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Separation of dipeptides by high-resolution gas chromatography on a fused silica capillary column after trimethylsilylation
ANALYTICAL
BIOCHEMISTRY
108,
269-273 (1980)
Separation of Dipeptides by High-Resolution Gas Chromatography on a Fused Silica Capillary Column after Trimethylsilylation MIRALDIZDAROGLU**'AND *Food
Engineering
Laboratory, U. S. Army Natick 01760, and TNational Bureau
MICHAELG.
Research &Development of Standards, Washington,
SIMIC? Command, Natick, D. C. 20234
Massachusetts
Received May 14, 1980 Separation of trimethylsilyl derivatives of over 50 dipeptides was achieved by highresolution gas chromatography using a fused silica capillary column coated with methyl silicone liquid phase. Excellent peak symmetry and reproducibility were obtained. Several pairs of sequence isomeric dipeptides could also be well separated. Application of this approach to peptide sequencing by means of the dipeptidyl aminopeptidase-gas chromatography/mass spectrometry method is also discussed.
The method proposed by McDonald et al. (1) for determining the amino acid sequence of peptides involves their hydrolysis into dipeptides with dipeptidyl aminopeptidase I (DAP I).2 A second DAP I digestion of the peptide after removal of its amino-terminal amino acid by the Edman degradation (2) leads to a new set of dipeptides. Identification of all the dipeptides in the two mixtures is required to determine the sequence of the original peptide (3,4). Sequence analysis of peptides has also been described utilizing a mixture of DAP I and DAP IV (5). This method eliminates most of the difficulties with enzyme specificity when either enzyme is used alone (5,6). The identification of the released dipeptides, however, requires efficient methods for separation of the digestion mixtures into their components. Dipeptides have been analyzed by ionexchange, paper, and thin-layer chromatography (7 - 13). High-performance liquid 1 Correspondence should be sent to this author. Present address: University of Maryland, Baltimore County, Chemistry Department, Baltimore, Maryland 21228. ’ Abbreviations used: DAP, dipeptidyl aminopeptidase; hplc, high-performance liquid chromatography; gc, gas chromatography; ms, mass spectrometry; TMS, trimethylsilyl; BSTFA, bis(trimethylsilyl)trifluoracetamide; WCOT, wall-coated open tubular. 269
chromatography (hplc) has also been used for separation of dipeptides (14-21). Gas chromatography (gc) combined with mass spectrometry (ms) seems to be the most effective technique for separation and identification of dipeptides because of its speed, high sensitivity, and ease of identification. Dipeptides, however, must be derivatized prior to their gc analysis. Various derivatization methods have been suggested and successfully used for this purpose (5,6,2234). Trimethylsilylation seems to be the most useful derivatization method since it can be easily carried out and gives derivatives with good chromatographic properties and easily interpretable mass spectra (33). This method has been widely used for gc analysis of amino acids (35,36) and dipeptides (23,25,29,33,34) utilizing various silylation reagents. The papers listed above on the gc analysis of dipeptides have reported separations using conventional packed columns. Gas chromatography with capillary columns (high-resolution gas chromatography), however, offers great advantages over gc with packed columns for separating very complex mixtures (37-40). Moreover, recent advances in column technology have brought into existence capillary 0003-2697/80/160269-05$02.00/O Copyright All rights
0 1980 by Academic Press, Inc. of reproduction in any form reserved.
