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Molecular weight determination of peptides by high-performance gel permeation chromatography
ANALYTICAL
BIOCHEMISTRY
Molecular
133,288-291
(1983)
Weight Determination Gel Permeation W.O.
of Peptides by High-Performance Chromatography’
RICHTER,B.JACOB,ANDP.
SCHWANDT
Department of Internal Medicine II, University of Munich, Klinikum Grosshadem, Marchionistrasse 15, D-8000, Munich 70, West Germany Received December 14, 1982 A method for molecular weight determination of small peptides using Bio-Sil TSK 20 and Bio-Gel TSK 125 columns is described. The TSK 20 column provided a good separation of the standard peptides in the range from lOOO-10,000 with an accuracy of less than 5% from the calculated regression line. Two combined TSK 125 columns ahowed a reliable molecular weight determination in the range from 800 to 3500. KEYWORDS: molecular weight determination; peptides; HPLC, guanidine hydrochloride.
The exact molecular weight determination of small amounts of peptides, especially in the range between 1000 and 7000 often raises problems: gel filtration (1,2) and ultracentrifugation (3,4) are not suitable for very small amounts of the peptides, additionally these methods are limited in their separational capacity in the lower molecular range. The latter is also true for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (5). Size exclusion high-performance liquid chromatography is increasingly being used for separation of proteins based on the molecular weight ( 10). By chemical modifications of the silica gels a higher mechanical stability and an excellent recovery have been achieved (11-13). We developed a procedure which allows the molecular weight determination of peptides between 1000 and 10,000. MATERIAL
AND
METHODS
The peptides used as standards are listed in Table 1. Grade I guanidine hydrochloride was obtained from Sigma, Munich, West Germany, indole and acetic acid from Merck, ’ Supported by Deutsche Forschungsgemeinschaft (SFB 5 l/C-34). 0003-2697183 $3.00 Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved.
288
TABLE
1
LISTOFFWTIDESUSEDASINTERNALSTANDARD
Number 1 2 3 4 5 6 7 8 9 10 11 12
13 14 15 16 17 18
:i 21
Substance Bovine serum albumin” My oglobin ’ Lysozyme” Cytochrome c’ Porcine &lipotropind Trypsin inhibitor” ACTH porcine” @-endorphin human b Insulin B chain’ Glucagonn ACTH l-24’ Insulin A chain” Gramicidine” Alpha-MSH b Bacitracin” Actinomycin C” Bradykinin ’ LRF’ PZ-Pro-Leu-Gly-Pro-D-Argo Cathepsin D” DNP-Alanine”
Molecular weight
69,000 17,800 14,400 12,300 9,200
6,‘3OfJ 4,500 3,466 3,495 3,483 2,934 2,532
2,0@3 1,665 1,450 1,280 1,060 1,182 777 849 255
a Purchased from Serva, Heidelberg, W. Germany. b Purchased from Bachem, Burgdorf, Switzerland. cGift from Hoechst, Frankfurt, W. Germany. d Prepared as previously described ( 14).
MOLECULAR
WEIGHT
OF PEPTIDES BY LIQUID
Darmstadt, W. Germany. Water was glassdistilled from the deionized water supply of the laboratory. Detection was performed at 279 nm in an Uvicord S photometer connected with a two-channel recorder 22 10 (LKB, Munich, W. Germany) and a HewlettPackard integrator 3380 A. The columns used were: two Bio-Sil TSK 125 (No. 22032 and 22023) and one Bio-Gel TSK 20 (No. 144 15), 300 X 7.5 mm i.d., the TSK 125 contained silica modified with silanoyl groups, the TSK 20 silica with hydroxylated polyethers (BioRad, Munich, W. Germany). The filtered (0.15 pm) guanidine hydrochloride was fed to an Altex 110 A pump equipped with a special pressure filter. The flow rate was 1.0 ml/min. The columns were equilibrated with the eluant for 2 h. For longer storage periods the columns were filled with methanol. The experiments were performed at 23°C either with a TSK I25 column, two connected TSK 125 columns or one TSK 20 column. Samples (5-50 pg) were dissolved in 6 M guanidine hydrochloride, the injection volume was 0.1 ml.
5-
289
CHROMATOGRAPHY
* BSA
EACITRiC~L;;~~iN BRADYKININ
c
I, “13
U
15
ELUTION
16
17
18 ml
VOLUME
FIG.2. Calibration plots of peptides using the TSK 125 column (y = 7.89 - 0.283x, r = 0.98).
