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Preparation of Insoluble Transmembrane Peptides: Glycophorin-A, Prion (110–137), and FGFR (368–397)
Analytical Biochemistry 272, 270 –274 (1999) Article ID abio.1999.4182, available online at http://www.idealibrary.com on
Preparation of Insoluble Transmembrane Peptides: Glycophorin-A, Prion (110–137), and FGFR (368–397) Kerney Jebrell Glover, Paul M. Martini, Regitze R. Vold, and Elizabeth A. Komives 1 Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0601
Received April 23, 1999
A general procedure for the reliable preparation of large quantities of insoluble transmembrane peptides has been developed. Optimal couplings were obtained during synthesis by using high-temperature couplings in conjunction with O-(7-azabenzotriazol-1-yl)-1,1,3,3tetramethyluronium hexafluorophosphate (HATU) activation. Improved purification schemes were developed that use reverse- phase HPLC on a C1 column and elution with a 2:1 mixture of 1-propanol:1-butanol. Using these methods three very different transmembrane peptides all longer than 25 amino acids have been prepared: glycophorin-A, prion (110 –137), and fibroblast growth factor receptor (368 –397). © 1999 Academic Press
Transmembrane domains are involved in a variety of cellular processes including receptor dimerization and channel formation (1). Due to their extreme hydrophobicity, studies on these peptides have been hindered by their physical characteristics. Recent advancements in the field of “native-like” membrane environments have now made it possible to structurally characterize peptides derived from these domains using biophysical techniques (2). Reliable methods to prepare large quantities of highly purified transmembrane peptides are therefore needed. This requires both an efficient synthetic methodology and an effective general purification scheme. Standard synthesis methods for peptides are often inadequate for transmembrane peptides which contain high numbers of b-branched amino acids and have a strong propensity for secondary structure and for oligomerization. We present in this report the combined
use of an efficient activation chemistry utilizing O-(7azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) 2 combined with elevated synthesis temperatures (3, 4). Although there have been a number of reports describing the purification of individual hydrophobic peptides by HPLC, none has been widely applicable. The majority of these methods deal with peptides which are labeled as hydrophobic but in fact are fully soluble in aqueous buffers and can be purified using a C4 or C18 reverse-phase column in conjunction with an acetonitrile (or 2-propanol) gradient. The few papers which address extremely hydrophobic peptides often use harsh conditions which greatly shorten column life or utilize detergents which are difficult to remove from the purified peptide (5). One purification of glycophorin-A has been reported using a C4 column (6). This protocol called for a single step of greater than 80% organic solvent. Under these conditions the peptide was eluted but not effectively purified. Modification of this procedure to include authentic gradient elution resulted in excessive peak broadening which masked any separation. Here we present the reliable purification of long-chain insoluble hydrophobic peptides using a reverse-phase C1 column and an elution solvent composed of a 2:1 (v/v) mixture of 1-propanol:1-butanol. MATERIALS AND METHODS
Peptide synthesis. Peptides were synthesized on a Milligen 9050 peptide synthesizer using standard solid-phase methods. Synthesis was carried out at 50°C using a water-jacketed column and 60-min coupling cycles. All amino acids were free carboxylic acids with a fluorenyl-methoxycarbonyl (FMOC) blocker on the 2
1
To whom correspondence should be addressed. Fax: (619) 8220079. E-mail: [email protected].
270
Abbreviations used: HATU, O-(7-azabenzotriazol-1-yl)-1,1,3,3tetramethyluronium hexafluorophosphate; HFIP, hexafluoro-2-propanol; TFA, trifluoroacetic acid.
0003-2697/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
PREPARATION OF INSOLUBLE TRANSMEMBRANE PEPTIDES
271
Yields of these peptides suffered due to difficulties in coupling of b-branched amino acids and steric interactions of the growing chains. To overcome these difficulties, we have optimized several parameters which are essential for obtaining high yields with a minimal percentage of deletion products. A combination of HATU activation, 60-min couplings, and a synthesis temperFIG. 1. Peptide sequences of prion (110 –137), glycophorin-A, and FGFR (368 –397).
