- Email: [email protected]
[6] Manual Edman sequencing techniques for proteins and peptides at the nanomole level
[6]
MANUAL EDMAN SEQUENCING TECHNIQUES
27
was about 12 pmol (230 rig) of interferon. Even though the response per mole for ammonia is close to one-thousandth that of a typical amino acid, the ammonia peak is the largest one in the chromatogram. Analysis of the basic amino acids would not be possible if ninhydrin or o-phthalaldehyde were used for detection, owing to the tremendous ammonia peak that would ensue. With this procedure it is not necessary to extract the protein from the gel in order to carry out amino acid analysis. Extraction from the gel typically gives poor recovery of protein and introduces amino acid contamination. This procedure offers a unique opportunity to characterize a protein. In many instances gel electrophoresis is the simplest method for separating a protein from a mixture, and it is applicable at the low microgram level. In our studies on purified human fibroblast interferon, a minor contaminant of about 40,000 daltons was detected on gels. Amino acid analysis revealed it to be a dimer of interferon by its identity in composition with the 20,000-dalton band. 2 2 H. J. Friesen, S. Stein, M. Evinger, P. C. Familletti, J. Moschera, J. Meienhofer, J. Shively, a n d S. Pestka, Arch. Biochem. Biophys. 206, 432 (1981).
[6] M a n u a l E d m a n S e q u e n c i n g T e c h n i q u e s f o r P r o t e i n s a n d Peptides at the Nanomole Level B y W A R R E N P. L E V Y
A major obstacle in determining the primary sequence of proteins has been the availability of sufficient amounts of pure material for sequencing. Standard techniques for automatic or manual sequencing that use the Edman reaction 1 require milligram amounts of protein. Purification protocols for many proteins yield only microgram amounts of homogeneous material, necessitating the development of new methods for sequence analysis. The manual dansyl-Edman technique has been applied successfully to peptides 2"3 and intact proteins 4 at the nanomole level. However, this technique requires the removal of an aliquot of the peptide at each cycle for ' P. E d m a n , Acta Chem. Scand. 4, 283 (1950). C. J. Bruton and B. S. Hartley, J. Mol. Biol. 52, 165 (1970). a W. R. Gray and J. F. Smith, Anal. Biochem. 33, 36 (1970). 4 A. M. Weiner, T. Platt, and K. Weber, J. Biol. Chem. 247, 3242 (1972).
METHODS IN ENZYMOLOGY,VOL. 79
Copyright © 1981by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181979-~
28
CHROMATOGRAPHY AND ANALYSIS OF INTERFERONS
[6]
hydrolysis and subsequent analysis, increasing the repetitive losses norreally encountered in Edman sequencing. The tremendous sensitivity of the method, which has been the major reason for its widespread use, has been approached by recent improvements in the analysis of subnanomolar quantities of phenylthiohydantoin (PTH) amino acids. 5-7 Automatic spinning cup sequenators have been successfully remodeled for microsequence analysis after extraordinary investments of time, manpower, and resources, s-l° Although these automatic methods are clearly required for long peptides or intact proteins, a simple manual technique would be beneficial in the analysis of small peptides or of the first few residues of a large peptide. We report here a manual microsequencing strategy that has been used to determine the primary sequence of nanomolar and subnanomolar quantities of small peptides (nine amino acids or less) produced by tryptic digestions of human leukocyte interferon.11 An excellent theoretical analysis of the Edman chemistry and typical manual sequencing procedures has already been published ~ and will not be repeated here. Materials Polypropylene microcentrifuge tubes (1.5 ml; Walter Sarstedt, Inc., Princeton, New Jersey) are used exclusively throughout this