20 column the retention times of the standards from 1000 to about 10,000 were inversely correlated to the logarithms of their molecular weights. The TSK 125 column provided a good separation in the molecular weight range from 1000 to 17,800, but the correlation was not as good as with the TSK 20 column. On this column none of the peptides deviated more than 5% of its molecular weight from the calculated regression line, the within-day RESULTS AND DISCUSSION variation of the molecular weight was in the range from 1 to 2%. The TSK 125 column The elution positions of the peptide stanallowed the molecular weight estimation in dards using 6 M guanidine hydrochloride as eluant are shown for the TSK 20 (Fig. 1) and the range from 1000 to 10,000 with an accuracy of ?15% and up to 17,800 of +20%, the TSK 125 columns (Fig. 2). On the TSK the within-day variation was in the range from 0.9 to 2.2%. Since in these experiments the correlation between retention times and molecular weights was better in the lower range . BSA (<4000), we combined two TSK 125 columns for the separation of peptides in the range from 800 to 3500 (Fig. 3). Z” The peptides were, with exception of LRF (1 O%), in the range of +5% of the calculated regression line, the within-day precision was 1 to 1.9%. For all the determinations the within-day precision for the retention time of each stanELUTION VOLUME dard was between 0.1 and 0.44% (Tables 2 and 3), corresponding to a variation in the FIG. 1. Calibration plots of peptides using the TSK 20 molecular weight from 0.9 to 2.2%. The day column (eluant guanidine hydrochloride, flow rate .l ml/ min Lv = 6.987 - 0.576x, r = 0.9971. to day variation of the standards was maxi-
5 1
r
290
RICHTER,
JACOB, AND SCHWANDT
1
. DNP-ALANINE
2.5 0zli
17
18
19
ELUTION
20
21
VOLUME
22
(ml)
FIG. 3. Calibration plots of peptides in the lower molecular weight range, using two combined TSK 125 columns 0; = 6.14 - 0.154x, r = 0.991).
mally 0.63% (results of 5 peptides are shown in Table 3). Figure 4 demonstrates the separation of 7 peptides in the molecular weight range from 1060 to 3500 using the TSK 20 where the determination is most difficult because of the short retention time. This figure as well as Table 2 demonstrate that peptides differing by only 150 to 200 in their molecular weights can be separated and their difference in retention times exceeds by far the standard de-
viation. On the other hand, peptides with nearly identical molecular weights had the same retention time. As an indicator for nonspecific absorptions we used the hydrophobic substance indole which was eluted at the same retention time as other markers for total eluation volume (e.g., acetic acid, DNP-alanine). Peak symmetry (Fig. 4) may be taken as a further argument against nonspecific interaction. Since the within-day variation of the system
TABLE 2 RETENTION
TIMES (s) OF FIVE PEPTIDES OBTAINED
WITH BDGEL
TSK
20 COLUMN
Molecular weight
1 2 3 4
5 Mean value _+ SD
1280
1450
2934
9700
12,300
403.2 404.4 403.2 403.8 402.0
396.6 397.8 396.6
366.0 364.8 364.2 366.6 366.6
315.0 314.4 315.0 314.4 315.0
310.2
403.3
-t 0.88
396.0 396.0 396.6
-t 0.73
365.6
-+ 1.08
314.1
f 0.33
309.6 309.6 310.8 309.6
309.9 f 0.53
MOLECULAR
WEIGHT
OF PEPTIDES
BY LIQUID
291
CHROMATOGRAPHY
TABLE 3 MEAN
RETENTION
TIME”
(SD)
OF FIVE PEPTIDES (WITHIN-DAY
AND DAY TO DAY VARIATION)
Molecular weight
Day 1 Day 2 Day 3
1280
1450
2532
6ooo
14,300
1032 f 3 1029.6 + 2.8 1031.4 f 3.2
1010.4 f 3.2 1008.6 + 2.9 1011 f 1.8
942 + 3.4 936 + 2.7 936 + 3.2
878 + 3.4 879.2 + 3.2 879.1 + 3.3
813 f 3.2 810 f 3.6 805.2 f 3.2
Note. Results obtained with TSK 125 column (mean of 5 determinations). a In seconds.