270
DIZDAROGLU
columns drawn from fused silica (41). These columns seem to be superior to glass capillary columns in terms of flexibility, overall inertness, and efficiency (41). In this paper we describe the separation of trimethylsilyl (TMS) derivatives of dipeptides by high-resolution gas chromatography using a fused silica capillary column. MATERIALS
AND METHODS
Chemicals and reagents. Dipeptides were purchased from Sigma and Research Plus Laboratories. Bis(trimethylsilyl)trifluoroacetamide (BSTFA) and acetonitrile were obtained from Pierce Chemical Company. The test mixture for column evaluation was purchased from Alltech Association. Trimethylsilylation. A sample of ca. 0.5 mg of each dipeptide was placed in a Tefloncapped Hypo-Vial (Pierce Chemical Co,) and trimethylsilylated with 0.4 ml of a mixture of BSTFA and acetonitrile (1: 1) by heating for 30 min at 120°C in a sand bath. Gas chromatography. A Hewlett-Packard Model 5880A microprocessor controlled gas chromatograph equipped with a flame ionization detector was used. The injection port and the detector were maintained at 250°C. Helium was used as the carrier gas at an inlet pressure of 0.75 bar. The split ratio was 1:lOO and the flow through the column 0.6 ml/min. Separations were obtained by using a fused silica capillary column (12 m, 0.2 mm i.d.) coated (WCOT) with SP-2100 (methyl silicone liquid phase). It was purchased from Hewlett-Packard. The measured efficiency of the column was ca. 4200 plates per meter based on pentadecane peak at 130°C (k’ = 5.13; linear velocity, ,% = 35.7 cm/s). The ratio of 2,6dimethylphenol to 2,6-dimethylaniline (42) was 1.006. RESULTS AND DISCUSSION
Figure 1 shows the separation of TMS derivatives of over 50 dipeptides. Peak identification is given in Table I. Some im-
AND SIMIC
purities were noted when running individual standard compounds; these appeared at several places on Fig. 1 (such as between peaks 6 and 7). Excellent peak symmetry and reproducibility were obtained for all dipeptides except for those containing histidine and tryptophan. Derivatives of such dipeptides have been reported to be the least volatile and most troublesome compounds for gc analysis (32,33). Under our experimental conditions tailing peaks were obtained for TMS derivatives of such compounds (peaks 34,44,52, and 55). It is interesting to note that L-His-Gly (peak 34) and L-His-L-Ala (peak 44) produced tailing peaks whereas Gly-L-His (peak 53) and L-Ala-L-His (peak 45) showed no tailing behavior. L-Trp-Gly (peak 50) gave a symmetrical peak. It has been reported that derivatives of dipeptides containing arginine were not sufficiently volatile for gc analysis and did not elute from the gc column (29,32,33). Such compounds could be chromatographed after conversion of arginine to ornithine (33). Dipeptides containing arginine were not studied in this work. Dipeptides containing glycine as the first amino acid gave two peaks due to the substitution of the a-amino group of glycine by one or two TMS groups (33,35,43). The second peak corresponds to the derivative with two TMS groups. In some cases the first peak disappears completely as in the case of Gly-L-Val (peak 23) and Gly-LHis (peak 53) or decreases as in the case of Gly-L-Thr (peaks 6 and 20) and Gly-L-Ile (peaks 13 and 26) after storage of the sample a few hours at room temperature. Dipeptides containing glycine as the second amino acid gave only one peak. L-AlaL-Asn also formed two derivatives (peaks 19 and 26). This is probably due to the trimethylsilylation of the o-amino group of asparagine to different extents. This amino acids has been reported to yield two peaks after trimethylsilylation (36). DL-Ala-DL-Ser and DL-Leu-DL-Phe
271
CAPILLARY GAS CHROMATOGRAPHY OF DIPEPTIDES
5
10
15
20
25
min 230
160
100
lC
FIG. 1. Separation of TMS derivatives of dipeptides by high-resolution gc. Column; fused silica SP2100, 12 m, 0.2 mm i.d. programmed 4Vmin from 100 to 160°C 6”Cimin from 160 to 230°C and maintained at 230°C for 15 min. For other column details see Materials and Methods. Peak identification is given in Table 1. Identification was achieved by comparison of retention times of individually injected compounds. Each peak corresponds to about O.l- 1 ng of dipeptide.