procedure to minimize losses of peptides due to adsorption. Ultraviolet-absorbing impurities are removed from the tubes by washing with acid. Tubes are soaked in 2 N HC1 for at least 4 hr, rinsed in ultrapure water, and airdried. The polypropylene tubes are preferable to acid-washed glass vials, which have been shown to adsorb large quantities of peptides and to inhibit the extraction and washing steps by selectively adsorbing the emulsified droplets produced during vigorous mixing. All manipulations of liquids are done with polypropylene micropipette tips (Fisher Scientific Co., Springfield, New Jersey). Phenylisothiocyanate (PITC), trifluoroacetic acid (TFA), and N,Ndimethyl-N-allylamine (DMAA) are Sequenal grade from Pierce Chemical C. L. Zimmerman, E. Apella, and J. J. Pisano, Anal. Biochem. 77, 569 (1977). M. Abrahamsson, K. Gr6ningsson, and S. Castensson, J. Chromatogr. 154, 313 (1978). A. J. Bhown, J. E. Mole, A. Weissinger, and J. C. Bennett, J. Chromatogr. 148, 532 (1978). 8 B. Wittmann-Liebold, Hoppe Seyler's Z. Physiol. Chem. 354, 1415 (1973). a M. W. Hunkapiller and L. E. Hood, Biochemistry 17, 2124 (1978). 10 M. W. Hunkapiller and L. E. Hood, Science 207, 523 (1980). 1~ W. P. Levy, M. Rubinstein, J. Shively, U. Del Valle, C.-Y. Lai, J. Moschera, L. Brink, L. Gerber, S. Stein, and S. Pestka, in preparation. tz G. E. Tarr, this series, Vol. 47, p. 335.
[6]
MANUAL EDMAN SEQUENCING TECHNIQUES
29
Co., Rockford, Illinois. Heptane, ethyl acetate, and acetonitrile are from Burdick and Jackson Laboratories, Inc., Muskegon, Michigan. Pyridine is distilled over ninhydrin. Water is purified by glass distillation followed by filtration through charcoal and mixed-bed deionizer cartridges (Hydro Service and Supplies, Inc., Durham, North Carolina). It has been our experience that the use of ultrapure solvents is the most important prerequisite for reproducible results. Phenylthiohydantoin amino acids are identified by high-performance liquid chromatography (HPLC) with a system (Altex Scientific, Berkeley, California) consisting of an Ultrasphere ODS column (5/zm), Model 420 microprocessor, two Model 110-A pumps, and a Model 153 UV detector. Samples are dried under vacuum with a Speed Vac concentrator (Model SVC100, Savant Instruments, Inc., Hicksville, New York). All centrifugation steps are for 1 min in an Eppendorf microcentrifuge (Model 5412, Brinkmann Instruments, Inc., Westbury, New York). Procedures
Sample Preparation. The tryptic peptide (1-10 nmol) is transferred to an acid-washed 1.5-ml polypropylene microcentrifuge tube. Norleucine (25-50 nmol) is added as a carrier and internal standard to monitor the efficiency of the reactions. The sample is dried under vacuum, and the tube is flushed with argon gas. Nitrogen gas can be used also, but argon is heavier than air and will remain in the tube when the cap is removed. Peptides have been stored for at least 2 weeks under these conditions with no observable loss in coupling efficiency. PITC Coupling. The peptide is suspended in 40/xl of DMAA buffer (15 ml of pyridine, 1.18 ml of DMAA, 10 ml of H20, pH adjusted to 9.5 with TFA). This buffer provides a higher yield of coupled peptides than others that have been examined. Triethylamine and trimethylamine give inferior yields, presumably owing to the presence of decomposition products that accumulate upon storage and may inhibit the reaction. The vial is flushed with argon gas, and immediately thereafter 3 ~1 of PITC are added. The reaction proceeds for 30 min at 50°. Excess reagent is removed by washing twice with 150 ~1 of heptaneethyl acetate (10 : 1) and once with 200/zl of heptane-ethyl acetate (2 : 1). Vigorous mixing facilitates removal of the excess reagent and by-products of the reaction. Centrifugation is required for adequate separation of the two phases. The organic phase is discarded after each wash, and the aqueous phase is dried under vacuum after washing is complete. Cleavage. Trifluoroacetic acid is used for cleavage. Anhydrous HC1 has been suggested as a better reagent, 12 but we have found TFA to be adequate for most peptides. Twenty microliters of TFA are added to the
