,31 The large peak at the beginning of the chromatogram shows an 18-mer peptide used to elute rhodopsin from the affinity column. It is not present with samples that were not affinity purified. Other immunoaffinity elution methods that use high or low pH are also available,36 but they are unfavorable for rhodopsin and the other retinal photopigments since they can alter the integrity of the protein. Similar to previous findings, the sample was not free of contaminants that constitute some of the other well-shaped peaks. However, they are well separated and do not interfere with the detection of the CNBr peptides. Figure 3 shows the selected ion chromatograms for the observed CNBr fragments. Two peaks (fragments 8 and 9) appear bimodal presumably due to two major conformations of these fragments in solution; this was observed with all of the sample types. Two of the smallest hydrophilic fragments (fragments 12 and 16) did not form well-separated peaks because they elute very close to the injection solvent peak and are not well resolved by the C4 column.8 Fragment 13, although more hydrophobic, was also not well resolved because it coelutes with several other peaks. (36) Vijayalakshmi, M. A. Appl. Biochem. Biotechnol. 1998, 75, 93-102.
Table 1. Cyanogen Bromide Fragments of Bovine Rhodopsin
fragment 1a,b 2b,d,e 3 4 5 6 7g 8g 9 10h 11 12 13 14 15i 16 17j
residues
expected mass [M + H]1+
1 2-39 40-44 45-49 50-86 87-143 144-155 156-163 164-183 184-207 208-253 254-257 258-288 289-308 309 310-317 318-348
144.1c 6497.8 520.3 588.4 4241.4 6353.3 1374.7 862.5 2159.1 3005.3 5315.8 427.3 3580.8 2198.2 102.1 1097.5 3599.9
observed mass
observed charge state
retention time (min)
6514f 520 588 4241 6370f 1374 878f 2175f 3005 5316 428 3598f 2198
+1 +1 +1 +3 +4 +2 +1 +2 +3 +3 +1 +3 +2
26 7 20 42 49 13 18 21 19 42 2 43 29
1098 3600
+2 +2
1 56
a Not detected with LC/MS/MS. b Observed with MALDI-TOF as incomplete cleavage after the first methionine. c With acetylation. d Observed with MALDI-TOF. e The most abundant native glycoform; the recombinant pigment had non-native glycosylation. f Observed with oxidated tryptophan. g Also observed with homoserine C terminal ending. h Also observed with pyroglutamic acid as the first amino acid. i Not detected. j With palmitylated cysteines.
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Figure 2. Base peak chromatogram of bovine rhodopsin expressed in COS cells. The CNBr cleavage fragments and the peptide used for the elution of the protein from the antibody column are indicated. The additional peaks are contaminants.
Figure 3. Selected ion chromatograms from ESI-MS detection of peaks eluting from HPLC separation of CNBr fragments of rhodopsin expressed in COS cells. The fragment numbers and the detected ions are indicated. The ions correspond to the most abundant charge states.
