Anal. Chem. 2008, 80, 2590-2598
Membrane Protein Separation and Analysis by Supercritical Fluid Chromatography-Mass Spectrometry Xu Zhang, Mark Scalf, Michael S. Westphall, and Lloyd M. Smith*
Department of Chemistry, University of WisconsinsMadison, 1101 University Avenue, Madison, Wisconsin 53706
Membrane proteins comprise 25-30% of the human genome and play critical roles in a wide variety of important biological processes. However, their hydrophobic nature has compromised efforts at structural characterization by both X-ray crystallography and mass spectrometry. The detergents that are generally used to solubilize membrane proteins interfere with the crystallization process essential to X-ray studies and cause severe ion suppression effects that hinder mass spectrometric analysis. In this report, the use of supercritical fluid chromatography-mass spectrometry for the separation and analysis of integral membrane proteins and hydrophobic peptides is investigated. It is shown that detergents are rapidly and effectively separated from the proteins and peptides, yielding them in a state suitable for direct mass spectrometric analysis. Membrane proteins carry out many essential cellular functions in processes such as cell signaling (G-protein coupled receptors (GPCRs)), cell-cell interactions (integrins and adhesion proteins), the intracellular compartmentalization of organelles (kinaseanchoring proteins), ion and solute transport (potassium channels), and energy generation (bacteriorhodopsin, ATP synthase).1 Characterizing the membrane proteome is thus critical to understanding their role in these fundamental biological processes. However, their hydrophobic nature has made them notoriously difficult to study.2 The two major methods employed in membrane proteomics are gel-based mass spectrometry2-8 and liquid chromatography * To whom correspondence should be addressed. Phone: 608-263-2594. Fax: 608-265-6780. E-mail:
[email protected]. (1) Alberts, B.; Johnson, A.; Lewis, J.; Raff, M.; Roberts, K.; Walter, P. Molecular Biology of the Cell; Garland Science: New York, 2002. (2) Santoni, V.; Molloy, M.; Rabilloud, T. Electrophoresis 2000, 21, 1054-1070. (3) Santoni, V.; Kieffers, S.; Desclaux, D.; Masson, F.; Rabilloud, T. Electrophoresis 2000, 21, 3329-3344. (4) Ferro, M.; Seigneurin-Berny, D.; Rolland, N.; Chapel, A.; Slavi, D.; Garin, J.; Joyard, J. Electrophoresis 2000, 21, 3517-3526. (5) Henningsen, R.; Gale, B. L.; Straub, K. M.; Denagel, D. C. Proteomics 2002, 2, 1479-1488. (6) Quach, T. T.; Li, N.; Richards, D. P.; Zheng, J.; Keller, B. O.; Li, L. J. Proteome Res. 2003, 2, 543-552. (7) Molloy, M. P.; Herbert, B. R.; Williams, K. L.; Gooley, A. A. Electrophoresis 1999, 20, 701-704. (8) Vener, A. V.; Harms, A.; Sussman, M. R.; Vierstra, R. D. J. Biol. Chem. 2001, 276 (10), 6959-6966.
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(LC)-tandem mass spectrometry (MS/MS).9-14 In both cases the proteins are typically solubilized using detergents, followed by either enzymatic or chemical digestion. The digestion products are then fractionated by either two-dimensional (2-D) gel electrophoresis or LC, followed by mass spectrometric (MS) analysis. Due to their hydrophobic nature, integral membrane proteins are prone to irreversibly aggregate, adhere to sample handling surfaces such as tube and pipet walls, and bind to chromatographic columns during this process. In addition, the detergent solutions necessary for their solubilization interfere with MS analysis, primarily by causing severe ion suppression which interferes with the ability to obtain analyte ion signal. These issues have presented a tremendous obstacle to the MS analysis of membrane proteins. Several alternative approaches have been investigated for the analysis of enriched membrane fractions. One approach used organic solvents15,16 to solubilize the membrane proteins, whereas another approach utilized organic acids,17 both being compatible with subsequent proteolytic digestion/chemical cleavage, separation, and analysis by LC-MS/MS. However, the highly hydrophobic integral membrane proteins were still generally not amenable to these approaches. In addition, the identification of post-translational modifications was not possible because of low sequence coverage in the MS/MS analysis of the protein digests. In recent work, two approaches have been introduced that are well-suited to the analysis of integral membrane proteins. The introduction of MS-compatible surfactants,18,19 which are either acid-degraded before analysis or HPLC-separated from the peptides, has increased protein solubilization and proteolytic ef(9) Han, D. K.; Eng, J.; Zhou, H.; Aebersold, R. Nat. Biotechnol. 