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Separation of phenylthiohydantoin-amino acids by high-pressure liquid chromatography for sequence determination of radiolabeled proteins
ANALYTICAL
BIOCHEMISTRY
87, 66-70
(1978)
Separation of Phenylthiohydantoin-Amino High-Pressure Liquid Chromatography Sequence Determination of Radiolabeled Proteins ANNE
DEVILLERS-THIERY
The Rockefeller
University
Acids by for
AND GCJNTER BLOBEL New
York,
NeM,
York
10021
Received August 29, 1977; accepted January 20, 1978 Published procedures for the separation of ethyl acetate-extractable phenylthiohydantoin-amino acids by high-pressure liquid chromatography with organic solvent gradients have been modified to facilitate sequence determination of proteins labeled with several radioactive amino acids at a time.
Several laboratories have recently determined the amino-terminal sequence of proteins which had been synthesized in vitro by translation of mRNA in a cell-free system (l-9). In most cases, translation was in the presence of 19 cold amino acids and a single radioactive amino acid. The radiolabeled protein was then subjected to consecutive Edman degradations. Discrete peaks of radioactivity were released from the radiolabeled protein after certain of the cycles of the Edman degradation. However, to determine amino acid residues for all of the positions in an amino-terminal sequence (e.g., of up to 50 residues in length) by this procedure may require as many as 20 separate syntheses, each of them with a different radioactive amino acid. An alternative approach has been taken (1) by translating mRNA in the presence of as many as 18 radioactive amino acids and by separating the phenylthiohydantoin (PTH) derivatives resulting from each Edman degradation by thin-layer chromatography. However, this approach was not wholly satisfactory, primarily because of difficulties in quantitating the data. Recently high-pressure liquid chromatography (HPLC) has been introduced as an alternative to thin-layer or gas chromatography, commonly used for the analysis of PTHs. In this paper we describe our modifications of published procedures (10,ll) which resulted in an improved separation of PTHs.
0003-26971781087 Copyright All rights
l-0066$02.00/0
0 1978 by Academic Press. Inc. of reproduction in any form reserved.
66
PHENYLTHIOHYDANTOIN-AMINO
METHODS
ACIDS
SEPARATION
67
AND REAGENTS
High-pressure liquid chromatography. Analyses were performed with a high-pressure liquid chromatography system (Waters Associates, ALC/ GPC 204 model) equipped with a solvent programmer (model 660) and U6K injector. Separation of PTHs was carried out at room temperature on IO-pm silica beads packed in a stainless steel tube (300-mm x 3.9-mm i.d.), available from Waters Associates (PPorasil P/N 27477). Samples in 25 ~1 of ethyl acetate were injected into the injection loop before the column, without release of pressure in the column, and were eluted at 500 psi by a gradient of organic solvents. Two sets of conditions were used. Under Condition 1, Solvent A was isooctane and Solvent B was a mixture of methanol:2-propanol (9:ll). A 20-min concave gradient (Curve 8 of solvent programmer) from 5 to 70% of Solvent B with a flow rate of 1.5 ml/min was used. Under Condition 2, Solvent A was a mixture of isooctane:chloroform (7:3) and Solvent B was methanol: isooctane:chloroform (15:42.5:42.5). A 20-min concave gradient (Curve 9 of solvent programmer) from 1 to 100% of Solvent B with a flow rate of 1.5 mlimin was used. Eluted PTHs were detected by their uv absorption at 254 nm in a lo-p.1 flow cell. At the end of elution, final conditions were run for 5 to 10 min, the gradient was then reversed to initial conditions in 5 min, and the column was allowed to reequilibrate to initial conditions for another 5 min. Reagents. Ethyl acetate, methanol, 2-propanol, chloroform, and isooctane (2,2,4-trimethylpentane) were purchased from Burdick and Jackson, Muskegon, Mich. PTH-amino acids were from Pierce, Rockford, Ill. RESULTS
Several procedures have been published for the separation of PTHs by HPLC. Zimmerman et al. (12) were able to separate all 20 PTHs in a single run in less than 20 min using a gradient with a mixture of aqueous and organic solvents and a temperature of 62°C for elution. This elegant procedure, however, presents problems when subsequent counting of each of the collected PTHs is required, as is the case in sequencing of radiolabeled proteins. Since each PTH is eluted and collected with a different amount of aqueous solvent, counting would require liquid scintillation cocktails accommodating water, and appropriate quench corrections. To eliminate these problems it would be desirable to separate PTHs with gradients of organic solvents; these can be evaporated, leaving behind the PTHs as a solid and thus simplifying subsequent counting procedures. HPLC procedures for the separation of ethyl acetateextractable PTHs using organic solvent gradients have been published. In the procedure of Matthews et al. (10) a silica column kept at 40°C and a hexane-methanol-Zpropanol gradient has been used for separation of
68
DEVILLERS-THIERY
AND BLOBEL
/
1
‘;
MINUTES
FIG. 1. Elution profile of ethyl acetate-extractable (see Methods and Reagents section).