30
CHROMATOGRAPHY AND ANALYSIS OF INTERFERONS
[6]
RESOLUTION OF PHENYLTHIOHYDANTOIN (PTH) AMINO ACIDS BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHYa
PTH amino acid
Elution time (min)
Asp Ash Glu Ser Gin Thr His Gly Arg Ala Tyr Met Vai Pro Trp Phe Ile Lys Leu
3.8 6.5 7.0 7.4 8.0 8.5 9.0 9.4 12.7 13.0 15.1 20.2 20.5 20.7 23.4 25.5 26.3 26.6 27.6
a The gradient buffers are 10 mM sodium acetate, pH 4.5 (buffer A) and 5 mM sodium acetate, pH 4.5, 50% acetonitrile (buffer B). The PTH amino acids are eluted with a linear gradient of 40 to 75% B for 12.5 min followed by isocratic elution at 75% B for 17.5 min. The column temperature is maintained at 62L These conditions provide a maximum sensitivity of approximately 20 pmol. vial, a n d t h e s a m p l e is i n c u b a t e d at 50 ° f o r 20 m i n . T h e s a m p l e is t h e n dried under vacuum. Extraction. F o r t y m i c r o l i t e r s o f 30% p y r i d i n e a r e a d d e d to d i s s o l v e t h e s a m p l e , a n d t h e a n i l i n o t h i a z o l i n o n e ( A T Z ) a m i n o a c i d is s e p a r a t e d f r o m t h e r e s i d u a l p e p t i d e b y e x t r a c t i n g t h r e e t i m e s w i t h 150-/~1 p o r t i o n s o f b e n z e n e - - e t h y l a c e t a t e ( 1 : 2 ) . C e n t r i f u g a t i o n is r e q u i r e d a f t e r m i x i n g to provide adequate phase separation.
[7]
SEQUENCING WITH SPINNINCrCUP SEQUENATOR
31
The organic layers from each extraction are pooled and dried under vacuum. The aqueous layer containing the peptide is supplemented with 25-50 nmol of norleucine and dried under vacuum. After flushing with argon gas, a second cycle of Edman degradation can be performed as described above. Conversion. The dried organic phase containing the ATZ amino acid is suspended in 40/~1 of 1 N HCI. Conversion of the ATZ amino acid to the PTH derivative of the amino acid is complete after 10 min at 80°. The PTH amino acid is removed from the aqueous phase by three extractions with 50-/xl portions of ethyl acetate. The ethyl acetate layers are pooled, dried under vacuum, and suspended in 30 #1 of acetonitrile. Identification of PTH Amin O Acid. The PTH amino acids are identified with an HPLC system described under Materials. The individual PTH amino acids are resolved within 30 min under our experimental conditions (see the table). Discussion The microsequencing strategy described here has been used to determine the complete primary sequence of approximately 1-nmol amounts of two pentapeptides produced by tryptic digestion of human leukocyte interferon, and partial sequence data on a number of larger peptides. H This methodology is generally applicable for microsequencing of nanomole and subnanomole amounts of peptides. Acknowledgments The author would like to thank S. Stein and S. Udenfriend for use of their facilities, J. Moschera for the HPLC system, L. Gerber and L. Brink for expert technical assistance, S. Kimura, M. Rubinstein, and R. Lewis for helpful suggestions, and S. Pestka for support of this research.
[7] S e q u e n c e D e t e r m i n a t i o n s o f P r o t e i n s a n d P e p t i d e s a t t h e Nanomole and Subnanomole Level with a Modified Spinning Cup Sequenator By JOHN E. SHIVELY
The most widely used approach to determine extended amino acid sequences of proteins or peptides is the Edman degradation scheme/ which was published in automated form in 1967 by Edman and Begg. 2 The P. E d m a n , Acta Chem. Scand. 4, 283 (1950). 2 p. E d m a n an d G. Begg, Eur. J. Biochem. 1, 80 (1967).