gave two peaks which were completely separated (peaks 15, 16 and peaks 39, 41, respectively). In the case of these compounds a trimethylsilylation of the amino groups of alanine and leucine to different extents cannot be expected because of the steric hindrance and it has not been observed before with the free amino acids. Carboxyl groups and the OH group of serine are substituted by only one TMS group. A recent hplc investigation of DL,DLdipeptides (21) showed that DL-Ala-DL-Ser and DL-Leu-DL-phe gave two peaks which correspond to DL-, LD- and DD-, IL-isomers, respectively. Based on these results, the occurrence of two gc peaks can possibly be explained by separation of optical isomers of these dipeptides. However, this explanation cannot be established without examining all possible four optical isomers, which were not available to us. Separation of sequence isomeric dipeptides. The resolution of the sequence isomerit dipeptides is very important for the
sequencing of peptides. In this paper, several pairs of sequence isomeric dipeptides were examined and found to be separable. A complete separation of the pairs L-Ala-L-Leu (peak 8)/L-Leu-L-Ala (peak 9) and L-Ser-L-Ala (peak 14)/~~Ala-DL-Ser (peaks 15 and 16) was obtained. The other pairs were well resolved (see peak 28/29,31/32, 37138, 44145, and 471 48 in Fig. 1 and Table 1). Sequence isomeric dipeptides containing glycine were not separable (peaks 3, 11, and 21). However, they can be easily distinguished since the isomer containing glycine as the first amino acid gives two derivatives (compare peaks 3 and 24, 11 and 36, 21 and 43) or one derivative with two TMS groups at the aamino group (compare peaks 2 and 23, 34 and 53). CONCLUSIONS
The results obtained in this paper clearly demonstrate that complex mixtures of di-
272
DIZDAROGLU
AND SIMIC
TABLE PEAK
Peak No. 1 2 3
Dipeptide
4 5 6 7 8 9 10 11 12 13 14 15 16 17
IDENTIFICATION
Peak No.
Gly-Gly L-Val-Gly Gly-L-Leu L-Leu-Gly L-Ala-L-Ala Gly-L-Ser Gly-L-Thr L-Ala-L-Val L-Ala-r-Leu L-Ala-L-Be L-Leu-L-Ala cu-L-Asp-Gly Gly-L-Met L-Met-Gly Gly-r-Asp Gly-L-Be L-val-L-val L-Ser-L-Ala DL-Ala-DL-Ser DL-Ala-DL-Ser L-Ala-t-Thr
18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
peptides can be separated into their components by capillary gas chromatography after trimethylsilylation. The method offers great advantages in terms of speed, sensitivity, peak symmetry, reproducibility and separation of sequence isomers. It can be a useful tool for sequence analysis of peptides by using the DAP-gc/ms method. ACKNOWLEDGMENT M.D. acknowledges the Fellowship granted by National Academy of Sciences/National Research Council.
REFERENCES 1. McDonald, J. K., Zeitman, B. B., Reilly, T. J., and Ellis, S. (1969) J. Biol. Chem. 244, 2693-2709. 2. Edman, P. (1956) Acta Chem. Stand. 10, 761-768. 3. McDonald, J. K., Callahan, P. X., and Ellis, S. (1972) in Methods in Enzymology (Hirs, C. H. W., and Timasheff, S. N., eds.), Vol. 25, pp. 272-298, Academic Press, New York. 4. Lindley, H. (1972) Biochem. J. 126, 683-688. 5. Caprioli, R. M., and Seifert, W. E. (1975)Biochem. Biophys.
Res.
Commun.
64,295-303.
1 IN FIG.
1
Dipeptide Gly-L-Ser L-Ala-L-Asn Gly -L-Thr Gly-L-Phe L-Phe-Gly Gly - L-Glu Gly-L-Val L-Leu-L-Leu Gly-L-Leu L-Ala-L-Asp Gly-L-Be L-Ala-t.-Asn L-Leu-L-Ser L-Ala-L-Met L-Met-r-Ala LrAla- L-Glu t.-Phe-r-Ala L-Ala-L-Phe y-L-Glu-L-Leu Gly-~-Asp L-His-Gly
Peak No.
Dipeptide
35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55
L-Ser-L-Met Gly-L-Met L-Phe - r-Val L-Val-L-Phe DL-Leu-DL-Phe Gly - L-Glu DL-Leu-DL-Phe L-Ser-L-Phe Gly - L-Phe L-His-L-Ala L-Ala-L-His L-Met-L-Met L-Ala- L-Tyr L-Tyr- L-Ala L-Tyr-Gly L-Trp-Gly L-Met-L-Phe r-His-L-Ser Gly-~-His L-Phe-t.-Phe L-Trp-L-Ala
Seifert, W. E., and Caprioli, R. M. (1978) Biochemistry 17, 436-441. 7. Manning, J. M., and Moore, S. (1%8) J. Biol. Chem. 243, 5591-5597. 8. Callahan, P. X., Shepard, J. A., Reilly, T. J., McDonald, J. K., and Ellis, S. (1970) Anal. 6.
Biochem. 9.
38, 330-356.
Heatcote, J. G., Washington, R. .I., Keogh, B. J., and Glanville, R. W. (1972) J. Chromatogr. 65, 397-405.