2001, 19, 946951. (10) Loo, R. R. O.; Dales, N.; Andrews, P. C. Protein Sci. 1994, 3, 1975-1983. (11) Rosinke, B.; Strupat, K.; Hillenkamp, F.; Rosenbusch, J.; Dencher, N.; Kruger, U.; Galla, H.-J. J. Mass Spectrom. 1995, 30, 1462-1468. (12) Wu, C. C.; Yates, J. R. Nat. Biotechnol. 2003, 21, 262-267. (13) Molloy, M. P. Anal. Biochem. 2000, 280, 1-10. (14) Buttner, K.; Bernhardt, J.; Scharf, C.; Schmid, R.; Mader, U.; Eymann, C.; Antelmann, H.; Volker, A.; Volker, U.; Hecker, M. Electrophoresis 2001, 22, 2908-2935. (15) Blonder, J.; Goshe, M. B.; Moore, R. J.; Pasa-Tolic, L.; Masselon, C. D.; Lipton, M. S.; Smith, R. D. J. Proteome Res. 2002, 1, 351-360. (16) Goshe, M. B.; Blonder, J.; Smith, R. D. J. Proteome Res. 2003, 2, 153161. (17) Washburn, M. P.; Wolters, D.; Yates, J. R., III. Nat. Biotechnol. 2001, 19, 242-247. (18) Chen, E. I.; Cociorva, D.; Norris, J. L.; Yates, J. R., III. J. Proteome Res. 2007, 6 (7), 2529-2538. (19) Norris, J. L.; Porter, N. A.; Caprioli, R. M. Anal. Chem. 2003, 75 (23), 66426647. 10.1021/ac702319u CCC: $40.75
© 2008 American Chemical Society Published on Web 02/28/2008
Figure 1. Schematic diagram of the SFC-ESI-MS system: (a) CO2 pump, (b) modifier pump, (c) six-port sample injector, (d) column, (e) UV detector, (f) low-dead-volume tee, (g) back-pressure regulator, (h) ESI-TOF mass spectrometer.
ficiency.18 The use of heated columns in reversed-phase chromatography20,21 has been shown to greatly increase the recovery of hydrophobic peptides from membrane proteins. In the present work the use of supercritical carbon dioxide as a mass spectrometry-compatible solvent for membrane proteins is explored. Supercritical carbon dioxide is a fluid with excellent dissolving power and complete transparency to a mass spectrometer.22 In recent years, supercritical fluid chromatography (SFC) has been exploited as an alternative to high-performance liquid chromatography (HPLC) because of its superior selectivity, higher efficiency, and speed.23,24 Fluids that are commonly used in SFC, such as CO2, are relatively nonpolar. As a result, SFC has been a technique traditionally used on relatively nonpolar compounds. SFC coupled to mass spectrometry (SFC-MS) has been one of the most successful applications of SFC.25 Both atmospheric pressure chemical ionization (APCI)26-29 and electrospray ionization (ESI)29-33 have been utilized to interface between SFC and a mass spectrometer. An additional advantage of SFC-MS is that it is much easier to evaporate a supercritical mobile phase into the MS source than most LC solvents. LC-MS is used extensively in a variety of proteomics applications for protein identification. However, some of its drawbacks, including the limited speed of LC separation and its limited utility for highly hydrophobic compounds, have been noted.9,11,12 SFC is faster than HPLC due to the lower viscosity of CO2 and higher diffusivity of solutes in carbon dioxide based fluids, which lead to more rapid separations and higher efficiencies. Because of these (20) Martosella, J.; Zolotarjova, N.; Liu, H. B.; Nicol, G.; Boyes, B. E. J. Proteome Res. 2005, 4 (5), 1522-1537. (21) Speers, A. E.; Blackler, A. R.; Wu, C. C. Anal. Chem. 2007, 79 (12), 46134620. (22) Willams, J. R.; Clifford, A. A.; Al-Saidi, S. H. R. Mol. Biotechnol. 2002, 22, 263-286. (23) Matsumoto, K.; Taguchi, M. Chromatogr. Sci. 1994, 65, 365-396. (24) Sadoun, F.; Virelizier, H.; Arpino, P. J. J. Chromatogr. 1993, 647, 351-359. (25) Smith, R. D.; Kalinoski, H. T.; Udseth, H. R. Mass Spectrom. Rev. 1987, 6, 445-496. (26) Sjoberg, P. J. R.; Markides, K. E. J. Chromatogr., A 1999, 855, 317-327. (27) Kalinoski, H. T.; Wright, B. W.; Smith, R. D. Biomed. Environ. Mass Spectrom. 1988, 15, 239. (28) Smith, R. D.; Kalinoski, H. T.; Udseth, H. R. Mass Spectrom. Rev. 1987, 6, 445-496. (29) Pinkston, J. D.; Chester, T. L. Anal. Chem. 1995, 67, 650A-656A. (30) Ventura, M. C.; Farrell, W. P.; Aurigemma, C. M.; Greig, M. J. Anal. Chem. 1999, 71, 2410-2416. (31) Sadoun, F.; Virelizier, H.; Arpino, P. J. J. Chromatogr. 1993, 647, 351-359. (32) Pinkston, J. D. Eur. J. Mass Spectrom. 2005, 11, 189-197. (33) Sjoberg, P. J. R.; Markides, K. E. J. Chromatogr., A 1997, 785, 101-110.
