- Email: [email protected]
Tryptic peptide mapping of picomolar quantities of protein labeled with the Bolton-Hunter reagent
ANALYTICAL
116, 473-479
BIOCHEMISTRY
(1981)
Tryptic Peptide Mapping of Picomolar Quantities of Protein Labeled with the Bolton-Hunter Reagent MARK *Department
A. FEITELSON,
of k4icrobiology
*-I FELIX
0. WETTSTEIN,**~
AND JACK G. STEVENS*
and Immunology. UCLA School of Medicine, University of California. Los Angeles, California Received
January
and tMolecular 90024
Biology
Institute.
5, 1981
Characterization of 5 to 25 pmol of purified proteins by tryptic peptide mapping has been accomplished using the Bolton-Hunter reagent (‘*‘I-3-[4-hydroxyphenyllpropionic acid Nhydroxysuccinimide ester). Radioacylation is followed by reaction with unlabeled ester and reductive methylation to ensure resistance of lysyl residues to trypsinization. Reduced and alkylated proteins are analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, trypsinized from individual gel slices, and mapped two-dimensionally on thin layers. The method permits peptide mapping of proteins with specific activities of I to 2 X IO4 cpm/ng, results in more spots (and often more structural information) than direct iodination procedures, and can be used for characterization of proteins that could not be biosynthetically labeled.
Peptide mapping of proteins has become an important tool in protein characterization in combination with analysis by sodium dodecyl sulfateepolyacrylamide gel electrophoresis. A special problem is posed when proteins of interest are derived from whole animals or for other reasons cannot be biosynthetically labeled to high specific activities and are available only in small quantities. Protein iodination has been used to increase the sensitivity of peptide mapping but has the disadvantage that only tyrosinecontaining peptides can be detected. We have developed an alternate approach employing ‘251-labeled 3-( 4-hydroxyphenyl)propionic acid N-hydroxysuccinimide ester ( Bolton-Hunter reagent) ( 1) which labels the N-terminus and the more common lysyl residues. MATERIALS
AND
METHODS
Preparation of the Bolton-Hunter reagent and acylation of model proteins. pHydroxyphenylpropionic ’ Present Department of Medicine,
address: Division of of Medicine, Stanford Palo Alto, California
acid
N-hydroxy-
Infectious University
Diseases, School
succinimide ester (0.2-0.5 /*g) (TAGIT, Calbiochem) dissolved in 1 to 2 J of dioxane (Matheson, Coleman, and Bell) was added to 50 /*I of 0.5 M sodium phosphate buffer (Sigma), pH 7.5, containing 0.5-1.0 mCi of ‘*‘I (New England Nuclear) and 100 pg of freshly prepared chloramine-T (Eastman). Iodination was carried out in a I-ml screwcap vial, at room temperature, on a magnetic stirrer. The labeled ester was immediately extracted by addition of 5 ~1 dimethylformamide and 100 ~1 of benzene (Baker), the aqueous phase removed, and the organic phase transferred to a 6 X 50-mm kimax vial (Kimble) and quickly dried over a gentle stream of air. To prevent excessive hydrolysis, labeling and extraction were carried out within 40 s ( 1). Protein (250 ng) [bovine serum albumin ( BSA,2 Miles), ovalbumin, bovine chymotrypsinogen, or hen egg lysozyme (HEL, Worthington)] in 2 to 2.5 ~1 of ice-cold sodium borate buffer (Sigma), pH 8.5, was added to the dried BoltonHunter reagent. The reaction was carried out for 1 h at 0°C and then at 4°C overnight. * Abbreviations used: H EL, hen egg lysozyme;
473
BSA, SDS,
bovine sodium
serum albumin; dodecyl sulfate.