amine. HATU was used for carboxylic activation during coupling. The PAL-PEG-PS solid support, HATU, and side-chain-protected amino acids were obtained from PE Biosystems (Framingham, MA). OmniSolv glass distilled dimethylformamide (DMF) was from EM Sciences (distributed by VWR, Los Angeles, CA). After coupling of all amino acids, the amino terminus was acetylated using a solution of dimethylformamide (87%), dichloromethane (10%), acetic anhydride (3%), and hydroxyazabenzotriazole (12.2 g). The resin was then washed with dichloromethane and dried overnight under vacuum. Peptides were cleaved from the resin using trifluoroacetic acid (90%), thioanisole (5%), ethanedithiol (3%), and anisole (2%). Cleavage from the resin yielded an amide on the carboxyl terminus. The slurry was filtered through glass wool and the filtrate poured into cold diethyl ether to induce precipitation of the peptide. After overnight at 220°C, the precipitate was collected on a sintered glass funnel, dissolved in hexafluoro-2-propanol (HFIP), and lyophilized. Yield of crude peptides was about 90% on average for a 0.20-mmol synthesis scale. Peptide purification. Crude peptide was dissolved in a solution of 80% H 2O, 20% acetic acid with heating should be at 50°C. Before injection the solution was centrifuged for 2 min at 14,000 rpm to remove particulates. The mixture was injected onto an 80 Å, 10-mm Waters Spherisorb C1 column (4.6 3 150 mm) at a flow rate of 1.00 ml/min. The column was maintained at 50°C by immersion in a constant temperature circulating water bath. The peptides were eluted with various gradient mixtures composed of solvent A (0.1% TFA) and solvent B [2:1 (v/v) solution of 1-propanol:1-butanol]. Both solutions were filtered, degassed through 0.2-mm membranes, and continuously sparged with helium. For each peptide, the major peak was collected and lyophilized to yield the pure product in 60% yield. Gradient elution conditions for each peptide are described later. RESULTS AND DISCUSSION
Peptide synthesis. We have synthesized several different long, insoluble hydrophobic peptides (Fig. 1).
FIG. 2. HPLC separation of the crude, synthetic peptides. All peptides were isolated on a Waters Spherisorb C1 (4.6 3 150 mm) analytical reverse-phase HPLC column at a flow rate of 1.00 ml/min using a gradient. (A) The gradient for prion (110 –137) is 100% solution A for 5 min and 0 –20% 1-propanol:1-butanol (2:1, v/v) over 15 min, and the pure peptide has a retention time of 10.2 min. (B) The gradient for glycophorin-A is 94% solution A for 5 min and 6 –36% 1-propanol:1-butanol (2:1, v/v) over 15 min, and the purified peptide has a retention time of 9.6 min. (C) The gradient for FGFR (368 –397) is 94% solution A for 5 min and 6 –36% 1-propanol:1butanol (2:1, v/v) over 15 min, and the purified peptide has a retention time of 10.2 min.
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ature of 50°C resulted in reproducibly high yield of the correct product (Fig. 2). Peptide purification. It is critical to load the peptide onto the column in a deaggregated state. Lyophilization of the crude peptide after dissolution in HFIP was found to be an essential first step in deaggregation. Immediately before loading on the column, the peptides were dissolved in a solution of 80% H 2O/20% acetic acid which was found to be ideal for deaggregating the peptide without sacrificing initial adsorption to the column. In addition, this solution must be kept at 50°C prior to injection to be optimally effective. For purification of these peptides, use of standard reverse-phase HPLC conditions and a C4 or C18 column in conjunction with a water/acetonitrile gradient is inadequate. Their employment results in either irreversible column binding or unworkably broad peaks. One attempt at addressing this concern is the substitution of isopropanol for acetonitrile (7). While this increase in solvent strength may work for a few borderline hydrophobic peptides, it was unsuccessful at eluting our peptides. Instead we found that employment of a reverse-phase C1 column in conjunction with a long-chain alcohol elution solvent was ideal for the purification and high recovery of these peptides. The C1 column contains only a single carbon moiety covalently bonded to a silica backbone, and as a result it is one of the least hydrophobic reverse-phase columns available. The 2:1 1-propanol:1-butanol solution has an extremely hydrophobic nature while retaining water miscibility which is perfectly suited to the elution of these sticky peptides. However, due to the viscous nature of this solvent system, the column was run at 50°C to minimize backpressure while simultaneously increasing peptide solubility. The starting solvent for HPLC purification was varied for each peptide to achieve maximum adsorption to the column. The prion peptide was injected onto the column in 100% solvent A. Although the prion peptide is only sparingly soluble in this solution, the combined effect of injection in 80% H 2O/20% acetic acid and adsorption to the column keeps the peptide from precipitating out as well as reaggregating. The percentage of solvent B was increased at a rate of 1.3%/min with the pure peptide then eluting around 7% (Fig. 3A). Reinjection of the collected prion peptide showed that purification had indeed been achieved by this gradient elution (Fig. 3B). At this percentage of solvent B, the peptide is very soluble and could be readily lyophilized. The glycophorin-A and FGFR peptides were significantly more hydrophobic than the prion peptide and were insoluble in solvent A. To avoid reaggregation of these peptides after injection, the column was equilibrated at 94% solution A and 6% solution B. At this
FIG. 3. Comparison of the HPLC trace for (A) the crude prion (110 –137) peptide with that of (B) the purified prion peptide. Both traces used the gradient described in the legend to Fig. 2.