METHODS IN ENZYMOLOGY, VOL. 79
Copyright © 1981by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181979-5
MANUAL EDMAN SEQUENCING TECHNIQUES
27
was about 12 pmol (230 rig) of interferon. Even though the response per mole for ammonia is close to one-thousandth that of a typical amino acid, the ammonia peak is the largest one in the chromatogram. Analysis of the basic amino acids would not be possible if ninhydrin or o-phthalaldehyde were used for detection, owing to the tremendous ammonia peak that would ensue. With this procedure it is not necessary to extract the protein from the gel in order to carry out amino acid analysis. Extraction from the gel typically gives poor recovery of protein and introduces amino acid contamination. This procedure offers a unique opportunity to characterize a protein. In many instances gel electrophoresis is the simplest method for separating a protein from a mixture, and it is applicable at the low microgram level. In our studies on purified human fibroblast interferon, a minor contaminant of about 40,000 daltons was detected on gels. Amino acid analysis revealed it to be a dimer of interferon by its identity in composition with the 20,000-dalton band. 2 2 H. J. Friesen, S. Stein, M. Evinger, P. C. Familletti, J. Moschera, J. Meienhofer, J. Shively, a n d S. Pestka, Arch. Biochem. Biophys. 206, 432 (1981).
[6] M a n u a l E d m a n S e q u e n c i n g T e c h n i q u e s f o r P r o t e i n s a n d Peptides at the Nanomole Level B y W A R R E N P. L E V Y
A major obstacle in determining the primary sequence of proteins has been the availability of sufficient amounts of pure material for sequencing. Standard techniques for automatic or manual sequencing that use the Edman reaction 1 require milligram amounts of protein. Purification protocols for many proteins yield only microgram amounts of homogeneous material, necessitating the development of new methods for sequence analysis. The manual dansyl-Edman technique has been applied successfully to peptides 2"3 and intact proteins 4 at the nanomole level. However, this technique requires the removal of an aliquot of the peptide at each cycle for ' P. E d m a n , Acta Chem. Scand. 4, 283 (1950). C. J. Bruton and B. S. Hartley, J. Mol. Biol. 52, 165 (1970). a W. R. Gray and J. F. Smith, Anal. Biochem. 33, 36 (1970). 4 A. M. Weiner, T. Platt, and K. Weber, J. Biol. Chem. 247, 3242 (1972).
METHODS IN ENZYMOLOGY,VOL. 79
Copyright © 1981by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181979-~
28
CHROMATOGRAPHY AND ANALYSIS OF INTERFERONS
[6]
hydrolysis and subsequent analysis, increasing the repetitive losses norreally encountered in Edman sequencing. The tremendous sensitivity of the method, which has been the major reason for its widespread use, has been approached by recent improvements in the analysis of subnanomolar quantities of phenylthiohydantoin (PTH) amino acids. 5-7 Automatic spinning cup sequenators have been successfully remodeled for microsequence analysis after extraordinary investments of time, manpower, and resources, s-l° Although these automatic methods are clearly required for long peptides or intact proteins, a simple manual technique would be beneficial in the analysis of small peptides or of the first few residues of a large peptide. We report here a manual microsequencing strategy that has been used to determine the primary sequence of nanomolar and subnanomolar quantities of small peptides (nine amino acids or less) produced by tryptic digestions of human leukocyte interferon.11 An excellent theoretical analysis of the Edman chemistry and typical manual sequencing procedures has already been published ~ and will not be repeated here. Materials Polypropylene microcentrifuge tubes (1.5 ml; Walter Sarstedt, Inc., Princeton, New Jersey) are used exclusively throughout this procedure to minimize losses of peptides