10. Giliberti,
F., and Niederwieser, A. (1972) J. 66, 261-276. 11. Howarth, C. (1972) J. Chromatogr. 67, 315-323. 12. Mattel, C., and Phelps, D. J. (1975) J. Chromatogr. Chromatogr.
115, 633-634.
13. Arendt, A., Kotodziejczyk, A., and Sokotowska, T. (1976) Chromatogruphia 9, 123- 126. 14. Hansen, J. J., Greibrokk, T., Currie, B. L., Johansson, K. N. G., and Folkers, K. (1977) J. Chromatogr. 135, 155-164. 15. Kikta, Jr., E. J., and Grushka, E. (1977) J. Chromatogr. 16.
135, 367-376.
Fong, G. W. K., and Grushka, Chromatogr.
17.
Molnar, I., and
18.
Lundanes,
E. (1977) J.
142, 299-309. Horvath, C. (1977) J. Chromatogr.
142, 623-640. Chromatogr. 19.
E., and Greibrokk,
T. (1978) J.
149, 241-254.
Hancock, W. S., Bishop, C. A., Prestige, R. L.,
CAPILLARY
20. 21. 22. 23.
GAS CHROMATOGRAPHY
Harding, D. R. K., and Heam, M. T. W. (1978) J. Chromatogr. 153, 391-398. Nakamura, H., Zimmerman, C. L., and Pisano, J. J. (1979) Anal. Biochem. 93, 423-429. Dizdaroglu, M., and Simic, M. G. (1980) J. Chromatogr. 195, 119- 126. Weygand, F., Kolb, B., Prox, A., Tilak, M. A., and Tomida, I. (l%O) 2. Physiol. Chem. 322, 38-51. Birkofer, L., Ritter, A., and Neuhausen, P. (1962) Justus
Liebigs
Ann.
Chem.
659,
190-199.
24. Weygand, R., and 18, 9325. Ruhlmann,
F., Prox, A., Jorgensen, E. C., Axen, Kirchner, P. (1963) Z. Naturforsch. B 104. K., Simon, H., and Becker, M. (1966) Chem. Ber. 99, 780-781. 26. Ovchinikov, Y. A., and Kiryushkin, A. A. (1972) FEBS
Let?.
21, 300-302.
27. Caprioli, R. M., Seifert, Jr., W. E., and Sutherland, D. E. (1973) Biochem. Biophys. Res. Commun.
S&67-75.
28. Lindley, H., and Davis, P. C. (1974) J. Chromatogr. 100, 117-121. 29. Kelley, J. A., Nau, H., Forster, H. J., and Biemann, K. (1975) Biomed. Mass Spectrom. 2, 313-325. 30.
Young, M. A., and Desiderio, D. M. (1976) Ann/. Biochem.
70, 110-123.
273
OF DIPEPTIDES
31. Schier, G. M., Bolton, P. D., and Halpem, B. (1976) Biomed. Mass Spectrom. 3, 32-40. 32. Frank, H., Haegele, K. D., and Desiderio, D. M. (1977) Anal. Chem. 49, 287-291. 33. Krutzsch, H. C., and Pisano, J. J. (1978) Biochemistry
17, 2791-2797.
H. C., and Kindt, T. J. (1979) Anal. Biochem. 92, 525-53 1. 35. Gehrke, C. W., Nakamoto, H., and Zumwalt, R. W. (1969) J. Chromatogr. 45, 24-51. 36. Gehrke, C. W., and Leimer, K. (1971)J. Chroma34.
Krutzsch,
togr.
57,219-238.
Schomburg, G., Husmann, H., and Weeke, F. (1974) J. Chromatogr. 99,63-79. 38. Schomburg, G., and Husmann, H. (1975) Chroma37.
tographia
8, 517-530.
Novotny, M. (1978) Ana/. Chem. 50, 16A-32A. 40. Freeman, R. R. (1979) High Resolution Gas Chromatography, p. 1, Part No. 5950-3562. Hewlett-Packard Publ., Palo Alto, Calif. 41. Dandeneau, R. D., and Zerenner, E. H. (1979) 39.
J. High Commun.
Resolut. Chromatogr. 2,351-355.
42. Grob, K., and 4, 422-424.
43. Bergstrom, (1970)
Chromatogr.