is minimal a recalibration during the day is not necessary. However, to exclude day to day variations an every day calibration with four different internal standards is recommended. The combination of the results obtained with the TSK 20 and two TSK 125 columns allows the molecular weight determination of small peptides with high accuracy. We have also
tested the separational capacity of several other gel permeation columns and different buffer systems (e.g., phosphate, acetate, fox-mate, sodium dodecyl sulfate, or guanidine hydrochloride), but no sufficient results were obtained. In conclusion, the HPLC methods described above are easy to perform, rapid and accurate for molecular weight determinations of small peptides down to 800. REFERENCES Whitaker, J. R. (1963) Anal. Chem. 35, 1950-1954. Andrews, P. (1964) B&hem. J. 91, 222-223. Yphantis, D. A. (1964) Biochemistry 3,297-302. Van Holde, K. E., and Baldwin, R. L. (1958) J. Phys. Chem.
62,734-743.
Weber, K., and Qsbom, M. (1969) J. Biol. Chem. 16,4406-4412. Niemann, M. A., Hollaway, W. L., and Mole, J. E. ( 1979) J. High Resolut. Commun. 2, 743-745.
_ _ ._
FIG. 4. Chromatograms of 7 peptides. Three with nearly identical molecular weights (h-&endorphin = 8, glucagon = 9, insulin B chain = 10); and insulin A chain = 12, bacitracin = 15, actinomycin C = 16, bradykinin = 17. (I) Injection. The peaks eluated between 9 and 10 min contain the salt of the peptide samples.
Chromatogr.
Chromatogr.
8. Hashimoto, T., Sax&, H., Aiura, M., and Kato, Y. (1978) J. Chromatogr. 160, 301-305. 9. Buchholz, K., Giidelmann, B., and Molnar, I. (1982) J. Chromatogr. 238, 193-202. 10. Rivier, J. E. (1980) J. Chromatogr. 202, 2 1 l-222. 11. Engelhardt, H., and Mathes, D. (1979) J Chromatogr. 185. 305-319. 12. Unger; K., Schick-Kalb, J., and Krebs, K. F. (1973) Chromatography
83, 5-9.
13. Regnier, F. E., and Gooding, K. M. (1980) 103, l2.5. 14. Schwandt, P., and Richter, W. 0. (1980) J. Clin. Chem. Clin. Biochem. 10, 736-737.
BIOCHEMISTRY
Molecular
133,288-291
(1983)
Weight Determination Gel Permeation W.O.
of Peptides by High-Performance Chromatography’
RICHTER,B.JACOB,ANDP.
SCHWANDT
Department of Internal Medicine II, University of Munich, Klinikum Grosshadem, Marchionistrasse 15, D-8000, Munich 70, West Germany Received December 14, 1982 A method for molecular weight determination of small peptides using Bio-Sil TSK 20 and Bio-Gel TSK 125 columns is described. The TSK 20 column provided a good separation of the standard peptides in the range from lOOO-10,000 with an accuracy of less than 5% from the calculated regression line. Two combined TSK 125 columns ahowed a reliable molecular weight determination in the range from 800 to 3500. KEYWORDS: molecular weight determination; peptides; HPLC, guanidine hydrochloride.
The exact molecular weight determination of small amounts of peptides, especially in the range between 1000 and 7000 often raises problems: gel filtration (1,2) and ultracentrifugation (3,4) are not suitable for very small amounts of the peptides, additionally these methods are limited in their separational capacity in the lower molecular range. The latter is also true for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (5). Size exclusion high-performance liquid chromatography is increasingly being used for separation of proteins based on the molecular weight ( 10). By chemical modifications of the silica gels a higher mechanical stability and an excellent recovery have been achieved (11-13). We developed a procedure which allows the molecular weight determination of peptides between 1000 and 10,000. MATERIAL
AND
METHODS
The peptides used as standards are listed in Table 1. Grade I guanidine hydrochloride was obtained from Sigma, Munich, West Germany, indole and acetic acid from Merck, ’ Supported by Deutsche Forschungsgemeinschaft (SFB 5 l/C-34). 0003-2697183 $3.00 Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved.