due to adsorption. Ultraviolet-absorbing impurities are removed from the tubes by washing with acid. Tubes are soaked in 2 N HC1 for at least 4 hr, rinsed in ultrapure water, and airdried. The polypropylene tubes are preferable to acid-washed glass vials, which have been shown to adsorb large quantities of peptides and to inhibit the extraction and washing steps by selectively adsorbing the emulsified droplets produced during vigorous mixing. All manipulations of liquids are done with polypropylene micropipette tips (Fisher Scientific Co., Springfield, New Jersey). Phenylisothiocyanate (PITC), trifluoroacetic acid (TFA), and N,Ndimethyl-N-allylamine (DMAA) are Sequenal grade from Pierce Chemical C. L. Zimmerman, E. Apella, and J. J. Pisano, Anal. Biochem. 77, 569 (1977). M. Abrahamsson, K. Gr6ningsson, and S. Castensson, J. Chromatogr. 154, 313 (1978). A. J. Bhown, J. E. Mole, A. Weissinger, and J. C. Bennett, J. Chromatogr. 148, 532 (1978). 8 B. Wittmann-Liebold, Hoppe Seyler's Z. Physiol. Chem. 354, 1415 (1973). a M. W. Hunkapiller and L. E. Hood, Biochemistry 17, 2124 (1978). 10 M. W. Hunkapiller and L. E. Hood, Science 207, 523 (1980). 1~ W. P. Levy, M. Rubinstein, J. Shively, U. Del Valle, C.-Y. Lai, J. Moschera, L. Brink, L. Gerber, S. Stein, and S. Pestka, in preparation. tz G. E. Tarr, this series, Vol. 47, p. 335.
[6]
MANUAL EDMAN SEQUENCING TECHNIQUES
29
Co., Rockford, Illinois. Heptane, ethyl acetate, and acetonitrile are from Burdick and Jackson Laboratories, Inc., Muskegon, Michigan. Pyridine is distilled over ninhydrin. Water is purified by glass distillation followed by filtration through charcoal and mixed-bed deionizer cartridges (Hydro Service and Supplies, Inc., Durham, North Carolina). It has been our experience that the use of ultrapure solvents is the most important prerequisite for reproducible results. Phenylthiohydantoin amino acids are identified by high-performance liquid chromatography (HPLC) with a system (Altex Scientific, Berkeley, California) consisting of an Ultrasphere ODS column (5/zm), Model 420 microprocessor, two Model 110-A pumps, and a Model 153 UV detector. Samples are dried under vacuum with a Speed Vac concentrator (Model SVC100, Savant Instruments, Inc., Hicksville, New York). All centrifugation steps are for 1 min in an Eppendorf microcentrifuge (Model 5412, Brinkmann Instruments, Inc., Westbury, New York). Procedures
Sample Preparation. The tryptic peptide (1-10 nmol) is transferred to an acid-washed 1.5-ml polypropylene microcentrifuge tube. Norleucine (25-50 nmol) is added as a carrier and internal standard to monitor the efficiency of the reactions. The sample is dried under vacuum, and the tube is flushed with argon gas. Nitrogen gas can be used also, but argon is heavier than air and will remain in the tube when the cap is removed. Peptides have been stored for at least 2 weeks under these conditions with no observable loss in coupling efficiency. PITC Coupling. The peptide is suspended in 40/xl of DMAA buffer (15 ml of pyridine, 1.18 ml of DMAA, 10 ml of H20, pH adjusted to 9.5 with TFA). This buffer provides a higher yield of coupled peptides than others that have been examined. Triethylamine and trimethylamine give inferior yields, presumably owing to the presence of decomposition products that accumulate upon storage and may inhibit the reaction. The vial is flushed with argon gas, and immediately thereafter 3 ~1 of PITC are added. The reaction proceeds for 30 min at 50°. Excess reagent is removed by washing twice with 150 ~1 of heptaneethyl acetate (10 : 1) and once with 200/zl of heptane-ethyl acetate (2 : 1). Vigorous mixing facilitates removal of the excess reagent and by-products of the reaction. Centrifugation is required for adequate separation of the two phases. The organic phase is discarded after each wash, and the aqueous phase is dried under vacuum after washing is complete. Cleavage. Trifluoroacetic acid is used for cleavage. Anhydrous HC1 has been suggested as a better reagent, 12 but we have found TFA to be adequate for most peptides. Twenty microliters of TFA are added to the