weight. Although the CNBr fragments could be identified from the selected ion chromatograms, their identity was further confirmed with MS/MS sequence data. As an example, the MS/ MS spectrum of fragment 9, containing an oxidized tryptophan and a pyridylethylated cysteine is shown in Figure 4. Rhodopsin has several posttranslational modifications. Two types of posttranslational modifications are not detectable with this methodology: the retinal chromophore, originally attached 4778
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to lysine 296, and the disulfide bridge between cysteines 110 and 187 are lost during sample preparation. The other posttranslational modifications are retained during the processing. However, only the presence of the two palmityl thioester substituents on cysteines 322 and 323 was confirmed by the data shown here. These two cysteines were palmitylated in all of the samples, including the pigment expressed in COS cells. Another modification, the phosphorylation of the rhodopsin C terminus has been reported in other cases, but in these samples, it was not present because the expression system does not contain rhodopsin kinase and the native pigments were dark-adapted. The other two modifications (glycosylation and acetylation) were not observed because CNBr fragments 1 and 2 were not observed in the HPLC-ESI-MS analysis. To obtain more information on these modifications, the collected 4-min fractions that constitute 90% of the HPLC eluent not injected into the LCQ mass spectrometer were further analyzed in a second experiment. The collected fractions were screened by MALDI-MS for the CNBr fragment 2, and the fractions that contained this fragment were deglycosylated. This experiment identified the position of the glycosylated fragment in the chromatogram and confirmed the presence and nature of the glycosylation. Although glycosylation has extensively been studied for the native bovine pigment,37,38 it was important to confirm the presence and nature of the glycosylation for the pigment that was expressed in COS cells. Previously, these cells were only analyzed by gel electrophoresis, which indicated irregular glycosylation.19,20 Therefore, the spectra of CNBr fragment 2 of the affinity-purified native and expressed pigments before and after deglycosylation were compared by MALDI-MS. The glycosylation of the expressed pigment was confirmed to be different from that of the native one. Although glycosylation is present, it is more heterogeneous and is probably composed of different glycans, since the specific spectral pattern of glycosylation characteristic to native rhodopsin was missing from the spectrum of the expressed pigment. The deglycosylation of the native and expressed pigments by N-glycanase-PLUS also shows differences. While the expressed pigment is completely deglycosylated, the same sample preparation for the native pigment achieved only partial deglycosylation (one glycan was completely removed but the other one was retained in 50% of the sample) and showed the pattern of the native glycosylation. Acetylation was also indicated, since both fragment 2 and the partially cleaved fragment 1-2 complex was detected and their mass difference corresponded to a methionine plus 42 Da, characteristic of the addition of an acetyl group. Together with this second experiment, more than 99% of the bovine rhodopsin primary structure was mapped. Excluding fragments 1 and 2, 90% of the sequence was covered with tandem mass spectra. These results confirm that the modified methodology for the mass spectrometric analysis of integral membrane proteins permits the analysis of subnanomolar amounts of protein. The results obtained with the modified methodology using 250 pmol of rhodopsin were comparable to the results obtained before with more than 1 order of magnitude larger amount of initial sample. It was also demonstrated that rhodopsin protein purified with (37) Duffin, K. L.; Lange, G. W.; Welply, J. K.; Florman, R.; O’Brien, P. J.; Dell, A.; Reason, A. J.; Morris, H. R.; Fliesler, S. J. Glycobiology 1993, 3, 365380. (38) Kean, E. L. Biochim. Biophys. Acta 1999, 1473, 272-285.
Figure 4. Amino acid sequence of CNBr fragment 9 of bovine rhodopsin expressed in COS cells. X stands for pyridylated cysteine and B for homoserine lactone. The sequence also contained an oxidated tryptophan. The amino acids detected individually are underlined.
affinity chromatography and recombinant rhodopsin expressed in COS cells can be analyzed and compared to native pigments. The modified methodology was utilized with the same chromatography system used in the earlier work with larger samples. It is likely that the sample requirement can be reduced still further by adapting the method for use with a capillary HPLC system. CONCLUSIONS The observation of 99% of the structure of the expressed bovine rhodopsin constitutes, to our knowledge, the first complete mass spectrometric mapping of a recombinant integral membrane protein isolated by affinity chromatography. The determination of almost the complete protein sequence in a single experiment provided not only the identification but also the location of the posttranslational modifications. The ability to analyze subnanomolar initial amounts paves the way for the application of the method to the analysis of other photopigments or integral membrane proteins that, due to their low concentration in the membranes, could not be previously
analyzed. The methodology should make it possible to analyze mutant recombinant membrane proteins produced by eukaryotic expression systems for the confirmation of mutation sites, purity, and relevant posttranslational modifications. ACKNOWLEDGMENT We thank Patrice Goletz for assistance in protein preparations and Dr. Kevin Schey for helpful discussions. Funding was provided by grants from NIH (EY-04939 and EY-08239) and the Office of Naval Research and an unrestricted grant to the Department of Ophthalmology from the Foundation for Prevention of Blindness. The mass spectrometric work was performed in the MUSC Mass Spectrometry Institutional Research Resource Facility.
Received for review July 11, 2001. Accepted September 5, 2001. AC015563N
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