288
TABLE
1
LISTOFFWTIDESUSEDASINTERNALSTANDARD
Number 1 2 3 4 5 6 7 8 9 10 11 12
13 14 15 16 17 18
:i 21
Substance Bovine serum albumin” My oglobin ’ Lysozyme” Cytochrome c’ Porcine &lipotropind Trypsin inhibitor” ACTH porcine” @-endorphin human b Insulin B chain’ Glucagonn ACTH l-24’ Insulin A chain” Gramicidine” Alpha-MSH b Bacitracin” Actinomycin C” Bradykinin ’ LRF’ PZ-Pro-Leu-Gly-Pro-D-Argo Cathepsin D” DNP-Alanine”
Molecular weight
69,000 17,800 14,400 12,300 9,200
6,‘3OfJ 4,500 3,466 3,495 3,483 2,934 2,532
2,0@3 1,665 1,450 1,280 1,060 1,182 777 849 255
a Purchased from Serva, Heidelberg, W. Germany. b Purchased from Bachem, Burgdorf, Switzerland. cGift from Hoechst, Frankfurt, W. Germany. d Prepared as previously described ( 14).
MOLECULAR
WEIGHT
OF PEPTIDES BY LIQUID
Darmstadt, W. Germany. Water was glassdistilled from the deionized water supply of the laboratory. Detection was performed at 279 nm in an Uvicord S photometer connected with a two-channel recorder 22 10 (LKB, Munich, W. Germany) and a HewlettPackard integrator 3380 A. The columns used were: two Bio-Sil TSK 125 (No. 22032 and 22023) and one Bio-Gel TSK 20 (No. 144 15), 300 X 7.5 mm i.d., the TSK 125 contained silica modified with silanoyl groups, the TSK 20 silica with hydroxylated polyethers (BioRad, Munich, W. Germany). The filtered (0.15 pm) guanidine hydrochloride was fed to an Altex 110 A pump equipped with a special pressure filter. The flow rate was 1.0 ml/min. The columns were equilibrated with the eluant for 2 h. For longer storage periods the columns were filled with methanol. The experiments were performed at 23°C either with a TSK I25 column, two connected TSK 125 columns or one TSK 20 column. Samples (5-50 pg) were dissolved in 6 M guanidine hydrochloride, the injection volume was 0.1 ml.
5-
289
CHROMATOGRAPHY
* BSA
EACITRiC~L;;~~iN BRADYKININ
c
I, “13
U
15
ELUTION
16
17
18 ml
VOLUME
FIG.2. Calibration plots of peptides using the TSK 125 column (y = 7.89 - 0.283x, r = 0.98).
20 column the retention times of the standards from 1000 to about 10,000 were inversely correlated to the logarithms of their molecular weights. The TSK 125 column provided a good separation in the molecular weight range from 1000 to 17,800, but the correlation was not as good as with the TSK 20 column. On this column none of the peptides deviated more than 5% of its molecular weight from the calculated regression line, the within-day RESULTS AND DISCUSSION variation of the molecular weight was in the range from 1 to 2%. The TSK 125 column The elution positions of the peptide stanallowed the molecular weight estimation in dards using 6 M guanidine hydrochloride as eluant are shown for the TSK 20 (Fig. 1) and the range from 1000 to 10,000 with an accuracy of ?15% and up to 17,800 of +20%, the TSK 125 columns (Fig. 2). On the TSK the within-day variation was in the range from 0.9 to 2.2%. Since in these experiments the correlation between retention times and molecular weights was better in the lower range . BSA (<4000), we combined two TSK 125 columns for the separation of peptides in the range from 800 to 3500 (Fig. 3). Z” The peptides were, with exception of LRF (1 O%), in the range of +5% of the calculated regression line, the within-day precision was 1 to 1.9%. For all the determinations the within-day precision for the retention time of each stanELUTION VOLUME dard was between 0.1 and 0.44% (Tables 2 and 3), corresponding to a variation in the FIG. 1. Calibration plots of peptides using the TSK 20 molecular weight from 0.9 to 2.2%. The day column (eluant guanidine hydrochloride, flow rate .l ml/ min Lv = 6.987 - 0.576x, r = 0.9971. to day variation of the standards was maxi-
5 1
r
290
RICHTER,
JACOB, AND SCHWANDT
1
. DNP-ALANINE
2.5 0zli
17
18
19
ELUTION
20
21
VOLUME
22
(ml)
FIG. 3. Calibration plots of peptides in the lower molecular weight range, using two combined TSK 125 columns 0; = 6.14 - 0.154x, r = 0.991).