Grob, G. (1971) Chromatogruphia
K., Giirtler, J., and Blomstrand,
Anal.
Biochem.
34,74-87.
R.
BIOCHEMISTRY
108,
269-273 (1980)
Separation of Dipeptides by High-Resolution Gas Chromatography on a Fused Silica Capillary Column after Trimethylsilylation MIRALDIZDAROGLU**'AND *Food
Engineering
Laboratory, U. S. Army Natick 01760, and TNational Bureau
MICHAELG.
Research &Development of Standards, Washington,
SIMIC? Command, Natick, D. C. 20234
Massachusetts
Received May 14, 1980 Separation of trimethylsilyl derivatives of over 50 dipeptides was achieved by highresolution gas chromatography using a fused silica capillary column coated with methyl silicone liquid phase. Excellent peak symmetry and reproducibility were obtained. Several pairs of sequence isomeric dipeptides could also be well separated. Application of this approach to peptide sequencing by means of the dipeptidyl aminopeptidase-gas chromatography/mass spectrometry method is also discussed.
The method proposed by McDonald et al. (1) for determining the amino acid sequence of peptides involves their hydrolysis into dipeptides with dipeptidyl aminopeptidase I (DAP I).2 A second DAP I digestion of the peptide after removal of its amino-terminal amino acid by the Edman degradation (2) leads to a new set of dipeptides. Identification of all the dipeptides in the two mixtures is required to determine the sequence of the original peptide (3,4). Sequence analysis of peptides has also been described utilizing a mixture of DAP I and DAP IV (5). This method eliminates most of the difficulties with enzyme specificity when either enzyme is used alone (5,6). The identification of the released dipeptides, however, requires efficient methods for separation of the digestion mixtures into their components. Dipeptides have been analyzed by ionexchange, paper, and thin-layer chromatography (7 - 13). High-performance liquid 1 Correspondence should be sent to this author. Present address: University of Maryland, Baltimore County, Chemistry Department, Baltimore, Maryland 21228. ’ Abbreviations used: DAP, dipeptidyl aminopeptidase; hplc, high-performance liquid chromatography; gc, gas chromatography; ms, mass spectrometry; TMS, trimethylsilyl; BSTFA, bis(trimethylsilyl)trifluoracetamide; WCOT, wall-coated open tubular. 269
chromatography (hplc) has also been used for separation of dipeptides (14-21). Gas chromatography (gc) combined with mass spectrometry (ms) seems to be the most effective technique for separation and identification of dipeptides because of its speed, high sensitivity, and ease of identification. Dipeptides, however, must be derivatized prior to their gc analysis. Various derivatization methods have been suggested and successfully used for this purpose (5,6,2234). Trimethylsilylation seems to be the most useful derivatization method since it can be easily carried out and gives derivatives with good chromatographic properties and easily interpretable mass spectra (33). This method has been widely used for gc analysis of amino acids (35,36) and dipeptides (23,25,29,33,34) utilizing various silylation reagents. The papers listed above on the gc analysis of dipeptides have reported separations using conventional packed columns. Gas chromatography with capillary columns (high-resolution gas chromatography), however, offers great advantages over gc with packed columns for separating very complex mixtures (37-40). Moreover, recent advances in column technology have brought into existence capillary 0003-2697/80/160269-05$02.00/O Copyright All rights
0 1980 by Academic Press, Inc. of reproduction in any form reserved.