advantages, SFC-MS has been utilized by several groups as an alternative to LC-MS for small molecule analysis.34-41 One particularly significant application of supercritical fluid separation techniques has been in the characterization of polymeric mixtures. Many papers have been published on SFC separations of polymer homologs, such as polystyrene,42 poly(methyl methacrylate),43 and polyisocyanates.44 Optimization of the operating conditions for SFC enabled the separation of polymeric complexes consisting of oligomers up to 10 000 Da. Several studies have also been done using SFC-MS for biopolymer analysis. Cyclosporin A, a hydrophobic cyclic peptide with a molecular mass of ∼1200 Da, was analyzed in one of the earliest reports of characterizing high molecular weight, biologically active compounds using SFC-chemical ionization mass spectrometry (CI-MS).45 Blackwell and Stringham46 studied the elution of a group of polypeptides containing four to nine amino acids on a divinylbenzene polymeric column with a modifier of either ethanol or 2-methoxyethanol, containing 50 mM of either methanesulfonic acid, trifluoromethanesulfonic acid, or heptadecafluorooctanesulfonic acid. They reported that the acidity of the additive was critical and that an acid stronger than trifluoroacetic acid (TFA) is required for elution of these peptides. Bradykinin, which contained the largest number of amino acid residues, was retained the longest and gave the broadest peak among the chosen peptides using the same chromatographic conditions. Gramicidin, a mixture of membrane-spanning peptides (A, B, C), was reported to be separated by SFC using an organic modifier (34) Combs, M. T.; Ashraf-Khorassani, M.; Taylor, L. T. J. Chromatogr. 1997, A785, 85-100. (35) Matsumoto, K.; Nugata, S.; Hattori, H.; Tsuge, S. J. J. Chromatogr. 1992, 605, 87-94. (36) Thomas, D.; Sim, P. G.; Benoit, F. Rapid Commun. Mass Spectrom. 1994, 8, 105-110. (37) Scalia, S.; Games, D. E. Org. Mass. Spectrom. 1992, 27, 1266-1270. (38) Via, J.; Taylor, L. T. Anal. Chem. 1994, 66, 1385-1395. (39) Jedrzejewski, P. T.; Taylor, L. T. J. Chromatogr. 1995, A703, 489-501. (40) Pinkston, J. D.; Baker, T. R. Rapid Commun. Mass Spectrom. 1995, 9, 10871094. (41) Berger, T. A.; Wilson, W. H. Anal. Chem. 1993, 65, 1451-1455. (42) Ute, K. Chromatogr. Sci. 1997, 75, 349-368. (43) Ute, K.; Miyatake, N.; Osugi, Y.; Hatada, K. Polym. J. 1993, 25, 1153. (44) Ute, K.; Asai, T.; Fukunishi, Y.; Hatada, K. Polym. J. 1995, 27, 445. (45) Kalinoski, H. T.; Wright, B. W.; Smith, R. D. Biomed. Environ. Mass Spectrom. 1988, 15, 239. (46) Blackwell, J. A.; Stringham, R. W. J. High Resolut. Chromatogr. 1999, 22, 74-78.