percentage of organic solvent, the peptides were soluble. A gradient of increasing solvent B at a rate of 2%/min eluted the pure glycophorin-A at 15% and the pure FGFR peptide at 16%. Mass spectral characterization of the synthetic peptides. Synthesis of the prion peptide was the least problematic. This peptide had the least number of b-branched amino acids and most likely adopts an extended structure in solution. It was also the least hydrophobic as indicated by its solubility in 0.1% TFA. The electrospray mass spectrum of this peptide (Fig. 4A) shows that the crude product was already fairly pure due to the optimal synthesis conditions employed. Purification on C1 resulted in a product that was .95% pure (Fig. 4B). Synthesis of glycophorin-A was more problematic due to the presence of 13 b-branched amino acids and its high helical propensity as well as its potential for oligomerization. The electrospray mass spectrum of the crude product showed three major products, with
PREPARATION OF INSOLUBLE TRANSMEMBRANE PEPTIDES
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FIG. 4. Electrospray mass spectra of (A) crude and (B) purified prion (110 –137) peptide for which the expected mass is 2533 amu. Both MH 1 and MNa 1 ions were observed.
FIG. 5. Electrospray mass spectra of (A) crude and (B) purified glycophorin-A peptide for which the expected mass is 3568 amu. Both MH 1 and MNa 1 ions were observed.
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FIG. 6. Electrospray mass spectra of (A) crude and (B) purified FGFR (368 –397) peptide for which the expected mass is 3347 amu. Both MH 1 and MNa 1 ions were observed.
the desired product representing less than 30% of the total (Fig. 5A). Purification of glycophorin-A required three critical developments. First, we discovered that lyophilization of the crude peptide from HFIP deaggregated the peptide and enabled dissolution of the peptide in 20% acetic acid. Second, use of the C1 column enabled efficient elution of the peptide compared to irreversible retention on C4. Finally, we identified an HPLC solvent system in which the peptide was soluble and yet was retained on the column. The resulting purified glycophorin-A was .95% pure as assessed by electrospray mass spectrometry (Fig. 5B). To assess the generality of the synthesis and purification schemes, we applied them to a third peptide, FGFR (368 –397). Comparison of the mass spectra of the crude (Fig. 6A) and purified peptide (Fig. 6B) shows that both the synthetic scheme and the purification scheme resulted in high yields of the desired product despite the presence of eight b-branched amino acids and a high predicted helical propensity. The methods presented here are therefore reliable and general for obtaining large quantities of highly purified long, insoluble transmembrane peptides.
ACKNOWLEDGMENTS We thank Jennifer A. Whiles and Jennifer A. Hauer for helpful discussions and Steven Berkus for laboratory assistance. We also thank Dr. Larry Gross (UCSD) for mass spectral assistance. K.J.G. is supported by an NIH Heme Training Grant.
REFERENCES 1. MacKenzie, K. R., Prestegard, J. H., and Engelman, D. M. (1997) Science 276, 131–133. 2. Vold, R. R., Prosser, R. S., and Deese, A. J. (1997) J. Biomol. NMR 9, 329 –335. 3. Lloyd, D. H., Petrie, G. M., Noble, R. L. N., and Tam, J. P. (1989) in Peptides: Chemistry, Structure and Biology (Rivier, J. E., and Marshall, G. R., Eds.), ESCOM, Leiden. 4. Rabinovich, A. K., and Rivier, J. E. (1994) in Peptides: Chemistry, Structure and Biology (Hodges, R. S., and Smith, J. A., Eds.), ESCOM, Leiden. 5. Tomich, J. M., Carson, L. W., Kanes, K. J., Vogelaar, N. J., Emerling, M. R., and Richards, J. H. (1988) Anal. Biochem. 174, 197–203. 6. Smith, S. O., Jonas, R., Braiman, M., and Bormann, B. J. (1994) Biochemistry 33, 6334 – 6341. 7. Lew, S., and London, E. (1997) Anal. Biochem. 251, 113–116.