FTH-amino
acids under Condition 1
PTHs in 40 min. We modified this method and achieved an improved separation in 20 min and at room temperature with a gradient of isooctane-methanol-2-propanol. Figure 1 shows the elution profile of ethyl acetate-extractable PTHs. It can be seen that some PTHs coelute as one peak (Leu-Ile, Phe-Met, and Glu-Asp) while others are not separated from each other to baseline (Val-Pro, Gly-Thr-Trp, and Ser-Tyr). Using the same column but a modified gradient of isooctane-chloroform-methanol (11) yielded an elution profile of ethyl acetate-extractable PTHs shown in Fig. 2. Under these conditions Pro, Leu, Ile, Val, and Ser are now clearly separated. However, Trp and Lys coelute as one peak which in turn is not separated to baseline from Gly. Furthermore Tyr-Thr and Met-Phe are not separated from each other to baseline. Again the acidic residues are poorly resolved and may spill over into the Gln and Asn peaks. DISCUSSION
High-sensitivity sequencing of radiolabeled proteins containing several radioactive amino acid residues requires separation of PTHs and subsequent liquid scintillation counting for identification and quantitation. Neither gas chromatography nor amino acid analysis lend themselves readily to simultaneous or subsequent liquid scintillation counting. Thin-layer chroma-
PHENYLTHIOHYDANTOIN-AMINO
ACIDS SEPARATION
N I-
,-
I;
F 0 MINUTES
FIG. 2. Elution profile of ethyl acetate-extractable (see Methods and Reagents section).
PTH-amino
acids under Condition 2
tography and in particular the recently introduced HPLC, on the other hand, yield fractions which can be counted readily. In this paper we report modifications of published procedures for the separation of PTHs using HPLC. Since separation was performed with gradients of organic solvents, it is possible to evaporate the solvent; liquid scintillation counting of the remaining dried ITH fraction simplifies subsequent counting procedures by eliminating the need for elaborate quench correction schemes due to the presence of different quantities of solvents in each PTH fraction. In those cases where PTHs coelute as a pair or where they are not separated from each other to baseline, synthesis of the protein with isotopically different amino acids (35Sor IT vs 3H) and subsequent detection by double-label counting procedures can be used. Alternatively, it is possible to synthesize protein with one radioactive amino acid of an unresolved pair at a time. Application of these procedures should permit sequence determination of proteins synthesized with several radiolabeled
70
DEVILLERS-THIERY
AND BLOBEL
amino acids at a time and should therefore require only a few rounds of synthesis to assign residues to all positions. ACKNOWLEDGMENTS We thank H. Desruisseaux for her technical assistance. This work was supported by grant CA 12413 from the National Cancer Institute, Public Health Service, Department of Health, Education, and Welfare.
REFERENCES 1. Devillers-Thiery, A., Kindt, T., Scheele, G., Blobel, G. (1975) Proc. Nat. Acad. Sci. USA 72, 5016-5020. 2. Burstein, Y., and Schechter, I. (1975) Proc. Nat. Acad. Sci. USA 74, 716-720. 3. Kemper, B., Habener, J. F., Ernst, M. D., Potts, J. T., and Rich, A. (1976) Biochemistry 15, 15-19.
4. Chan, S. J., Keim, P., and Steiner, D. F. (1976) Proc. Nat. Acad. Sci. USA 73, 19641968. 5. Maurer, R. A., Gorski, J., and McKean, D. J. (1977) Biochem. J. 161, 189-192. 6. Inouye, S., Wang, S., Sekizawa, J., Halegoua, S., and Inouye, M. (1977) Proc. Nat. Acad. Sci. USA 74, 1004-1008. 7. Strauss, A. W., Donohue, A. M., Bennett, C. D., Rodkey, J. A., and Albert% A. W. (1977) Proc. Nat. Acad. Sci. USA 74, 1358-1362. 8. Yu, S., and Redman, C. (1977) Biochem. Biophys. Res. Commun. 76,469-476. 9. Shields, D., and Blobel, G. (1977) Proc. Nat. Acad. Sci. USA 74, 2059-2063. 10. Matthews, E. W., Byfield, P. G. H., and McIntyre, I. (197S)J. Chromatogr. 110, 369373.
11. Bridgen, J., Graffeo, A. P., Karger, B. L., and Waterfield, M. D. (1975) in Instrumentation in Amino Acid Sequence Analysis (Perham, R. N., ed.), p. 140, Academic Press, London. 12. Zimmerman, C. L., Appella, E., and Pisano, J. J. (1977) Anal. Biochem. 77, 569-573.