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Figure 2. (A) UV chromatogram obtained for the SFC separation of gramicidin from 1% Triton X-100 detergent; (B) ESI-TOF mass spectrum at the retention time of 5.56 min showing gramicidin A, B, and C; (C) MALDI-TOF mass spectrum of SFC-extracted gramicidin. Note that the ESI ions (B) are approximately 28 Da heavier than the MALDI ions (C). This is likely due to formylation of the peptides in the ESI analysis, since 0.1% formic acid was added to increase the ionization efficiency.
consisting of methanol containing either 2% water, 0.01 M acetic acid (AA), and 0.4% isopropylamine or 0.5% trifluoroethanol on a cyano column.47 In this study it was detected by both UV and ESI-MS. A commercially available peptide standard mixture, Sigma H2016, consisting of five peptides (angiotensin II, Gly-Tyr, Leu (47) Bolanos, B.; Greig, M.; Ventura, M.; Farrell, W.; Aurigemma, C. M.; Li, H.; Quenzer, T. L.; Tivel, K.; Bylund, J. M. R.; Tran, P.; Pham, C.; Phillipson, D. Int. J. Mass Spectrom. 2004, 238, 85-97.
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enkephalin, Met enkephalin, and Val-Tyr-Val), has also been reported to be separated and analyzed by SFC-ESI-MS47 with a modifier of methanol containing 2% water, 0.01 M acetic acid (AA), and 0.4% isopropylamine. A relatively large peptide, sauvagine, containing a variety of acidic and basic residues, has been reported to be eluted in SFC48 using a CO2/methanol mobile phase with (48) Zheng, J.; Pinkston, J. D.; Zoutendam, P. H.; Taylor, L. T. Anal. Chem. 2006, 78, 1535-1545.
Figure 3. Direct-infusion ESI-TOF mass spectrum of a 9.3 µM solution of bacteriorhodopsin/lipids (3:1 by weight) in CHCl3/MeOH/1% formic acid.
TFA as an additive on a 2-ethylpyridine-bonded silica column. This study48 represents the most ambitious work in the SFC-MS separation and detection of peptides previous to the work described here. Zheng et al.48 were able to separate and detect peptides over a wide range of polarities and over 4500 in molecular weight. Although some successes in peptide analyses have been reported, as reviewed above, SFC-MS has not been applied to either large proteins or membrane proteins. Considering the advantages that SFC can bring to polymer and biopolymer separations, the structural insights that can be provided by mass spectrometry, and with the expectation of improved selectivity and sensitivity, we explore here the use of SFC-MS as an alternative approach to membrane proteome analysis. EXPERIMENTAL SECTION Materials. SFC-grade carbon dioxide was from Linde gas (Wilmington, DE). Burdick and Jackson (Muskegon, MI) HPLCgrade methanol was used for the mobile-phase modifier. Trifluoroacetic acid (97+%, ACS grade) and formic acid (96+%, ACS grade) were obtained from Alfa Aesar (Ward Hill, MA). 2, 2, 2,Trifluoroethanol (TFE), Triton X-100, sodium dodecyl sulfate (SDS), sorbitol, sucrose, tricine-NaOH, Hepes buffer, 2-[Nmorpholino] ethanesulfonic acid (MES)-KOH, CHCl3, MgCl2, NaCl, and sodium EDTA were all purchased from Sigma-Aldrich (St. Louis, MO). Octylthioglucoside (OTG) and decyl-maltoside were obtained from Pierce (Rockford, IL). Platinum wire (99.95%, 0.368 mm diameter) was obtained from Alfa Aesar (Ward Hill, MA). Miracloth was acquired from Calbiochem (San Diego, CA). Gramicidin, cytochrome c, and bacteriorhodopsin were purchased from Sigma-Aldrich (St. Louis, MO). Modified trypsin was purchased from Promega (Madison, WI). Leaves from spinach obtained from local market sources were used for preparation of PS II-enriched membranes.