0003-2697/81/140473-07$02.00/O
474
FEITELSON,
WETTSTEIN,
The protein was then further acylated at 0°C for 3 h with 100 /lg TAGIT added in 5 ~1 0.1 M sodium borate buffer or in 1 ~1 dioxane. Several radioacylation reactions were carried out with commercially available Bolton-Hunter reagent (New England Nuclear) for comparison. Reductive methylation, alkylation, and analysis by gel electrophoresis. Reductive methylation was essentially as described by Means and Feeney (2). After protein acylation, the pH was adjusted to 9.0 with 1.0 M sodium hydroxide. Cold, freshly prepared reagents in 0.1 M sodium borate buffer, pH 9.0, were added at 0°C as follows: 1 ~1 of guanidine-HCl (10 M), 1 ~1 sodium borohydride ( 10 mg/ml), and finally, 5 additions of 1 ~1 each of a formaldehyde solution (2 ~1 of 37% formaldehyde (Baker, analyzed reagent) in 150 ~1 0.1 M borate buffer) spaced at 5-min intervals. The sequence of borohydride and formaldehyde addition was repeated using a new batch of borohydride. Following the two cycles of reductive methylation, an additional l-111 portion of freshly prepared sodium borohydride was added to ensure complete reduction of all formaldehyde. With the exception of formaldehyde, all reagents used for reductive methylation were purchased from Sigma. The solution containing the acylated and reductively methylated protein was made up to 2% with sodium dodecyl sulfate (SDS, Bio-Rad) and 2 M with urea (Mallinckrodt) and incubated in boiling water for 2 min. The pH was then adjusted to 8.0 with 1 M HCl (Baker) and the protein reduced in 0.025 M dithiothreitol (Sigma), at 37°C for 1 h and alkylated in 0.075 M iodoacetamide (Sigma) at room temperature for 45 min. After alkylation, the labeled and modified proteins were separated from reagents by chromatography on P-4 (Bio-Rad) in 0.1 M ammonium bicarbonate (Matheson, Coleman, and Bell), 0.1% SDS, lyophilized, and stored at 4°C. Discontinuous SDS-electrophoresis was carried out by the method of Laemmli (3),
AND
STEVENS
using l-mm thick slab gels, consisting of a 15-cm 15% separating gel and a 4-cm 5% stacking gel. Lyophilized samples were made to 2% with SDS, 4 M with urea, 0.001% with bromophenol blue tracking dye, and boiled for 2 min. Electrophoresis was carried out at 30 V for the first half hour and 60 V for approximately 16-18 h until the dye was within 0.5 cm of the bottom of the gel. The separating gels were fixed with 10% acetic acid, 25% isopropanol overnight, dried under vacuum, and the protein bands located by autoradiography. Trypsinization andpeptide mapping. Protein bands were cut out, dried overnight under high vacuum, and hydrated with 0.5 ml of ammonium bicarbonate (0.2 M), pH 8.0, containing 25 hg of trypsin (chymotrypsinfree, Calbiochem). The heavy paper (Whatman 3MM) on which gels were dried down upon was scraped off of each dried-down, protein-containing gel slice to permit greater access of trypsin to the protein. Proteolysis of gel slices was carried out at 25 or 37°C overnight with gentle rocking of the tube rack. The supernatants were lyophilized and redigested to completion in 20 ~1 of 0.05 M sodium borate buffer, pH 8.5, with 25 pg trypsin. Finally, the peptides were spotted on 20 X 20-cm thin-layer cellulose plates [ (0.1 -mm cellulose on glass or 0.15-mm cellulose on polyethylene-terephthalate (Brinkman)]. About 1 ~1 of 0.3 mg/ml pyronine Y tracking dye (Eastman Organic Chemicals) was spotted just below the peptide sample. Immediately before electrophoresis, the plates were dampened with a spray of 0.8% acetic acid, 0.2% formic acid (Baker), pH 2, and placed inside a Plexiglas trough fitted with a cooling plate maintained at 15°C. The plates were connected to the electrode compartments containing 8% acetic acid, 2% formic acid with a double layer of Whatman 3MM paper, and then overlaid with 100 to 200 ml of n-heptane (Baker, analyzed reagent) to prevent water evaporation. Since n-heptane is flammable, the trough accommodating the n-heptane-overlaid, thin-layer
