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Capillary zone electrophoresis for the analysis of peptides synthesized by recombinant DNA technology
Analytica Chimica Acta, 213 (1988) 215-219 Elsevier Science Publishers B.V., Amsterdam -
215 Printed in The Net,herlands
Short Communication
CAPILLARY ZONE ELECTROPHORESIS FOR THE ANALYSIS OF PEPTIDES SYNTHESIZED BY RECOMBINANT DNA TECHNOLOGY
H. LijDI* and E. GASSMANN Central Analytical Department, Ciba-Geigy Ltd., Base1 (Switzerland) H. GROSSENBACHER
and W. MARK1
Biotechnology Department, Ciba-Geigy Ltd., Base1 (Switzerland) (Received 10th March 1988)
Summary. Capillary zone electrophoresis is applied to investigate the recombinant insulin-like growth factor and recombinant hirudin. During the production of these peptides in S. cereuisiae, byproducts with small variations in the structure of the polypeptide chain are obtained. The different peptides are separated in a fused silica capillary and detected on-column by ultraviolet absorption or fluorescence. Separation times are lo-40 min. The excellent separation efficiencies obtained indicate that capillary zone electrophoresis is complementary to liquid chromatography in the analysis of these peptides.
Electrophoretic separations are powerful techniques for the analysis of biologically important macromolecules. Gel electrophoresis is a standard technique for this class of substances. The method is labour-intensive, but excellent resolution is achieved by combining it sequentially with other separation techniques. In contrast, the application of high-performance liquid chromatography (HPLC ) in the analysis of biomacromolecules is limited by slow mass transfer between mobile and stationary phases. In recent years, capillary zone electrophoresis (CZE) has proved to be powerful for the analysis of proteins [1,2]. The combination of electro-osmotic flow and the differences in mobility allow the detection of positively charged, neutral and negatively charged species passing through the same on-column detector. The absence of a hydrodynamic flow theoretically permits diffusionlimited band-broadening [ 11, leading to the reported outstanding separation efficiencies [ 2-51. Because of the small variations in the primary or secondary structure of peptides and proteins, only minute changes in the overall charge of the biomolecule are observed. To detect these differences in charge, a highresolution electrophoretic method is necessary. In this paper, CZE is used to separate peptides synthesized by recombinant DNA technology, in which the analysis of small variations in the structure is of fundamental interest.
0003-2670/88/$03.50
0 1988 Elsevier Science Publishers B.V.
216
Experimental Carbonic anhydrase (EC. 4.2.1.1) A and B, /?-lactogiobulin A, P-lactoglobulin B and sleep-inducing peptide (T1762; nine amino acids) were purchased from Sigma Chemical Company. Peptide (l-29aa) was synthesized by CibaGeigy. Both r-IGF I and r-hirudin are produced in S. cereuisiae; r-hirudin is a product of Ciba-Geigy and PLANTORGAN (Bad-Zwischenahn, F.R.G.). rIGF I is produced in collaboration with Chiron (Emeryville, CA). Chemicals were analytical grade and obtained either from Fluka or Merck. The fully automated apparatus is shown in Fig. 1 (cf. [ 31). The fused silica capillary column is 75-105 cm long (0.075 mm i.d. ). The sample is injected by using the electro-osmotic flow to transfer a small volume into the capillary. Injection is done with a Gilson autosampler (M221). First, the capillary and electrode are moved into the sample solution and high voltage is turned on for a short time, typically 5 s at 5 kV; then the electrode and capillary are moved back to the electrolyte vessel and the voltage (30 kV) is applied for electrophoresis. The electrical field is 250-300 V cm-l with a measured current of 15-30 PA. Ultraviolet absorption of substances is detected by using an oncolumn flow cell, which is connected with optical fibers to a spectrometer (Kratos Spectroflow 783). The principle is shown in the inset of Fig. 1. Alternatively, an on-column laser-induced fluorescence detector can be used with an intracavity frequency-doubled argon-ion laser (Spectra-Physics 2020,
piiiiiq
1gI
/ 1UV-Detector / rigzr-l
Fig. 1. A schematic diagram of the CZE apparatus. The dotted lines are connections to and from the computer. Inset is an enlarged view of the optical detection principle.