A tryptic digestion reaction was performed on a 1 mg/mL aqueous solution of cytochrome c as described elsewhere.49 PS II complex was purified from 100 g of 1-2 cm long depetiolated leaves as described elsewhere.50,51 The final PS II pellet was resuspended using a glass homogenizer to 1.1 mg chl/ mL final concentration in 10% surfactant stock solution (OTG/ decyl-maltoside/SDS, 4.5:4.5:1 by weight) and stored at -20 °C. The PS II product was characterized by SDS-PAGE. A series of bands below 60 kDa were observed, corresponding to the published molecular weights of the proteins that constitute the core of PS II. SFC/UV Instrumentation. The analytical SFC system (Figure 1) was home-built with a Shimadzu SPD-20AV UV-vis spectrophotometric detector. UV detection was at 220 nm for gramicidin and the cytochrome c tryptic digest and at 280 nm for bacteriorhodopsin and photosystem II. A 260D ISCO syringe pump with series D pump controller was used as the CO2 pump. A Shimadzu LC-6A LC pump with SCL-6A system controller was used as the modifier pump. The chromatographic column was SFC cyanobonded silica, non-end-capped, (Princeton Chromatography Inc., Cranbury, NJ). The column dimensions were 5 or 15 cm in length as noted and 4.6 mm i.d., with a particle size of 5 µm and a pore size of 60 Å. Unless otherwise specified, chromatographic conditions were as follows: injection volume 10 µL, mobile-phase flow rate 1.5 mL/min (measured in the liquid state), column outlet pressure 140 bar, and column oven temperature 50 °C. The mobile-phase composition gradient was varied as needed. The modifier consisted of either methanol with 0.5%TFE, methanol with 0.1%TFA, or methanol with CHCl3 and 1% aqueous formic acid (4/4/1; v/v). Between each change of mobile-phase additive, the stationary phase was washed with pure methanol or methanol with CHCl3 (49) Flannery, A. V.; Beynon, R. J.; Bond, J. S. Proteolytic Enzymes: A Practical Approach; IRL Press: Oxford, U.K., 1989. (50) Peter, G. F.; Thornber, J. P. J. Biol. Chem. 1991, 266 (25), 16745-16754. (51) Ghanotakis, D. F.; Babcock, G. T. FEBS Lett. 1983, 153 (1), 231-234.
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Figure 4. SFC-ESI-MS spectra of bacteriorhodopsin (BR): (A) total ion chromatogram; (B) mass spectrum of BR at 1.0 min; (C) example mass spectrum of lipids at 2.7 min. The lipids eluted from time 1.1 through 3.5 min.
and 1% aqueous formic acid (4/4/1; v/v) at 1 mL/min for 60 min (∼20 column volumes) in order to purge previous additive solution from the column or to remove analyte that was strongly retained on the stationary phase. After this time period, the next additive solution was introduced to the SFC system, and the column was equilibrated again for 30 min prior to injection. SFC-MS Instrumentation. A low-dead-volume chromatographic tee was installed immediately after the SFC column, before 2594
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the UV detector and the back-pressure regulator (BPR) (see Figure 1). A 50 cm long, 65 µm i.d. capillary tube was installed after the tee to direct ∼1/10 of the flow to the electrospray source of an ABI Mariner orthogonal acceleration ESI time-of-flight mass spectrometer (Applied Biosystems, Foster City, CA). All samples were electrosprayed from a fused-silica capillary (90/20 µm o.d./ i.d., Polymicro Technologies, Phoenix, AZ). The spray tip potential was applied via a platinum wire installed in a three-way tee. The
Figure 5. (A) UV chromatogram obtained for the SFC separation of PS II; (B) ESI mass spectrum of PsbA in the collected fraction from 9 to 10 min; (C) ESI mass spectrum of PsbE in the collected fraction from 10 to 11 min.
electrospray capillary, orifice, and ring voltages were held at +3750, +35, and +240 V, respectively. The remainder of the effluent was either collected or directed to waste after the BPR. For collection, the effluent was directed into 1.5 mL Eppendorf tubes. The CO2 evaporated immediately, and methanol remained in the tubes. All spectra shown were smoothed using a nine-point Gaussian smoothing algorithm available in the software supplied with the instrument. Matrix-Assisted Laser Desorption Ionization Time-ofFlight Mass Spectrometry Analysis. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectra were
obtained from collected fractions using a Voyager-DE STR mass spectrometer (Applied Biosystems, Foster City, CA). One microliter aliquots of the samples were spotted directly onto the MALDI plate and mixed with 1 µL of sinapinic acid matrix solution. A pulsed nitrogen laser (337 nm) was used for desorption/ionization, and positive ion mass spectra were acquired in linear mode. The total acceleration voltage was 20 kV, grid voltage at 90%, guide wire voltage at 0.1%, and a delay time of 10 ns was used. External mass calibration was performed using insulin and angiotensin I. Spectra were obtained by averaging data from 50 laser shots. Analytical Chemistry, Vol. 80, No. 7, April 1, 2008
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Figure 6. (A) UV Chromatogram obtained for the SFC separation of tryptic peptides from cytochrome c; (B) ESI mass spectrum of the collected fraction from 3 to 4 min; (C) ESI mass spectrum of the collected fraction from 4 to 5 min.