PEPTIDE
MAPPING
WITH
BOLTON-HUNTER
plates was covered with a glass plate to reduce evaporation of the organic solvent; therefore, electrophoresis should be performed in a well-ventilated room or in a fume hood. After electrophoresis for 2 h at 400 V and 4-16 mA, the plates were dried overnight at room temperature or for at least 1 h under a heat lamp followed by 30 min at room temperature. Thin-layer chromatography was ,then carried out in the second dimension at room temperature for approximately 4 h using n-butanol:water:ethyl acetate:acetic acid:pyridine at the ratio 120:60:20:4: 1 (v/v). After chromatography, the plates were dried, wrapped tightly in cellophane (Saran Wrap), and autoradiographed for 2 to 10 h at -70°C using Kodak RP/RZ Omat X-ray film and DuPont Cronex Xtra Life (lighting plus) intensifying screen (4). A.11 reagents used for chromatography were obtained from Baker. Chloramirw-T iodination and peptide mapping of model proteins. Two hundred fifty nanograms each of BSA, ovalbumin, chymotrypsinogen, and HEL dissolved in 10 ~1 of 0.05 M sodium phosphate buffer, pH 7.5, was added to 50 ~1 of 0.5 M sodium phosphate buffer, pH 7.5, and 0.5-1.0 mCi ‘25I in a I-ml screwcap vial, at room temperature, on a magnetic stirrer (5). Iodination was initiated by the addition of 100 pg chloramine-T and terminated after 1 min by the addition of 200 fig sodium metabisulfite (Sigma). Ten microliters of glycerol (Baker) and SDS to final concentrations of 10 and l%, respectively, were added and the proteins separated from free “‘1 on a P-4 column as described above. Reduction, alkylation, gel electrophoresis, and tryptic peptide mapping were carried out as described above, except that the chromatography solvent used was n-butanol:pyridine:acetic acid:water in the ratio 15:9: 3:9 (v/v). RESULTS
AND
DISCUSSION
BSA, ovalbumin, chymotrypsinogen, and HEL have lbeen labeled with the Bolton-
REAGENT
475
AB
FIG. I. SDS-electrophoresis profile of the four model proteins labeled by radioacylation or direct iodination. BSA (68K), ovalbumin (43K). chymotrypsinogen (25K), and HEL (14K) were prepared as described under Materiak and Methods. Approximately 250 ng (5-25 pmol) of each protein labeled by either procedure was analyzed by slab gel electrophoresis. Dried gels were exposed for autoradiography 30 min as outlined under Materials and Methods. (A) Bolton-Hunter-reagent-labeled proteins; (B) chloramine-T-iodinated proteins.
Hunter reagent and analyzed by gel electrophoresis and tryptic peptide mapping. For comparison, these same proteins have been labeled by direct iodination using chloramine-T and then similarly analyzed. Results from SDS gels show that, with the exception of chymotrypsinogen, the mobilities of the proteins were reduced following acylation in comparison to direct iodination (Fig. 1). Although the reason for the anomalous behavior of chymotrypsin remains to be clarified, it is likely that the slower mobility of the other proteins is due to acylation, which
476
FEITELSON,
WETTSTEIN,
AND
STEVENS
A
c
FIG. 2. Tryptic peptide mapping of labeled proteins. Proteins labeled by either method were separated by gel electrophoresis (Fig. 1) and trypsinized as described under Materials and Methods. Approximately 50-759~ of the cpm in each protein band was recovered following trypsinization of each dried gel slice. Trypsinization of gel slices at 37’C resulted in slightly higher recoveries than at 25°C. After lyophilization and redigestion, approximately 10% of the recovered counts ( IO5 cpm) were used for peptide mapping. Plates were exposed for autoradiography for 10 h. Freshly prepared or commercially obtained Bolton-Hunter reagent gave identical results. Electrophoresis is from right to left and the direction of chromatography is up. (A,B) BSA; (C,D) ovalbumin; (E,F) bovine chymotrypsinogen; (G,H) HEL. (A,C,E,G) Chloramine-T-iodinated; (B,D,F,H) BoltonHunter-reagent-labeled.