Preparation of Insoluble Transmembrane Peptides: Glycophorin-A, Prion (110–137), and FGFR (368–397) Kerney Jebrell Glover, Paul M. Martini, Regitze R. Vold, and Elizabeth A. Komives 1 Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0601
Received April 23, 1999
A general procedure for the reliable preparation of large quantities of insoluble transmembrane peptides has been developed. Optimal couplings were obtained during synthesis by using high-temperature couplings in conjunction with O-(7-azabenzotriazol-1-yl)-1,1,3,3tetramethyluronium hexafluorophosphate (HATU) activation. Improved purification schemes were developed that use reverse- phase HPLC on a C1 column and elution with a 2:1 mixture of 1-propanol:1-butanol. Using these methods three very different transmembrane peptides all longer than 25 amino acids have been prepared: glycophorin-A, prion (110 –137), and fibroblast growth factor receptor (368 –397). © 1999 Academic Press
Transmembrane domains are involved in a variety of cellular processes including receptor dimerization and channel formation (1). Due to their extreme hydrophobicity, studies on these peptides have been hindered by their physical characteristics. Recent advancements in the field of “native-like” membrane environments have now made it possible to structurally characterize peptides derived from these domains using biophysical techniques (2). Reliable methods to prepare large quantities of highly purified transmembrane peptides are therefore needed. This requires both an efficient synthetic methodology and an effective general purification scheme. Standard synthesis methods for peptides are often inadequate for transmembrane peptides which contain high numbers of b-branched amino acids and have a strong propensity for secondary structure and for oligomerization. We present in this report the combined
use of an efficient activation chemistry utilizing O-(7azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) 2 combined with elevated synthesis temperatures (3, 4). Although there have been a number of reports describing the purification of individual hydrophobic peptides by HPLC, none has been widely applicable. The majority of these methods deal with peptides which are labeled as hydrophobic but in fact are fully soluble in aqueous buffers and can be purified using a C4 or C18 reverse-phase column in conjunction with an acetonitrile (or 2-propanol) gradient. The few papers which address extremely hydrophobic peptides often use harsh conditions which greatly shorten column life or utilize detergents which are difficult to remove from the purified peptide (5). One purification of glycophorin-A has been reported using a C4 column (6). This protocol called for a single step of greater than 80% organic solvent. Under these conditions the peptide was eluted but not effectively purified. Modification of this procedure to include authentic gradient elution resulted in excessive peak broadening which masked any separation. Here we present the reliable purification of long-chain insoluble hydrophobic peptides using a reverse-phase C1 column and an elution solvent composed of a 2:1 (v/v) mixture of 1-propanol:1-butanol. MATERIALS AND METHODS
Peptide synthesis. Peptides were synthesized on a Milligen 9050 peptide synthesizer using standard solid-phase methods. Synthesis was carried out at 50°C using a water-jacketed column and 60-min coupling cycles. All amino acids were free carboxylic acids with a fluorenyl-methoxycarbonyl (FMOC) blocker on the 2
1
To whom correspondence should be addressed. Fax: (619) 8220079. E-mail: [email protected].
270
Abbreviations used: HATU, O-(7-azabenzotriazol-1-yl)-1,1,3,3tetramethyluronium hexafluorophosphate; HFIP, hexafluoro-2-propanol; TFA, trifluoroacetic acid.
0003-2697/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
PREPARATION OF INSOLUBLE TRANSMEMBRANE PEPTIDES
271
Yields of these peptides suffered due to difficulties in coupling of b-branched amino acids and steric interactions of the growing chains. To overcome these difficulties, we have optimized several parameters which are essential for obtaining high yields with a minimal percentage of deletion products. A combination of HATU activation, 60-min couplings, and a synthesis temperFIG. 1. Peptide sequences of prion (110 –137), glycophorin-A, and FGFR (368 –397).