RESULTS AND DISCUSSION Purification of Peptides from Detergents. To investigate the utility of SFC to separate hydrophobic peptides from detergents, solutions of gramicidin were prepared in methanol at a concentration of 2 mg/mL and combined with varying amounts of detergent. The detergents chosen were SDS and Triton X-100, since they are widely used for membrane protein solubilization.2,11,12 The mixture was separated and analyzed by SFC-ESIMS, using methanol with 0.5% trifluoroethanol as the modifier. 2596 Analytical Chemistry, Vol. 80, No. 7, April 1, 2008
The modifier concentration was increased from 20% to 50% at 6%/ min, and then was held at 50% for 5 min. Figure 2A shows a plot of UV absorbance versus retention time for the separation of gramicidin in 1% Triton X-100. The three forms of gramicidin were baseline separated from each other in 6 min and were very well separated from the Triton X-100, which eluted significantly earlier than the peptides due to its greater hydrophobicity. Figure 2B shows the ESI mass spectrum obtained at a retention time of 5.56 min, which corresponds to the maximum
Table 1. PS II Core Proteins Eluted and Detected by SFC-ESI-MS (Adapted from Ref 58) PS II obsd by proteins SFC-ESI-MS PsbA PsbB PsbC PsbD PsbE PsbF PsbG PsbH PsbI PsbJ PsbK PsbL PsbM PsbN PsbO PsbP PsbQ PsbR PsbS PsbT PsbTa PsbUb PsbVb PsbW PsbX a
× × × × × × × × × × × × × ×
×
subunit Dl CP47 CP43 D2 R-cyt b559 β-cyt b559 open H protein I protein J protein K protein L protein M protein N protein 33 kDa protein 23 kDa protein 16 kDa protein 10 kDa protein Lhc-like protein ycf8 protein 5 kDa protein U protein cyt c550 W protein X protein
mass (kDa)
no. of transmembrane R-helices
38.021 56.278 50.066 39.418 9.255 4.409
5 6 6 5 1 1
7.697 4.195 4.116 4.283 4.366 3.755 4.722 26.539 20.210 16.523 10.236 21.705 3.849 3.283 ∼10 15.121 5.928 4.225
1 1 1 1 1 1 1 0 0 0 0 4 1 0 0 0 1 1
Foundexclusivelyinhigherplantsandalgae. b Foundincyanobacteria.
signal intensity for the most dominant of the three gramicidin species present, gramicidin A. For clarity, only the region of the spectrum corresponding to the predominant +2 charge state is shown. Each of the forms of gramicidin are observed to be approximately 28 Da heavier in the ESI analysis (Figure 2B) as compared to MALDI analysis (Figure 2C). This is probably due to formylation of the peptides in the ESI spectrum (Figure 2B) since the only difference in sample handling was the addition of 0.1% formic acid to increase ionization efficiency for the off-line ESI-TOF analysis. As expected, gramicidin A gave the highest MS signal (charge state ) +2, m/z 955.00). The other form of gramicidin A (charge state ) +2, m/z 961.87), which accounts for about 20% of the molecules, generates a peak at higher m/z with the expected lower intensity. Gramicidin B and gramicidin C, which elute earlier than gramicidin A, also show up in the mass spectrum at this time point as doubly charged ions of m/z 935.98 and 943.78, respectively. Their peak intensities are much lower than that of gramicidin A. Similar results were obtained for Triton X-100 concentrations from 0.5% to 5%, with the only significant difference being a decrease in the resolution of the Triton X-100 peaks at the highest concentration of 5% (data not shown). This shows that up to 5% of Triton X-100 does not adversely affect the separation and MS signal of the separated gramicidin. To evaluate the utility of SFC for the preparation of samples for MALDI analysis, fractions eluting from the UV detector were collected at 1 min intervals and MALDI-TOF mass spectra were obtained. Figure 2C shows the MALDI mass spectrum of the gramicidin fraction eluting between 5 and 6 min, with the singly charged ion for each of the gramicidin A (both forms), B, and C. As with the ESI analyses, MALDI analyses also showed similar results for Triton X-100 concentrations ranging from 0.5% to 5%.