BIOCHEMISTRY
87, 66-70
(1978)
Separation of Phenylthiohydantoin-Amino High-Pressure Liquid Chromatography Sequence Determination of Radiolabeled Proteins ANNE
DEVILLERS-THIERY
The Rockefeller
University
Acids by for
AND GCJNTER BLOBEL New
York,
NeM,
York
10021
Received August 29, 1977; accepted January 20, 1978 Published procedures for the separation of ethyl acetate-extractable phenylthiohydantoin-amino acids by high-pressure liquid chromatography with organic solvent gradients have been modified to facilitate sequence determination of proteins labeled with several radioactive amino acids at a time.
Several laboratories have recently determined the amino-terminal sequence of proteins which had been synthesized in vitro by translation of mRNA in a cell-free system (l-9). In most cases, translation was in the presence of 19 cold amino acids and a single radioactive amino acid. The radiolabeled protein was then subjected to consecutive Edman degradations. Discrete peaks of radioactivity were released from the radiolabeled protein after certain of the cycles of the Edman degradation. However, to determine amino acid residues for all of the positions in an amino-terminal sequence (e.g., of up to 50 residues in length) by this procedure may require as many as 20 separate syntheses, each of them with a different radioactive amino acid. An alternative approach has been taken (1) by translating mRNA in the presence of as many as 18 radioactive amino acids and by separating the phenylthiohydantoin (PTH) derivatives resulting from each Edman degradation by thin-layer chromatography. However, this approach was not wholly satisfactory, primarily because of difficulties in quantitating the data. Recently high-pressure liquid chromatography (HPLC) has been introduced as an alternative to thin-layer or gas chromatography, commonly used for the analysis of PTHs. In this paper we describe our modifications of published procedures (10,ll) which resulted in an improved separation of PTHs.
0003-26971781087 Copyright All rights
l-0066$02.00/0
0 1978 by Academic Press. Inc. of reproduction in any form reserved.
66
PHENYLTHIOHYDANTOIN-AMINO
METHODS
ACIDS
SEPARATION
67
AND REAGENTS
High-pressure liquid chromatography. Analyses were performed with a high-pressure liquid chromatography system (Waters Associates, ALC/ GPC 204 model) equipped with a solvent programmer (model 660) and U6K injector. Separation of PTHs was carried out at room temperature on IO-pm silica beads packed in a stainless steel tube (300-mm x 3.9-mm i.d.), available from Waters Associates (PPorasil P/N 27477). Samples in 25 ~1 of ethyl acetate were injected into the injection loop before the column, without release of pressure in the column, and were eluted at 500 psi by a gradient of organic solvents. Two sets of conditions were used. Under Condition 1, Solvent A was isooctane and Solvent B was a mixture of methanol:2-propanol (9:ll). A 20-min concave gradient (Curve 8 of solvent programmer) from 5 to 70% of Solvent B with a flow rate of 1.5 ml/min was used. Under Condition 2, Solvent A was a mixture of isooctane:chloroform (7:3) and Solvent B was methanol: isooctane:chloroform (15:42.5:42.5). A 20-min concave gradient (Curve 9 of solvent programmer) from 1 to 100% of Solvent B with a flow rate of 1.5 mlimin was used. Eluted PTHs were detected by their uv absorption at 254 nm in a lo-p.1 flow cell. At the end of elution, final conditions were run for 5 to 10 min, the gradient was then reversed to initial conditions in 5 min, and the column was allowed to reequilibrate to initial conditions for another 5 min. Reagents. Ethyl acetate, methanol, 2-propanol, chloroform, and isooctane (2,2,4-trimethylpentane) were purchased from Burdick and Jackson, Muskegon, Mich. PTH-amino acids were from Pierce, Rockford, Ill. RESULTS
Several procedures have been published for the separation of PTHs by HPLC. Zimmerman et al. (12) were able to separate all 20 PTHs in a single run in less than 20 min using a gradient with a mixture of aqueous and organic solvents and a temperature of 62°C for elution. This elegant procedure, however, presents problems when subsequent counting of each of the collected PTHs is required, as is the case in sequencing of radiolabeled proteins. Since each PTH is eluted and collected with a different amount of aqueous solvent, counting would require liquid scintillation cocktails accommodating water, and appropriate quench corrections. To eliminate these problems it would be desirable to separate PTHs with gradients of organic solvents; these can be evaporated, leaving behind the PTHs as a solid and thus simplifying subsequent counting procedures. HPLC procedures for the separation of ethyl acetateextractable PTHs using organic solvent gradients have been published. In the procedure of Matthews et al. (10) a silica column kept at 40°C and a hexane-methanol-Zpropanol gradient has been used for separation of
68
DEVILLERS-THIERY
AND BLOBEL
/
1
‘;
MINUTES
FIG. 1. Elution profile of ethyl acetate-extractable (see Methods and Reagents section).