217
Mountain View, CA) at 257 nm (10 mW) as an excitation source. The filtered output of the laser is focused on an optical fiber which carries the excitation light to the on-column flow cell having a volume of about 0.5 nl. The resulting fluorescence is collected at right angles by a second optical fiber and measured with a monochromator/photomultiplier combination [ 31. The signal is integrated with a HP-3393A integrator, plotted on a chart recorder (Linear), digitized with an A/D converter (Keithley MM-195-A) and stored in a computer (HP-217), which also controls the autosampler and the high-voltage power supply (fug HCN 3535000). Results and discussion Mixtures of proteins, peptides and amino acids. Figures 2 and 3 demonstrate the speed and resolution obtained for the analysis of mixtures of different biological molecules. In Fig. 2, the theoretical plate numbers, calculated from peak width, are 680 000 for tryptophan, 620 000 for peptide (l-29aa) and 490 000 for the sleep-inducing peptide. The separation of carbonic anhydrase and P-lactoglobulin is shown in Fig. 3. As reported previously by Lauer and McManigill [2], the isoenzymes carbonic anhydrase A and B, as well as /3lactoglobulin A and /3-lactoglobulin B, can be separated by CZE. Figures 2 and 3 suggest that examination of small variations in the structure of peptides synthesized by recombinant DNA technology should be possible.
Fig. 2. Electropherogram: (A) tryptophan (0.001%); (B) peptide (l-29 aa; 0.05%); (C) sleepinducing peptide (0.02%). Electrolyte buffer, pH 8.90: 20 mM Tris/7.2 mM NazB,07/0.68 mM EDTA. Injection, 3 kV for 3 s; length of capillary, 100 cm (75 cm to the detector); voltage for electrophoresis, 300 V cm-’ at 18 PA, fluorescence detection, excitation at 257 nm, emission at 349 nm. Fig. 3. Electropherogram: (CA A&B) carbonic anhydrase A and B (0.08% ); (BL A) B-lactoglobulin A (0.08% ); (BL B) P-lactoglobulin B (0.08% ). Conditions as for Fig. 2, except for the buffer which was 30 mM TRICINE (N- [tris(hydroxymethyl)methyl]glycine) 15 mM NazB407/0.2 mM SDS (sodium dodecyl sulphate) at pH 8.22.
218
Recombinant insulin-like growth factor (r-IGF I). The IGF I is a peptide with a single chain of 70 amino acids. The structure and function have been described by Rinderknecht and Humbel [ 61. During the production of r-IGF I in S. cereuisiak, several variations of the molecule are obtained. As deduced from the results of fast-atom-bombardment mass spectrometry (FAB/MS) [ 71, the disulfide bonds in a r-IGF byproduct are between the cysteines 6-47 and 4852 instead of 6-48 and 47-52, as observed in IGF I (Fig. 4). The separation of the two molecules is shown in Fig. 5. It should be noted that an electrolyte buffer with a high pH (11.1) is necessary to avoid adsorption of r-IGF on the walls of the capillary. This high pH induces a strong electro-osmotic flow, which prevents the complete separation of the two compounds, when a standard capillary is used (Figs. 2 and 3). The resolution is improved by using a capillary with a length of 105 cm to the detector instead of 75 cm. The total length increased from 100 cm to 120 cm (Fig. 5). Recombinant hirudin. Hirudin, a thrombin inhibitor, is a single-chain peptide with 65 amino acids [8,9]. During the production of r-hirudin in S. cereuisiae several degradation products are obtained. Amino acid sequence analysis of the two main degradation products revealed that amino acid 65 (glutamine) and subsequently amino acid 64 (leucine) are cleaved from the C-terminus [lo]. Although neutral amino acids are removed from r-hirudin, the resulting difference in the overall charge is sufficient to separate r-hirudin (l-65 aa) from r-hirudin (l-64 aa) and from r-hirudin (l-63 aa) (Fig. 6)) besides several other unknown byproducts. The separation of peptides shown in Figs. 5 and 6 was repeated using reversed-phase HPLC (not shown). The resolution obtained with CZE is com-
r-IGFI
r - IGF Byproduct
Fig. 4. Schematic view of the structures of r-IGF I and r-IgF byproduct.
219
B
2
4
6
B TIms
IB [In,“,
12
14
16
18
20
0
5
10
15
28 T,rnc
25
30
35
40
45
cm\n,
Fig. 5. Electropherogram: (A) r-IGF byproduct (0.1%); (B) r-IGF I (0.1%). The irregularities in the baseline between 14 and 20 min are due to the buffer used for the stock solutions of the peptides (20 mM TRICINE/lO mM Na2B,0,/1 mM EDTA, pH 8.2). Electrolyte buffer, pH 11.1: 10 mM CAPS (3-cyclohexylamino-1-propanesulfonicacid)/5 mM NazB107/l mM EDTA. Injection, 10 kV for 10 s; length of capillary, 120 cm (105 cm to the detector); voltage for electrophoresis, 250 V cm-’ at 25 @; detection at 215 nm. Fig. 6. Electropherogram: (65 aa) r-hirudin (1-65 aa; 0.15%); (64 aa) r-hirudin (l-64 aa; 0.15%); (63 aa) r-hirudin (l-63 aa; 0.15%); (TRP) tryptophan (0.01%). Other byproducts observed between 32 and 43 min are of unknown structure. Electrolyte buffer, pH 6.7: 16.7 mM PIPES [ 1,4-piperazinebis(ethanesulfonic acid) ] /12 mM Na,B107/l mM EDTA. Injection, 10 kV for 10 s; length of capillary, 100 cm (75 cm to detector); voltage for electrophoresis, 300 V cm-’ at 26 @; detection at 215 nm.