Substituting the detergent SDS for Triton X-100 gave the same results in both ESI and MALDI analyses (data not shown). Purification of a Membrane Protein: Bacteriorhodopsin. As a readily available and well-characterized membrane protein, bacteriorhodopsin (BR)52 was chosen as a model to investigate the utility of SFC for purifying and characterizing integral membrane proteins. It has been shown that good quality mass spectra can be obtained from acetone-precipitated BR using Fourier transform ion cyclotron resonance mass spectrometry (FTICR) MS53 and LC-MS.54,55 In the present work, the BR obtained from commercial sources was dissolved directly in CHCl3/MeOH/1% aqueous formic acid (4/4/1, v/v) without acetone precipitation or any additional purification. According to the sample data sheet, the BR sample is complexed with lipids at a 3:1 ratio by weight. The dissolved BR/lipid solution was directly infused into the mass spectrometer using a syringe pump, and the resulting electrospray mass spectrum is shown in Figure 3. Many high-intensity peaks appear at m/z less than 1000. These probably correspond to lipids and other contaminants in the sample that have higher ionization efficiencies than BR. The inset of Figure 3 shows an expanded mass spectrum for the mass range of m/z 1600-3500. Five charge states of BR are detected (+10 to +14). Figure 4 shows the SFC-ESI-MS spectra obtained from the same BR sample. Panel A is a plot of total ion current versus retention time, produced using the following mobile-phase composition gradient: 5% modifier in CO2 for 1 min after injection, modifier concentration increased to 50% at 5%/min and held for 5 min at 50%. The modifier consisted of methanol with CHCl3 and 1% aqueous formic acid (4/4/1; v/v). Panel B shows the mass spectrum obtained at a retention time of 1.0 min, which corresponds to purified BR. Nine charge states were observed for the protein. Panel C, obtained at a retention time of 2.7 min, shows an example mass spectrum of the lipids and other contaminants that eluted starting just after the BR (time 1.2 min) through 3.5 min. The fact that these small molecules were separated from the BR protein is probably what leads to the improved mass spectra of BR, as the lipids and other small molecules are no longer causing ion suppression of BR. These results show that the integral membrane protein, BR, is not only able to be purified and eluted using SFC but also can be detected by ESI-MS. Separation of Membrane Protein Complex: Photosystem II. Photosystem II (PS II) is a membrane protein complex located in the thylakoid membranes of plants, eukaryotic algae, and cyanobacteria.56 It contains both intrinsic and extrinsic polypeptides. Structurally, PS II is comprised of ∼25 polypeptides and is surrounded by a variety of chlorophyll a and chlorophyll b binding proteins.57,58 It catalyzes the light-driven oxidation of water to O2 and the reduction of plastoquinone. Reversed-phase HPLC analysis (52) Nelson, D. L.; Cox, M. M. Lehninger Principles of Biochemistry; Worth Publishers: New York, 2000. (53) Whitelegge, J. P.; Conklin, K. A.; Aguilera, R.; Fountain, A. G.; Faull, K. F. Proceedings of the 50th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, Florida, June 2-6, 2002. (54) Whitelegge, J. P.; Gundersen, C. B.; Faull, K. F. Protein Sci. 1998, 7, 14231430. (55) Whitelegge, J. P. Trends Anal. Chem. 2005, 24 (7), 576-582. (56) Zolla, L.; Timperio, A. M. Proteins: Struct., Funct., Genet. 2000, 41, 398406. (57) Calderone, V.; Trabucco, M.; Vujicic, A.; Battistutta, R.; Giacometti, G. M.; Andreucci, F.; Barbato, R.; Zanotti, G. EMBO Rep. 2003, 4 (9), 900-905.
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Table 2. List of Tryptic Cytochrome c Peptides Detected Using SFC-ESI-MS peptide sequence GDVEKGK HKTGPNLHGLFGRK GGKHK TGPNLHGLFGR MIFAGIKK KTGQAPGFSYTDANKNK CAQCHTVEKGGKHK MIFAGIKKK KIFVQKCAQCHTVEK MIFAGIK MIFAGIK 1Met-ox HKTGPNLHGLFGRK KYIPGTKMIFAGIK YIPGTKMIFAGIKK GGKHKTGPNLHGLFGR TGQAPGFSYTDANKNKGITWGEETLMEYLENPK IFVQK TGPNLHGLFGRK YIPGTK EDLIAYLK
charge state
theor m/z
244.22 261.22 264.20 292.12 390.24 584.98 304.15 306.15 328.14 410.18 346.19 376.24 390.24 795.62 391.24 392.23 396.20 396.20 419.17 530.22
+3 +6 +2 +4 +3 +2 +3 +6 +5 +4 +3 +5 +2 +1 +4 +4 +4 +4 +4 +7
244.8016 261.1518 263.6590 292.9115 390.2128 584.8153 303.1865 305.3238 328.7622 410.7008 345.8848 375.9977 390.2284 795.4439 391.2238 392.4829 396.4816 396.4816 419.7345 530.2550
738.39 634.54 649.03 678.56 964.78
+5 +1 +2 +1 +1
738.7549 634.3928 648.8628 678.3827 964.5355
obsd m/z