could add as much as several thousand daltons of molecular weight in each case. This potential change in mobility in gel electrophoresis has to be kept in mind when comparing acylated to nonacylated proteins. Peptide maps of Bolton-Hunter-reagentacylated and chloramine-T-iodinated proteins are presented in Fig. 2. The peptide maps of the Bolton-Hunter-reagent-labeled proteins (Figs. 2B, D, F, H) exhibit more
spots than those of chloramine-T-iodinated proteins, thus providing more detailed information. This should be the case, since primary amino groups are more frequent in most proteins than tyrosine residues, resulting in a larger number of peptides being labeled by acylation than by direct iodination. The theoretical number of labeled tryptic peptides for the two smaller proteins, bovine chymotrypsinogen and HEL, has been de-
PEPTIDE
MAPPING
WITH
G
BOLTON-HUNTER
REAGENT
477
H
FIG. 2 continued termined from their amino acid sequences (6) and is five for both when labeled by the method described here and four and three, respectively, when labeled by the ,chloramine-T method. The relatively large number of spots observed in the peptide maps of acylated protein digests could be accounted for by conditions dependent upon and independent from the radioacylation step. Some spots could have been generated as a consequence of the autocatalytic breakdown of trypsin, since during this process an enzymatically active species (pseudotrypsin) having chymotryptic-like activity is formed and may contribute up to several percent of
the total activity during proteolysis (7). Protein microheterogeneities resulting from the spontaneous deamination of asparagine and glutamine (8) and/or possible oxidation of cysteine to cysteic acid prior to reduction and alkylation (9) might also give rise to some of the faint spots on the autoradiograms of Fig. 2. The presence of these microheterogeneities and/or the action of pseudotrypsin is also compatible with the peptide maps of HEL and chymotrypsinogen labeled by direct iodination, where a number of minor spots are seen and the total number of spots on each map is slightly greater than the number predicted from the primary se-
478
FEITELSON.
WETTSTEIN.
quence of each protein. A major contributing factor to peptide heterogeneity in the Bolton-Hunter-reagent-labeled proteins is radioacylation itself. The large bulk and short half-life of the Bolton-Hunter reagent (halflife 9 min in aqueous media), combined with steric restrictions and/or differences in the intrinsic reactivities of epsilon amino groups, suggest that radioacylation does not go to completion. Residues neighboring epsilon amino groups may affect labeling, depending upon their charge and/or bulk. Since the extent of radioacylation versus hydrolysis is dependent upon the ratio of Bolton-Hunter reagent to protein as well as protein concentration (data not shown, lo), the reaction is driven as far to completion as possible by the addition of cold ester (TAGIT). The extent of chemical heterogeneity introduced into a protein as a result of radioacylation would vary depending upon these precise conditions of reaction. Further acylation with TAGIT serves to reproducibly limit this heterogeneity so that the presence and intensity of all the spots in a peptide map will remain uniform from experiment to experiment. In addition to causing small changes in the electrophoretic mobility of proteins on SDS gels and introducing chemical heterogeneity, acylation affects other properties of proteins and peptides. Acylation of epsilon and N-terminal amino groups removes the charge from those groups ( 11) which results in slightly retarded mobility of acylated peptides in relationship to unmodified peptides in the electrophoretic dimension. As pointed out under Materials and Methods, acylation changes the chromatographic properties of the tryptic peptides, since different chromatography solvents are employed for acylated and iodinated hydrolysates. Further, protein acylation results in derivatized lysyl residues which are resistant to trypsinization. This follows from studies in which replacement of the t amino hydrogens by methyl groups prevents trypsinization at lysyl peptide bonds (2,12). In our hands, a synthetic peptide with a single lysyl residue
AND
STEVENS