mally 0.63% (results of 5 peptides are shown in Table 3). Figure 4 demonstrates the separation of 7 peptides in the molecular weight range from 1060 to 3500 using the TSK 20 where the determination is most difficult because of the short retention time. This figure as well as Table 2 demonstrate that peptides differing by only 150 to 200 in their molecular weights can be separated and their difference in retention times exceeds by far the standard de-
viation. On the other hand, peptides with nearly identical molecular weights had the same retention time. As an indicator for nonspecific absorptions we used the hydrophobic substance indole which was eluted at the same retention time as other markers for total eluation volume (e.g., acetic acid, DNP-alanine). Peak symmetry (Fig. 4) may be taken as a further argument against nonspecific interaction. Since the within-day variation of the system
TABLE 2 RETENTION
TIMES (s) OF FIVE PEPTIDES OBTAINED
WITH BDGEL
TSK
20 COLUMN
Molecular weight
1 2 3 4
5 Mean value _+ SD
1280
1450
2934
9700
12,300
403.2 404.4 403.2 403.8 402.0
396.6 397.8 396.6
366.0 364.8 364.2 366.6 366.6
315.0 314.4 315.0 314.4 315.0
310.2
403.3
-t 0.88
396.0 396.0 396.6
-t 0.73
365.6
-+ 1.08
314.1
f 0.33
309.6 309.6 310.8 309.6
309.9 f 0.53
MOLECULAR
WEIGHT
OF PEPTIDES
BY LIQUID
291
CHROMATOGRAPHY
TABLE 3 MEAN
RETENTION
TIME”
(SD)
OF FIVE PEPTIDES (WITHIN-DAY
AND DAY TO DAY VARIATION)
Molecular weight
Day 1 Day 2 Day 3
1280
1450
2532
6ooo
14,300
1032 f 3 1029.6 + 2.8 1031.4 f 3.2
1010.4 f 3.2 1008.6 + 2.9 1011 f 1.8
942 + 3.4 936 + 2.7 936 + 3.2
878 + 3.4 879.2 + 3.2 879.1 + 3.3
813 f 3.2 810 f 3.6 805.2 f 3.2
Note. Results obtained with TSK 125 column (mean of 5 determinations). a In seconds.
is minimal a recalibration during the day is not necessary. However, to exclude day to day variations an every day calibration with four different internal standards is recommended. The combination of the results obtained with the TSK 20 and two TSK 125 columns allows the molecular weight determination of small peptides with high accuracy. We have also
tested the separational capacity of several other gel permeation columns and different buffer systems (e.g., phosphate, acetate, fox-mate, sodium dodecyl sulfate, or guanidine hydrochloride), but no sufficient results were obtained. In conclusion, the HPLC methods described above are easy to perform, rapid and accurate for molecular weight determinations of small peptides down to 800. REFERENCES Whitaker, J. R. (1963) Anal. Chem. 35, 1950-1954. Andrews, P. (1964) B&hem. J. 91, 222-223. Yphantis, D. A. (1964) Biochemistry 3,297-302. Van Holde, K. E., and Baldwin, R. L. (1958) J. Phys. Chem.
62,734-743.
Weber, K., and Qsbom, M. (1969) J. Biol. Chem. 16,4406-4412. Niemann, M. A., Hollaway, W. L., and Mole, J. E. ( 1979) J. High Resolut. Commun. 2, 743-745.
_ _ ._
FIG. 4. Chromatograms of 7 peptides. Three with nearly identical molecular weights (h-&endorphin = 8, glucagon = 9, insulin B chain = 10); and insulin A chain = 12, bacitracin = 15, actinomycin C = 16, bradykinin = 17. (I) Injection. The peaks eluated between 9 and 10 min contain the salt of the peptide samples.
Chromatogr.
Chromatogr.
8. Hashimoto, T., Sax&, H., Aiura, M., and Kato, Y. (1978) J. Chromatogr. 160, 301-305. 9. Buchholz, K., Giidelmann, B., and Molnar, I. (1982) J. Chromatogr. 238, 193-202. 10. Rivier, J. E. (1980) J. Chromatogr. 202, 2 1 l-222. 11. Engelhardt, H., and Mathes, D. (1979) J Chromatogr. 185. 305-319. 12. Unger; K., Schick-Kalb, J., and Krebs, K. F. (1973) Chromatography
83, 5-9.
13. Regnier, F. E., and Gooding, K. M. (1980) 103, l2.5. 14. Schwandt, P., and Richter, W. 0. (1980) J. Clin. Chem. Clin. Biochem. 10, 736-737.