coupled with electrospray-ionization mass spectrometry has been used to analyze acetone-precipitated PS II.59 In this case, thylakoid membrane proteins were first stripped of lipids, chlorophyll, and other pigments by precipitation with acetone and then dissolved in 60% formic acid prior to LC-MS analysis. Eighteen core protein subunits of PS II were eluted and identified. However, the very hydrophobic proteins eluted from the column with lower efficiency so that a substantial proportion remained bound to the column. This necessitated a large amount of sample as well as the use of a second gradient with isopropyl alcohol to elute the more hydrophobic PS II core proteins and peptides including D1, D2, CP43, CP47, and PsbM. The use of the second gradient both substantially increased the time and complexity of the separation process and also produced poorer resolution for these more hydrophobic proteins. The PS II complex was chosen as a more challenging system for analysis by SFC-MS. About 50 µg of the extracted proteins in surfactant stock solution (OTG/decyl-maltoside/SDS, 4.5:4.5:1 by weight) was injected and run on the SFC-MS. Figure 5A shows a chromatogram obtained for the SFC separation of PS II. The mobile-phase composition gradient was as follows: 5% modifier in CO2 for 1 min after injection, modifier concentration increased to 50% at 5%/min and held for 5 min at 50%. The modifier consisted of methanol with CHCl3 and 1% aqueous formic acid (4/4/1; v/v). Because the signal-to-noise ratio (S/N) was too low to obtain high-quality mass spectra with direct ESI-MS analysis, samples were collected into vials at 1 min intervals, and off-line electrospray mass spectra of each fraction were obtained. Again, (58) Barber, J.; Nield, J.; Zheleva, D.; Hankamer, B. Physiol. Plant. 1997, 100 (4), 817-827. (59) Gomez, S. M.; Nishio, J. N.; Faull, K. F.; Whitelegge, J. P. Mol. Cell. Proteomics 2002, 1 (1), 46-59.
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detergent eluted the earliest, followed by a total of 15 proteins eluted from SFC and detected by ESI-TOF MS. Table 1 lists the PS II proteins detected by off-line ESI-TOF MS. Parts B and C of Figure 5 give two examples of the mass spectra obtained, corresponding to the PS II core proteins, PsbA (38 kDa) and PsbE (9.3 kDa). It is noteworthy that these relatively large hydrophobic proteins were able to be separated and eluted for off-line ESI analysis in only 15 min in a very straightforward and simple gradient and that the same gradient was also useful for much smaller hydrophobic peptides. This work shows that it is possible to perform the rapid separation and mass analysis of the components of a membrane protein complex using SFC-MS. Separation of Cytochrome c Tryptic Digest. Another area of interest is the analysis of protein tryptic digests, which is the mainstay technique of modern protein identification by mass spectrometry. A tryptic digest of cytochrome c, a relatively small protein of molecular weight 12 327 Da, is a common standard sample used to evaluate system performance. This standard was chosen to evaluate the use of SFC for the separation and mass analysis of peptide digests. Figure 6A shows the UV chromatogram obtained for the digested cytochrome c. The separation required approximately 7 min, and as before, 1 min fractions were collected and mass analyzed off-line by ESI-TOF MS. Figure 6B shows an example of the ESI analysis of the fraction collected from 3 to 4 min, and Figure 6C shows an example of ESI analysis of the fraction collected from 4 to 5 min. The peptide masses that were observed are shown in Table 2 and correspond to over 90% sequence coverage. CONCLUSIONS The research presented here addresses a critical problem in membrane proteomics, namely, the efficient extraction of membrane proteins in a form suitable for chromatography and MS analysis. Gramicidin and bacteriorhodopsin were purified from detergents and lipids by SFC, and high-quality on-line mass spectra were acquired. Photosystem II was also investigated by SFCMS, and a total of 16 out of 26 core proteins eluted in 15 min. These results show that SFC-MS provides an attractive means to purify, separate, and rapidly characterize membrane proteins. SFC-MS may also have utility in the MS analysis of protein tryptic digests, where in addition to providing a rapid means of separation, it also may offer a normal-phase elution that provides an excellent complement to standard reversed-phase elutions for the 2-D separation of complex peptide mixtures. Further work is needed to explore these potential applications of SFC-MS. ACKNOWLEDGMENT This work was supported by NIH Grant GM074234 and NHLBI Proteomics Center Contract N01-HV-28182. We thank Dr. Teshik Yoon for providing access to his SFC instrument before our own instrument had been built.
Received for review November 9, 2007. Accepted January 15, 2008. AC702319U