modified by acylation failed to cleave in the presence of trypsin (data not shown). This result is consistent with the presence of t amino hydrogens for recognition by the active site of trypsin (12). Since not all of the lysyl residues may become fully acylated, reductive methylation is employed to modify any remaining free amino groups in the protein, thereby ensuring that all of the lysyl residues become resistant to trypsinization. Reductive methylation, then, helps to prevent ambiguities in the number of spots seen on peptide maps by eliminating lysyl residues as substrates for trypsin. Proteolysis, then, occurs only at arginyl and alkylated cysteinyl residues. Since cysteinyl residues alkylated by iodoacetamide, but not by iodoacetic acid, are susceptible substrates for trypsin (12), use of the former reagent is advantageous in creating another site for trypsinization in the absence of susceptible lysyl residues. The presence of this additional cleavage site increases the number of labeled peptides and thus provides more information. Alkylation is also important in preventing reformation of disulfide bonds, thereby facilitating complete tryptic cleavage. In addition to reductive methylation and alkylation, P-4 chromatography prior to gel electrophoresis improved the quality and reproducibility of this method. This step was found useful to separate labeled material which moved in a broad peak in electrophoresis with the mobility of proteins in the molecular weight range of 10,000 on 15% gels. This material is not completely washed out during fixation with acetic acid and isopropanol. Finally, the quality of peptide mapping was increased during thinlayer electrophoresis by the addition of a heptane overlay, which prevented evaporation of water from the plate, helped maintain a cool, constant temperature over the plate, and gave better separation of peptides in the first dimension. The method herein is capable of labeling proteins to high specific activities and pos-
PEPTIDE
MAPPING
WITH
BOLTON-HUNTER
sesses many advantages over the direct iodination of proteins with chloramine-T. IJpon labeling the ester with 0.5 mCi of ‘251, approximately 10% of the label (0.05 mCi) becomes incorporated. Approximately 90% of the Bolton-Hunter reagent hydrolyses during the protein-labeling step. Even so, the specific activities of the model proteins herein are l-2 X lo4 cpm/ng. When the same proteins were labeled by chloramine-T, the specific activities were five- to sixfold higher. The use of greater quantities or higher specific activities of Bolton-Hunter reagent could be employed if the quantities of protein available warrant it. It is possible that in some systems radioacylation may result in extremely complicated patterns and that direct iodination may be preferable. However, in virus protein systems analyzed thus far, up to 80 spots in peptide maps have been clearly discerned using this approach (13; Feitelson et al., unpublished work).. The more uniform distribution of label in radioacylated proteins permits evaluation of differences in sequence or posttranslational modification which may well be missed by direct iodination by virtue of the smaller subset of labeled peptides. Furthermore, radioacylation can be used to generate maps of proteins completely lacking tyrosinle. The chemical conditions for labeling are milder; they do not include the oxidizing and reducing agents used in direct iodination, which can lead to artifactual aggregation of proteins ( 14). Direct iodination of proteins after their separation by gel electrophoresis (15) circumvents this problem, but larger quantities of protein are needed to permit the detection of proteins by staining. The method herein, then, provides a procedure for the molecular characterization of picomole quantities of protein which cannot be labeled by biosynthetic incorporation of amino acids. This situation may be encountered in the characterization of cell surface
REAGENT
479
receptors or antigens, for virus- and/or parasite-associated proteins in systems lacking tissue culture, and other proteins of high biological activity when available only from whole animals. ACKNOWLEDGMENTS This research was supported by grant No. CA- I8 15 1 from the National Cancer Institute, National Institutes of Health, the California Institute for Cancer Research, and the Cancer Research Coordinating Committee of the University of California, M.A.F. was a recipient of a National Research Service Award CA-9030.