parable to that of HPLC. However, a higher detection limit is obtained in CZE, because of the small volumes involved. An on-column detector with improved sensitivity has already been described [ 111.Therefore CZE is a powerful technique which is complementary to HPLC for the analysis of peptides synthesized by recombinant DNA technology. Further applications include the characterization of new batches, quality control and identification of byproducts. REFERENCES
8 9 10 11
J.W. Jorgensen and K.D. Lukacs, Science, 222 (1983) 266. H.H. Lauer and D. McManigill, Anal. Chem., 58 (1986) 166. P. Gozel, E. Gassmann, H. Michelsen and R.N. Zare, Anal. Chem., 59 (1987) 44. S. Terabe, K. Otauka and T. Ando, Anal. Chem., 57 (1985) 834. R.E.P. Mikkers, F.M. Evaeraerts andTh.P.E.M. Verheegen, J. Chromatogr., 169 (1979) 11. E. Rinderknecht and R.E. Humbel, J. Biol. Chem., 253 (1978) 2769. F. Raschdorf, R. Dahinden, W. Miirki, J.P. Merryweather and W.J. Richter, 192nd ACS National Meeting, Anaheim, CA, 1986. J. Dodt, H.-P. Mtiller, U. Seemtiler and J.-Y. Chang, FEBS L&t., 165 (1984) 180. J. Dodt, U. Seemtiler, R. Maschler and H. Fritz, Biol. Chem. Hoppe-Seyler, 366 (1985) 379. H. Grossenbacher, J.A.L. Auden, K. Bill, M. Liersch, W. MBirkiand R. Maschler, Thromb. Res., VII (Suppl.) (1987) 34. T.G. Nolan and N.J. Dovichi, Anal. Chem., 59 (1987) 2803.
215 Printed in The Net,herlands
Short Communication
CAPILLARY ZONE ELECTROPHORESIS FOR THE ANALYSIS OF PEPTIDES SYNTHESIZED BY RECOMBINANT DNA TECHNOLOGY
H. LijDI* and E. GASSMANN Central Analytical Department, Ciba-Geigy Ltd., Base1 (Switzerland) H. GROSSENBACHER
and W. MARK1
Biotechnology Department, Ciba-Geigy Ltd., Base1 (Switzerland) (Received 10th March 1988)
Summary. Capillary zone electrophoresis is applied to investigate the recombinant insulin-like growth factor and recombinant hirudin. During the production of these peptides in S. cereuisiae, byproducts with small variations in the structure of the polypeptide chain are obtained. The different peptides are separated in a fused silica capillary and detected on-column by ultraviolet absorption or fluorescence. Separation times are lo-40 min. The excellent separation efficiencies obtained indicate that capillary zone electrophoresis is complementary to liquid chromatography in the analysis of these peptides.
Electrophoretic separations are powerful techniques for the analysis of biologically important macromolecules. Gel electrophoresis is a standard technique for this class of substances. The method is labour-intensive, but excellent resolution is achieved by combining it sequentially with other separation techniques. In contrast, the application of high-performance liquid chromatography (HPLC ) in the analysis of biomacromolecules is limited by slow mass transfer between mobile and stationary phases. In recent years, capillary zone electrophoresis (CZE) has proved to be powerful for the analysis of proteins [1,2]. The combination of electro-osmotic flow and the differences in mobility allow the detection of positively charged, neutral and negatively charged species passing through the same on-column detector. The absence of a hydrodynamic flow theoretically permits diffusionlimited band-broadening [ 11, leading to the reported outstanding separation efficiencies [ 2-51. Because of the small variations in the primary or secondary structure of peptides and proteins, only minute changes in the overall charge of the biomolecule are observed. To detect these differences in charge, a highresolution electrophoretic method is necessary. In this paper, CZE is used to separate peptides synthesized by recombinant DNA technology, in which the analysis of small variations in the structure is of fundamental interest.
0003-2670/88/$03.50
0 1988 Elsevier Science Publishers B.V.