270
DIZDAROGLU
columns drawn from fused silica (41). These columns seem to be superior to glass capillary columns in terms of flexibility, overall inertness, and efficiency (41). In this paper we describe the separation of trimethylsilyl (TMS) derivatives of dipeptides by high-resolution gas chromatography using a fused silica capillary column. MATERIALS
AND METHODS
Chemicals and reagents. Dipeptides were purchased from Sigma and Research Plus Laboratories. Bis(trimethylsilyl)trifluoroacetamide (BSTFA) and acetonitrile were obtained from Pierce Chemical Company. The test mixture for column evaluation was purchased from Alltech Association. Trimethylsilylation. A sample of ca. 0.5 mg of each dipeptide was placed in a Tefloncapped Hypo-Vial (Pierce Chemical Co,) and trimethylsilylated with 0.4 ml of a mixture of BSTFA and acetonitrile (1: 1) by heating for 30 min at 120°C in a sand bath. Gas chromatography. A Hewlett-Packard Model 5880A microprocessor controlled gas chromatograph equipped with a flame ionization detector was used. The injection port and the detector were maintained at 250°C. Helium was used as the carrier gas at an inlet pressure of 0.75 bar. The split ratio was 1:lOO and the flow through the column 0.6 ml/min. Separations were obtained by using a fused silica capillary column (12 m, 0.2 mm i.d.) coated (WCOT) with SP-2100 (methyl silicone liquid phase). It was purchased from Hewlett-Packard. The measured efficiency of the column was ca. 4200 plates per meter based on pentadecane peak at 130°C (k’ = 5.13; linear velocity, ,% = 35.7 cm/s). The ratio of 2,6dimethylphenol to 2,6-dimethylaniline (42) was 1.006. RESULTS AND DISCUSSION
Figure 1 shows the separation of TMS derivatives of over 50 dipeptides. Peak identification is given in Table I. Some im-
AND SIMIC
purities were noted when running individual standard compounds; these appeared at several places on Fig. 1 (such as between peaks 6 and 7). Excellent peak symmetry and reproducibility were obtained for all dipeptides except for those containing histidine and tryptophan. Derivatives of such dipeptides have been reported to be the least volatile and most troublesome compounds for gc analysis (32,33). Under our experimental conditions tailing peaks were obtained for TMS derivatives of such compounds (peaks 34,44,52, and 55). It is interesting to note that L-His-Gly (peak 34) and L-His-L-Ala (peak 44) produced tailing peaks whereas Gly-L-His (peak 53) and L-Ala-L-His (peak 45) showed no tailing behavior. L-Trp-Gly (peak 50) gave a symmetrical peak. It has been reported that derivatives of dipeptides containing arginine were not sufficiently volatile for gc analysis and did not elute from the gc column (29,32,33). Such compounds could be chromatographed after conversion of arginine to ornithine (33). Dipeptides containing arginine were not studied in this work. Dipeptides containing glycine as the first amino acid gave two peaks due to the substitution of the a-amino group of glycine by one or two TMS groups (33,35,43). The second peak corresponds to the derivative with two TMS groups. In some cases the first peak disappears completely as in the case of Gly-L-Val (peak 23) and Gly-LHis (peak 53) or decreases as in the case of Gly-L-Thr (peaks 6 and 20) and Gly-L-Ile (peaks 13 and 26) after storage of the sample a few hours at room temperature. Dipeptides containing glycine as the second amino acid gave only one peak. L-AlaL-Asn also formed two derivatives (peaks 19 and 26). This is probably due to the trimethylsilylation of the o-amino group of asparagine to different extents. This amino acids has been reported to yield two peaks after trimethylsilylation (36). DL-Ala-DL-Ser and DL-Leu-DL-Phe
271
CAPILLARY GAS CHROMATOGRAPHY OF DIPEPTIDES
5
10
15
20
25
min 230
160
100
lC
FIG. 1. Separation of TMS derivatives of dipeptides by high-resolution gc. Column; fused silica SP2100, 12 m, 0.2 mm i.d. programmed 4Vmin from 100 to 160°C 6”Cimin from 160 to 230°C and maintained at 230°C for 15 min. For other column details see Materials and Methods. Peak identification is given in Table 1. Identification was achieved by comparison of retention times of individually injected compounds. Each peak corresponds to about O.l- 1 ng of dipeptide.