REFERENCES I. Bolton, A. E., and Hunter, W. M. (1973) Biochem. J. 133, 529-538. 2. Means, G. E., and Feeney, R. E. (1968) Biochemistry 7, 2192-2201. 3. Laemmli, U. K. (I 970) Nature (London] 227,680685. 4. Swanstrom, R.. and Shank, P. R. (1978) Anal. Biochem. 86, 184- 192. 5. Hunter, W. M. (1978) in Handbook of Experimental Immunology (Weir, D. M., ed.), 3rd ed., p. 14, Blackwell, Oxford. 6. Dickerson, R. E., and Geis, 1. (1969) The Structure and Action of Proteins, Harper & Row, New York. 1. Keil-Dlouha. V.. Zylber, N., Imhoff, J. M., and Keil, B. (1971) FEBS Letters 16, 291-295. 8. Robinson, A. B. (I 974). Proc. Nat. Acad. Sci. USA 71, 885-888. 9. O’Farrell, P. H. (1975). J. Biol. Chem. 250,40074021. IO. Bolton, A. E., Bennie, J. G., and Hunter, W. M. ( 1976) in Proteins and Related Subjects; Protides of Biological Fluids (Peeters, H., ed.), Vol. 24, p. 687, Pergamon, Elmsford, New York. I I. Wood, F. T., Wu, M. M., and Gerhart. J. C. (1975) Anal. Biochem. 69, 339-349. 12. Gorecki, M., and Shalatin. Y. (1967) Biochem. Biophys. Res. Commun. 29, 189-193. M. A., Marion, P. L., and Robinson, 13. Feitelson. W. S. (1981) J. Viral. 39, 447-454. 14. Spira. G., Estes, M. K., Dreesman, G. R., Butel, J. S.. and Rawls, W. E. (1974) Intervirology 3, 220-231. 15. Elder, J. H., Pickett II, R. A., Hampton, J., and Lerner, R. A. ( 1977) J. Biol. Chem. 252,6510-6515.
116, 473-479
BIOCHEMISTRY
(1981)
Tryptic Peptide Mapping of Picomolar Quantities of Protein Labeled with the Bolton-Hunter Reagent MARK *Department
A. FEITELSON,
of k4icrobiology
*-I FELIX
0. WETTSTEIN,**~
AND JACK G. STEVENS*
and Immunology. UCLA School of Medicine, University of California. Los Angeles, California Received
January
and tMolecular 90024
Biology
Institute.
5, 1981
Characterization of 5 to 25 pmol of purified proteins by tryptic peptide mapping has been accomplished using the Bolton-Hunter reagent (‘*‘I-3-[4-hydroxyphenyllpropionic acid Nhydroxysuccinimide ester). Radioacylation is followed by reaction with unlabeled ester and reductive methylation to ensure resistance of lysyl residues to trypsinization. Reduced and alkylated proteins are analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, trypsinized from individual gel slices, and mapped two-dimensionally on thin layers. The method permits peptide mapping of proteins with specific activities of I to 2 X IO4 cpm/ng, results in more spots (and often more structural information) than direct iodination procedures, and can be used for characterization of proteins that could not be biosynthetically labeled.