216
Experimental Carbonic anhydrase (EC. 4.2.1.1) A and B, /?-lactogiobulin A, P-lactoglobulin B and sleep-inducing peptide (T1762; nine amino acids) were purchased from Sigma Chemical Company. Peptide (l-29aa) was synthesized by CibaGeigy. Both r-IGF I and r-hirudin are produced in S. cereuisiae; r-hirudin is a product of Ciba-Geigy and PLANTORGAN (Bad-Zwischenahn, F.R.G.). rIGF I is produced in collaboration with Chiron (Emeryville, CA). Chemicals were analytical grade and obtained either from Fluka or Merck. The fully automated apparatus is shown in Fig. 1 (cf. [ 31). The fused silica capillary column is 75-105 cm long (0.075 mm i.d. ). The sample is injected by using the electro-osmotic flow to transfer a small volume into the capillary. Injection is done with a Gilson autosampler (M221). First, the capillary and electrode are moved into the sample solution and high voltage is turned on for a short time, typically 5 s at 5 kV; then the electrode and capillary are moved back to the electrolyte vessel and the voltage (30 kV) is applied for electrophoresis. The electrical field is 250-300 V cm-l with a measured current of 15-30 PA. Ultraviolet absorption of substances is detected by using an oncolumn flow cell, which is connected with optical fibers to a spectrometer (Kratos Spectroflow 783). The principle is shown in the inset of Fig. 1. Alternatively, an on-column laser-induced fluorescence detector can be used with an intracavity frequency-doubled argon-ion laser (Spectra-Physics 2020,
piiiiiq
1gI
/ 1UV-Detector / rigzr-l
Fig. 1. A schematic diagram of the CZE apparatus. The dotted lines are connections to and from the computer. Inset is an enlarged view of the optical detection principle.
217
Mountain View, CA) at 257 nm (10 mW) as an excitation source. The filtered output of the laser is focused on an optical fiber which carries the excitation light to the on-column flow cell having a volume of about 0.5 nl. The resulting fluorescence is collected at right angles by a second optical fiber and measured with a monochromator/photomultiplier combination [ 31. The signal is integrated with a HP-3393A integrator, plotted on a chart recorder (Linear), digitized with an A/D converter (Keithley MM-195-A) and stored in a computer (HP-217), which also controls the autosampler and the high-voltage power supply (fug HCN 3535000). Results and discussion Mixtures of proteins, peptides and amino acids. Figures 2 and 3 demonstrate the speed and resolution obtained for the analysis of mixtures of different biological molecules. In Fig. 2, the theoretical plate numbers, calculated from peak width, are 680 000 for tryptophan, 620 000 for peptide (l-29aa) and 490 000 for the sleep-inducing peptide. The separation of carbonic anhydrase and P-lactoglobulin is shown in Fig. 3. As reported previously by Lauer and McManigill [2], the isoenzymes carbonic anhydrase A and B, as well as /3lactoglobulin A and /3-lactoglobulin B, can be separated by CZE. Figures 2 and 3 suggest that examination of small variations in the structure of peptides synthesized by recombinant DNA technology should be possible.
Fig. 2. Electropherogram: (A) tryptophan (0.001%); (B) peptide (l-29 aa; 0.05%); (C) sleepinducing peptide (0.02%). Electrolyte buffer, pH 8.90: 20 mM Tris/7.2 mM NazB,07/0.68 mM EDTA. Injection, 3 kV for 3 s; length of capillary, 100 cm (75 cm to the detector); voltage for electrophoresis, 300 V cm-’ at 18 PA, fluorescence detection, excitation at 257 nm, emission at 349 nm. Fig. 3. Electropherogram: (CA A&B) carbonic anhydrase A and B (0.08% ); (BL A) B-lactoglobulin A (0.08% ); (BL B) P-lactoglobulin B (0.08% ). Conditions as for Fig. 2, except for the buffer which was 30 mM TRICINE (N- [tris(hydroxymethyl)methyl]glycine) 15 mM NazB407/0.2 mM SDS (sodium dodecyl sulphate) at pH 8.22.