amine. HATU was used for carboxylic activation during coupling. The PAL-PEG-PS solid support, HATU, and side-chain-protected amino acids were obtained from PE Biosystems (Framingham, MA). OmniSolv glass distilled dimethylformamide (DMF) was from EM Sciences (distributed by VWR, Los Angeles, CA). After coupling of all amino acids, the amino terminus was acetylated using a solution of dimethylformamide (87%), dichloromethane (10%), acetic anhydride (3%), and hydroxyazabenzotriazole (12.2 g). The resin was then washed with dichloromethane and dried overnight under vacuum. Peptides were cleaved from the resin using trifluoroacetic acid (90%), thioanisole (5%), ethanedithiol (3%), and anisole (2%). Cleavage from the resin yielded an amide on the carboxyl terminus. The slurry was filtered through glass wool and the filtrate poured into cold diethyl ether to induce precipitation of the peptide. After overnight at 220°C, the precipitate was collected on a sintered glass funnel, dissolved in hexafluoro-2-propanol (HFIP), and lyophilized. Yield of crude peptides was about 90% on average for a 0.20-mmol synthesis scale. Peptide purification. Crude peptide was dissolved in a solution of 80% H 2O, 20% acetic acid with heating should be at 50°C. Before injection the solution was centrifuged for 2 min at 14,000 rpm to remove particulates. The mixture was injected onto an 80 Å, 10-mm Waters Spherisorb C1 column (4.6 3 150 mm) at a flow rate of 1.00 ml/min. The column was maintained at 50°C by immersion in a constant temperature circulating water bath. The peptides were eluted with various gradient mixtures composed of solvent A (0.1% TFA) and solvent B [2:1 (v/v) solution of 1-propanol:1-butanol]. Both solutions were filtered, degassed through 0.2-mm membranes, and continuously sparged with helium. For each peptide, the major peak was collected and lyophilized to yield the pure product in 60% yield. Gradient elution conditions for each peptide are described later. RESULTS AND DISCUSSION
Peptide synthesis. We have synthesized several different long, insoluble hydrophobic peptides (Fig. 1).
FIG. 2. HPLC separation of the crude, synthetic peptides. All peptides were isolated on a Waters Spherisorb C1 (4.6 3 150 mm) analytical reverse-phase HPLC column at a flow rate of 1.00 ml/min using a gradient. (A) The gradient for prion (110 –137) is 100% solution A for 5 min and 0 –20% 1-propanol:1-butanol (2:1, v/v) over 15 min, and the pure peptide has a retention time of 10.2 min. (B) The gradient for glycophorin-A is 94% solution A for 5 min and 6 –36% 1-propanol:1-butanol (2:1, v/v) over 15 min, and the purified peptide has a retention time of 9.6 min. (C) The gradient for FGFR (368 –397) is 94% solution A for 5 min and 6 –36% 1-propanol:1butanol (2:1, v/v) over 15 min, and the purified peptide has a retention time of 10.2 min.
272
GLOVER ET AL.
ature of 50°C resulted in reproducibly high yield of the correct product (Fig. 2). Peptide purification. It is critical to load the peptide onto the column in a deaggregated state. Lyophilization of the crude peptide after dissolution in HFIP was found to be an essential first step in deaggregation. Immediately before loading on the column, the peptides were dissolved in a solution of 80% H 2O/20% acetic acid which was found to be ideal for deaggregating the peptide without sacrificing initial adsorption to the column. In addition, this solution must be kept at 50°C prior to injection to be optimally effective. For purification of these peptides, use of standard reverse-phase HPLC conditions and a C4 or C18 column in conjunction with a water/acetonitrile gradient is inadequate. Their employment results in either irreversible column binding or unworkably broad peaks. One attempt at addressing this concern is the substitution of isopropanol for acetonitrile (7). While this increase in solvent strength may work for a few borderline hydrophobic peptides, it was unsuccessful at eluting our peptides. Instead we found that employment of a reverse-phase C1 column in conjunction with a long-chain alcohol elution solvent was ideal for the purification and high recovery of these peptides. The C1 column contains only a single carbon moiety covalently bonded to a silica backbone, and as a result it is one of the least hydrophobic reverse-phase columns available. The 2:1 1-propanol:1-butanol solution has an extremely hydrophobic nature while retaining water miscibility which is perfectly suited to the elution of these sticky peptides. However, due to the viscous nature of this solvent system, the column was run at 50°C to minimize backpressure while simultaneously increasing peptide solubility. The starting solvent for HPLC purification was varied for each peptide to achieve maximum adsorption to the column. The prion peptide was injected onto the column in 100% solvent A. Although the prion peptide is only sparingly soluble in this solution, the combined effect of injection in 80% H 2O/20% acetic acid and adsorption to the column keeps the peptide from precipitating out as well as reaggregating. The percentage of solvent B was increased at a rate of 1.3%/min with the pure peptide then eluting around 7% (Fig. 3A). Reinjection of the collected prion peptide showed that purification had indeed been achieved by this gradient elution (Fig. 3B). At this percentage of solvent B, the peptide is very soluble and could be readily lyophilized. The glycophorin-A and FGFR peptides were significantly more hydrophobic than the prion peptide and were insoluble in solvent A. To avoid reaggregation of these peptides after injection, the column was equilibrated at 94% solution A and 6% solution B. At this
FIG. 3. Comparison of the HPLC trace for (A) the crude prion (110 –137) peptide with that of (B) the purified prion peptide. Both traces used the gradient described in the legend to Fig. 2.