30
CHROMATOGRAPHY AND ANALYSIS OF INTERFERONS
[6]
RESOLUTION OF PHENYLTHIOHYDANTOIN (PTH) AMINO ACIDS BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHYa
PTH amino acid
Elution time (min)
Asp Ash Glu Ser Gin Thr His Gly Arg Ala Tyr Met Vai Pro Trp Phe Ile Lys Leu
3.8 6.5 7.0 7.4 8.0 8.5 9.0 9.4 12.7 13.0 15.1 20.2 20.5 20.7 23.4 25.5 26.3 26.6 27.6
a The gradient buffers are 10 mM sodium acetate, pH 4.5 (buffer A) and 5 mM sodium acetate, pH 4.5, 50% acetonitrile (buffer B). The PTH amino acids are eluted with a linear gradient of 40 to 75% B for 12.5 min followed by isocratic elution at 75% B for 17.5 min. The column temperature is maintained at 62L These conditions provide a maximum sensitivity of approximately 20 pmol. vial, a n d t h e s a m p l e is i n c u b a t e d at 50 ° f o r 20 m i n . T h e s a m p l e is t h e n dried under vacuum. Extraction. F o r t y m i c r o l i t e r s o f 30% p y r i d i n e a r e a d d e d to d i s s o l v e t h e s a m p l e , a n d t h e a n i l i n o t h i a z o l i n o n e ( A T Z ) a m i n o a c i d is s e p a r a t e d f r o m t h e r e s i d u a l p e p t i d e b y e x t r a c t i n g t h r e e t i m e s w i t h 150-/~1 p o r t i o n s o f b e n z e n e - - e t h y l a c e t a t e ( 1 : 2 ) . C e n t r i f u g a t i o n is r e q u i r e d a f t e r m i x i n g to provide adequate phase separation.
[7]
SEQUENCING WITH SPINNINCrCUP SEQUENATOR
31
The organic layers from each extraction are pooled and dried under vacuum. The aqueous layer containing the peptide is supplemented with 25-50 nmol of norleucine and dried under vacuum. After flushing with argon gas, a second cycle of Edman degradation can be performed as described above. Conversion. The dried organic phase containing the ATZ amino acid is suspended in 40/~1 of 1 N HCI. Conversion of the ATZ amino acid to the PTH derivative of the amino acid is complete after 10 min at 80°. The PTH amino acid is removed from the aqueous phase by three extractions with 50-/xl portions of ethyl acetate. The ethyl acetate layers are pooled, dried under vacuum, and suspended in 30 #1 of acetonitrile. Identification of PTH Amin O Acid. The PTH amino acids are identified with an HPLC system described under Materials. The individual PTH amino acids are resolved within 30 min under our experimental conditions (see the table). Discussion The microsequencing strategy described here has been used to determine the complete primary sequence of approximately 1-nmol amounts of two pentapeptides produced by tryptic digestion of human leukocyte interferon, and partial sequence data on a number of larger peptides. H This methodology is generally applicable for microsequencing of nanomole and subnanomole amounts of peptides. Acknowledgments The author would like to thank S. Stein and S. Udenfriend for use of their facilities, J. Moschera for the HPLC system, L. Gerber and L. Brink for expert technical assistance, S. Kimura, M. Rubinstein, and R. Lewis for helpful suggestions, and S. Pestka for support of this research.
[7] S e q u e n c e D e t e r m i n a t i o n s o f P r o t e i n s a n d P e p t i d e s a t t h e Nanomole and Subnanomole Level with a Modified Spinning Cup Sequenator By JOHN E. SHIVELY
The most widely used approach to determine extended amino acid sequences of proteins or peptides is the Edman degradation scheme/ which was published in automated form in 1967 by Edman and Begg. 2 The P. E d m a n , Acta Chem. Scand. 4, 283 (1950). 2 p. E d m a n an d G. Begg, Eur. J. Biochem. 1, 80 (1967).
METHODS IN ENZYMOLOGY, VOL. 79
Copyright © 1981by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181979-5