218
Recombinant insulin-like growth factor (r-IGF I). The IGF I is a peptide with a single chain of 70 amino acids. The structure and function have been described by Rinderknecht and Humbel [ 61. During the production of r-IGF I in S. cereuisiak, several variations of the molecule are obtained. As deduced from the results of fast-atom-bombardment mass spectrometry (FAB/MS) [ 71, the disulfide bonds in a r-IGF byproduct are between the cysteines 6-47 and 4852 instead of 6-48 and 47-52, as observed in IGF I (Fig. 4). The separation of the two molecules is shown in Fig. 5. It should be noted that an electrolyte buffer with a high pH (11.1) is necessary to avoid adsorption of r-IGF on the walls of the capillary. This high pH induces a strong electro-osmotic flow, which prevents the complete separation of the two compounds, when a standard capillary is used (Figs. 2 and 3). The resolution is improved by using a capillary with a length of 105 cm to the detector instead of 75 cm. The total length increased from 100 cm to 120 cm (Fig. 5). Recombinant hirudin. Hirudin, a thrombin inhibitor, is a single-chain peptide with 65 amino acids [8,9]. During the production of r-hirudin in S. cereuisiae several degradation products are obtained. Amino acid sequence analysis of the two main degradation products revealed that amino acid 65 (glutamine) and subsequently amino acid 64 (leucine) are cleaved from the C-terminus [lo]. Although neutral amino acids are removed from r-hirudin, the resulting difference in the overall charge is sufficient to separate r-hirudin (l-65 aa) from r-hirudin (l-64 aa) and from r-hirudin (l-63 aa) (Fig. 6)) besides several other unknown byproducts. The separation of peptides shown in Figs. 5 and 6 was repeated using reversed-phase HPLC (not shown). The resolution obtained with CZE is com-
r-IGFI
r - IGF Byproduct
Fig. 4. Schematic view of the structures of r-IGF I and r-IgF byproduct.
219
B
2
4
6
B TIms
IB [In,“,
12
14
16
18
20
0
5
10
15
28 T,rnc
25
30
35
40
45
cm\n,
Fig. 5. Electropherogram: (A) r-IGF byproduct (0.1%); (B) r-IGF I (0.1%). The irregularities in the baseline between 14 and 20 min are due to the buffer used for the stock solutions of the peptides (20 mM TRICINE/lO mM Na2B,0,/1 mM EDTA, pH 8.2). Electrolyte buffer, pH 11.1: 10 mM CAPS (3-cyclohexylamino-1-propanesulfonicacid)/5 mM NazB107/l mM EDTA. Injection, 10 kV for 10 s; length of capillary, 120 cm (105 cm to the detector); voltage for electrophoresis, 250 V cm-’ at 25 @; detection at 215 nm. Fig. 6. Electropherogram: (65 aa) r-hirudin (1-65 aa; 0.15%); (64 aa) r-hirudin (l-64 aa; 0.15%); (63 aa) r-hirudin (l-63 aa; 0.15%); (TRP) tryptophan (0.01%). Other byproducts observed between 32 and 43 min are of unknown structure. Electrolyte buffer, pH 6.7: 16.7 mM PIPES [ 1,4-piperazinebis(ethanesulfonic acid) ] /12 mM Na,B107/l mM EDTA. Injection, 10 kV for 10 s; length of capillary, 100 cm (75 cm to detector); voltage for electrophoresis, 300 V cm-’ at 26 @; detection at 215 nm.
parable to that of HPLC. However, a higher detection limit is obtained in CZE, because of the small volumes involved. An on-column detector with improved sensitivity has already been described [ 111.Therefore CZE is a powerful technique which is complementary to HPLC for the analysis of peptides synthesized by recombinant DNA technology. Further applications include the characterization of new batches, quality control and identification of byproducts. REFERENCES
8 9 10 11
J.W. Jorgensen and K.D. Lukacs, Science, 222 (1983) 266. H.H. Lauer and D. McManigill, Anal. Chem., 58 (1986) 166. P. Gozel, E. Gassmann, H. Michelsen and R.N. Zare, Anal. Chem., 59 (1987) 44. S. Terabe, K. Otauka and T. Ando, Anal. Chem., 57 (1985) 834. R.E.P. Mikkers, F.M. Evaeraerts andTh.P.E.M. Verheegen, J. Chromatogr., 169 (1979) 11. E. Rinderknecht and R.E. Humbel, J. Biol. Chem., 253 (1978) 2769. F. Raschdorf, R. Dahinden, W. Miirki, J.P. Merryweather and W.J. Richter, 192nd ACS National Meeting, Anaheim, CA, 1986. J. Dodt, H.-P. Mtiller, U. Seemtiler and J.-Y. Chang, FEBS L&t., 165 (1984) 180. J. Dodt, U. Seemtiler, R. Maschler and H. Fritz, Biol. Chem. Hoppe-Seyler, 366 (1985) 379. H. Grossenbacher, J.A.L. Auden, K. Bill, M. Liersch, W. MBirkiand R. Maschler, Thromb. Res., VII (Suppl.) (1987) 34. T.G. Nolan and N.J. Dovichi, Anal. Chem., 59 (1987) 2803.