percentage of organic solvent, the peptides were soluble. A gradient of increasing solvent B at a rate of 2%/min eluted the pure glycophorin-A at 15% and the pure FGFR peptide at 16%. Mass spectral characterization of the synthetic peptides. Synthesis of the prion peptide was the least problematic. This peptide had the least number of b-branched amino acids and most likely adopts an extended structure in solution. It was also the least hydrophobic as indicated by its solubility in 0.1% TFA. The electrospray mass spectrum of this peptide (Fig. 4A) shows that the crude product was already fairly pure due to the optimal synthesis conditions employed. Purification on C1 resulted in a product that was .95% pure (Fig. 4B). Synthesis of glycophorin-A was more problematic due to the presence of 13 b-branched amino acids and its high helical propensity as well as its potential for oligomerization. The electrospray mass spectrum of the crude product showed three major products, with
PREPARATION OF INSOLUBLE TRANSMEMBRANE PEPTIDES
273
FIG. 4. Electrospray mass spectra of (A) crude and (B) purified prion (110 –137) peptide for which the expected mass is 2533 amu. Both MH 1 and MNa 1 ions were observed.
FIG. 5. Electrospray mass spectra of (A) crude and (B) purified glycophorin-A peptide for which the expected mass is 3568 amu. Both MH 1 and MNa 1 ions were observed.
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FIG. 6. Electrospray mass spectra of (A) crude and (B) purified FGFR (368 –397) peptide for which the expected mass is 3347 amu. Both MH 1 and MNa 1 ions were observed.
the desired product representing less than 30% of the total (Fig. 5A). Purification of glycophorin-A required three critical developments. First, we discovered that lyophilization of the crude peptide from HFIP deaggregated the peptide and enabled dissolution of the peptide in 20% acetic acid. Second, use of the C1 column enabled efficient elution of the peptide compared to irreversible retention on C4. Finally, we identified an HPLC solvent system in which the peptide was soluble and yet was retained on the column. The resulting purified glycophorin-A was .95% pure as assessed by electrospray mass spectrometry (Fig. 5B). To assess the generality of the synthesis and purification schemes, we applied them to a third peptide, FGFR (368 –397). Comparison of the mass spectra of the crude (Fig. 6A) and purified peptide (Fig. 6B) shows that both the synthetic scheme and the purification scheme resulted in high yields of the desired product despite the presence of eight b-branched amino acids and a high predicted helical propensity. The methods presented here are therefore reliable and general for obtaining large quantities of highly purified long, insoluble transmembrane peptides.
ACKNOWLEDGMENTS We thank Jennifer A. Whiles and Jennifer A. Hauer for helpful discussions and Steven Berkus for laboratory assistance. We also thank Dr. Larry Gross (UCSD) for mass spectral assistance. K.J.G. is supported by an NIH Heme Training Grant.
REFERENCES 1. MacKenzie, K. R., Prestegard, J. H., and Engelman, D. M. (1997) Science 276, 131–133. 2. Vold, R. R., Prosser, R. S., and Deese, A. J. (1997) J. Biomol. NMR 9, 329 –335. 3. Lloyd, D. H., Petrie, G. M., Noble, R. L. N., and Tam, J. P. (1989) in Peptides: Chemistry, Structure and Biology (Rivier, J. E., and Marshall, G. R., Eds.), ESCOM, Leiden. 4. Rabinovich, A. K., and Rivier, J. E. (1994) in Peptides: Chemistry, Structure and Biology (Hodges, R. S., and Smith, J. A., Eds.), ESCOM, Leiden. 5. Tomich, J. M., Carson, L. W., Kanes, K. J., Vogelaar, N. J., Emerling, M. R., and Richards, J. H. (1988) Anal. Biochem. 174, 197–203. 6. Smith, S. O., Jonas, R., Braiman, M., and Bormann, B. J. (1994) Biochemistry 33, 6334 – 6341. 7. Lew, S., and London, E. (1997) Anal. Biochem. 251, 113–116.