FTH-amino
acids under Condition 1
PTHs in 40 min. We modified this method and achieved an improved separation in 20 min and at room temperature with a gradient of isooctane-methanol-2-propanol. Figure 1 shows the elution profile of ethyl acetate-extractable PTHs. It can be seen that some PTHs coelute as one peak (Leu-Ile, Phe-Met, and Glu-Asp) while others are not separated from each other to baseline (Val-Pro, Gly-Thr-Trp, and Ser-Tyr). Using the same column but a modified gradient of isooctane-chloroform-methanol (11) yielded an elution profile of ethyl acetate-extractable PTHs shown in Fig. 2. Under these conditions Pro, Leu, Ile, Val, and Ser are now clearly separated. However, Trp and Lys coelute as one peak which in turn is not separated to baseline from Gly. Furthermore Tyr-Thr and Met-Phe are not separated from each other to baseline. Again the acidic residues are poorly resolved and may spill over into the Gln and Asn peaks. DISCUSSION
High-sensitivity sequencing of radiolabeled proteins containing several radioactive amino acid residues requires separation of PTHs and subsequent liquid scintillation counting for identification and quantitation. Neither gas chromatography nor amino acid analysis lend themselves readily to simultaneous or subsequent liquid scintillation counting. Thin-layer chroma-
PHENYLTHIOHYDANTOIN-AMINO
ACIDS SEPARATION
N I-
,-
I;
F 0 MINUTES
FIG. 2. Elution profile of ethyl acetate-extractable (see Methods and Reagents section).
PTH-amino
acids under Condition 2
tography and in particular the recently introduced HPLC, on the other hand, yield fractions which can be counted readily. In this paper we report modifications of published procedures for the separation of PTHs using HPLC. Since separation was performed with gradients of organic solvents, it is possible to evaporate the solvent; liquid scintillation counting of the remaining dried ITH fraction simplifies subsequent counting procedures by eliminating the need for elaborate quench correction schemes due to the presence of different quantities of solvents in each PTH fraction. In those cases where PTHs coelute as a pair or where they are not separated from each other to baseline, synthesis of the protein with isotopically different amino acids (35Sor IT vs 3H) and subsequent detection by double-label counting procedures can be used. Alternatively, it is possible to synthesize protein with one radioactive amino acid of an unresolved pair at a time. Application of these procedures should permit sequence determination of proteins synthesized with several radiolabeled
70
DEVILLERS-THIERY
AND BLOBEL
amino acids at a time and should therefore require only a few rounds of synthesis to assign residues to all positions. ACKNOWLEDGMENTS We thank H. Desruisseaux for her technical assistance. This work was supported by grant CA 12413 from the National Cancer Institute, Public Health Service, Department of Health, Education, and Welfare.
REFERENCES 1. Devillers-Thiery, A., Kindt, T., Scheele, G., Blobel, G. (1975) Proc. Nat. Acad. Sci. USA 72, 5016-5020. 2. Burstein, Y., and Schechter, I. (1975) Proc. Nat. Acad. Sci. USA 74, 716-720. 3. Kemper, B., Habener, J. F., Ernst, M. D., Potts, J. T., and Rich, A. (1976) Biochemistry 15, 15-19.
4. Chan, S. J., Keim, P., and Steiner, D. F. (1976) Proc. Nat. Acad. Sci. USA 73, 19641968. 5. Maurer, R. A., Gorski, J., and McKean, D. J. (1977) Biochem. J. 161, 189-192. 6. Inouye, S., Wang, S., Sekizawa, J., Halegoua, S., and Inouye, M. (1977) Proc. Nat. Acad. Sci. USA 74, 1004-1008. 7. Strauss, A. W., Donohue, A. M., Bennett, C. D., Rodkey, J. A., and Albert% A. W. (1977) Proc. Nat. Acad. Sci. USA 74, 1358-1362. 8. Yu, S., and Redman, C. (1977) Biochem. Biophys. Res. Commun. 76,469-476. 9. Shields, D., and Blobel, G. (1977) Proc. Nat. Acad. Sci. USA 74, 2059-2063. 10. Matthews, E. W., Byfield, P. G. H., and McIntyre, I. (197S)J. Chromatogr. 110, 369373.
11. Bridgen, J., Graffeo, A. P., Karger, B. L., and Waterfield, M. D. (1975) in Instrumentation in Amino Acid Sequence Analysis (Perham, R. N., ed.), p. 140, Academic Press, London. 12. Zimmerman, C. L., Appella, E., and Pisano, J. J. (1977) Anal. Biochem. 77, 569-573.