gave two peaks which were completely separated (peaks 15, 16 and peaks 39, 41, respectively). In the case of these compounds a trimethylsilylation of the amino groups of alanine and leucine to different extents cannot be expected because of the steric hindrance and it has not been observed before with the free amino acids. Carboxyl groups and the OH group of serine are substituted by only one TMS group. A recent hplc investigation of DL,DLdipeptides (21) showed that DL-Ala-DL-Ser and DL-Leu-DL-phe gave two peaks which correspond to DL-, LD- and DD-, IL-isomers, respectively. Based on these results, the occurrence of two gc peaks can possibly be explained by separation of optical isomers of these dipeptides. However, this explanation cannot be established without examining all possible four optical isomers, which were not available to us. Separation of sequence isomeric dipeptides. The resolution of the sequence isomerit dipeptides is very important for the
sequencing of peptides. In this paper, several pairs of sequence isomeric dipeptides were examined and found to be separable. A complete separation of the pairs L-Ala-L-Leu (peak 8)/L-Leu-L-Ala (peak 9) and L-Ser-L-Ala (peak 14)/~~Ala-DL-Ser (peaks 15 and 16) was obtained. The other pairs were well resolved (see peak 28/29,31/32, 37138, 44145, and 471 48 in Fig. 1 and Table 1). Sequence isomeric dipeptides containing glycine were not separable (peaks 3, 11, and 21). However, they can be easily distinguished since the isomer containing glycine as the first amino acid gives two derivatives (compare peaks 3 and 24, 11 and 36, 21 and 43) or one derivative with two TMS groups at the aamino group (compare peaks 2 and 23, 34 and 53). CONCLUSIONS
The results obtained in this paper clearly demonstrate that complex mixtures of di-
272
DIZDAROGLU
AND SIMIC
TABLE PEAK
Peak No. 1 2 3
Dipeptide
4 5 6 7 8 9 10 11 12 13 14 15 16 17
IDENTIFICATION
Peak No.
Gly-Gly L-Val-Gly Gly-L-Leu L-Leu-Gly L-Ala-L-Ala Gly-L-Ser Gly-L-Thr L-Ala-L-Val L-Ala-r-Leu L-Ala-L-Be L-Leu-L-Ala cu-L-Asp-Gly Gly-L-Met L-Met-Gly Gly-r-Asp Gly-L-Be L-val-L-val L-Ser-L-Ala DL-Ala-DL-Ser DL-Ala-DL-Ser L-Ala-t-Thr
18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
peptides can be separated into their components by capillary gas chromatography after trimethylsilylation. The method offers great advantages in terms of speed, sensitivity, peak symmetry, reproducibility and separation of sequence isomers. It can be a useful tool for sequence analysis of peptides by using the DAP-gc/ms method. ACKNOWLEDGMENT M.D. acknowledges the Fellowship granted by National Academy of Sciences/National Research Council.
REFERENCES 1. McDonald, J. K., Zeitman, B. B., Reilly, T. J., and Ellis, S. (1969) J. Biol. Chem. 244, 2693-2709. 2. Edman, P. (1956) Acta Chem. Stand. 10, 761-768. 3. McDonald, J. K., Callahan, P. X., and Ellis, S. (1972) in Methods in Enzymology (Hirs, C. H. W., and Timasheff, S. N., eds.), Vol. 25, pp. 272-298, Academic Press, New York. 4. Lindley, H. (1972) Biochem. J. 126, 683-688. 5. Caprioli, R. M., and Seifert, W. E. (1975)Biochem. Biophys.
Res.
Commun.
64,295-303.
1 IN FIG.
1
Dipeptide Gly-L-Ser L-Ala-L-Asn Gly -L-Thr Gly-L-Phe L-Phe-Gly Gly - L-Glu Gly-L-Val L-Leu-L-Leu Gly-L-Leu L-Ala-L-Asp Gly-L-Be L-Ala-t.-Asn L-Leu-L-Ser L-Ala-L-Met L-Met-r-Ala LrAla- L-Glu t.-Phe-r-Ala L-Ala-L-Phe y-L-Glu-L-Leu Gly-~-Asp L-His-Gly
Peak No.
Dipeptide
35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55
L-Ser-L-Met Gly-L-Met L-Phe - r-Val L-Val-L-Phe DL-Leu-DL-Phe Gly - L-Glu DL-Leu-DL-Phe L-Ser-L-Phe Gly - L-Phe L-His-L-Ala L-Ala-L-His L-Met-L-Met L-Ala- L-Tyr L-Tyr- L-Ala L-Tyr-Gly L-Trp-Gly L-Met-L-Phe r-His-L-Ser Gly-~-His L-Phe-t.-Phe L-Trp-L-Ala
Seifert, W. E., and Caprioli, R. M. (1978) Biochemistry 17, 436-441. 7. Manning, J. M., and Moore, S. (1%8) J. Biol. Chem. 243, 5591-5597. 8. Callahan, P. X., Shepard, J. A., Reilly, T. J., McDonald, J. K., and Ellis, S. (1970) Anal. 6.
Biochem. 9.
38, 330-356.
Heatcote, J. G., Washington, R. .I., Keogh, B. J., and Glanville, R. W. (1972) J. Chromatogr. 65, 397-405.
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