Peptide mapping of proteins has become an important tool in protein characterization in combination with analysis by sodium dodecyl sulfateepolyacrylamide gel electrophoresis. A special problem is posed when proteins of interest are derived from whole animals or for other reasons cannot be biosynthetically labeled to high specific activities and are available only in small quantities. Protein iodination has been used to increase the sensitivity of peptide mapping but has the disadvantage that only tyrosinecontaining peptides can be detected. We have developed an alternate approach employing ‘251-labeled 3-( 4-hydroxyphenyl)propionic acid N-hydroxysuccinimide ester ( Bolton-Hunter reagent) ( 1) which labels the N-terminus and the more common lysyl residues. MATERIALS
AND
METHODS
Preparation of the Bolton-Hunter reagent and acylation of model proteins. pHydroxyphenylpropionic ’ Present Department of Medicine,
address: Division of of Medicine, Stanford Palo Alto, California
acid
N-hydroxy-
Infectious University
Diseases, School
succinimide ester (0.2-0.5 /*g) (TAGIT, Calbiochem) dissolved in 1 to 2 J of dioxane (Matheson, Coleman, and Bell) was added to 50 /*I of 0.5 M sodium phosphate buffer (Sigma), pH 7.5, containing 0.5-1.0 mCi of ‘*‘I (New England Nuclear) and 100 pg of freshly prepared chloramine-T (Eastman). Iodination was carried out in a I-ml screwcap vial, at room temperature, on a magnetic stirrer. The labeled ester was immediately extracted by addition of 5 ~1 dimethylformamide and 100 ~1 of benzene (Baker), the aqueous phase removed, and the organic phase transferred to a 6 X 50-mm kimax vial (Kimble) and quickly dried over a gentle stream of air. To prevent excessive hydrolysis, labeling and extraction were carried out within 40 s ( 1). Protein (250 ng) [bovine serum albumin ( BSA,2 Miles), ovalbumin, bovine chymotrypsinogen, or hen egg lysozyme (HEL, Worthington)] in 2 to 2.5 ~1 of ice-cold sodium borate buffer (Sigma), pH 8.5, was added to the dried BoltonHunter reagent. The reaction was carried out for 1 h at 0°C and then at 4°C overnight. * Abbreviations used: H EL, hen egg lysozyme;
473
BSA, SDS,
bovine sodium
serum albumin; dodecyl sulfate.
0003-2697/81/140473-07$02.00/O
474
FEITELSON,
WETTSTEIN,
The protein was then further acylated at 0°C for 3 h with 100 /lg TAGIT added in 5 ~1 0.1 M sodium borate buffer or in 1 ~1 dioxane. Several radioacylation reactions were carried out with commercially available Bolton-Hunter reagent (New England Nuclear) for comparison. Reductive methylation, alkylation, and analysis by gel electrophoresis. Reductive methylation was essentially as described by Means and Feeney (2). After protein acylation, the pH was adjusted to 9.0 with 1.0 M sodium hydroxide. Cold, freshly prepared reagents in 0.1 M sodium borate buffer, pH 9.0, were added at 0°C as follows: 1 ~1 of guanidine-HCl (10 M), 1 ~1 sodium borohydride ( 10 mg/ml), and finally, 5 additions of 1 ~1 each of a formaldehyde solution (2 ~1 of 37% formaldehyde (Baker, analyzed reagent) in 150 ~1 0.1 M borate buffer) spaced at 5-min intervals. The sequence of borohydride and formaldehyde addition was repeated using a new batch of borohydride. Following the two cycles of reductive methylation, an additional l-111 portion of freshly prepared sodium borohydride was added to ensure complete reduction of all formaldehyde. With the exception of formaldehyde, all reagents used for reductive methylation were purchased from Sigma. The solution containing the acylated and reductively methylated protein was made up to 2% with sodium dodecyl sulfate (SDS, Bio-Rad) and 2 M with urea (Mallinckrodt) and incubated in boiling water for 2 min. The pH was then adjusted to 8.0 with 1 M HCl (Baker) and the protein reduced in 0.025 M dithiothreitol (Sigma), at 37°C for 1 h and alkylated in 0.075 M iodoacetamide (Sigma) at room temperature for 45 min. After alkylation, the labeled and modified proteins were separated from reagents by chromatography on P-4 (Bio-Rad) in 0.1 M ammonium bicarbonate (Matheson, Coleman, and Bell), 0.1% SDS, lyophilized, and stored at 4°C. Discontinuous SDS-electrophoresis was carried out by the method of Laemmli (3),
AND
STEVENS
using l-mm thick slab gels, consisting of a 15-cm 15% separating gel and a 4-cm 5% stacking gel. Lyophilized samples were made to 2% with SDS, 4 M with urea, 0.001% with bromophenol blue tracking dye, and boiled for 2 min. Electrophoresis was carried out at 30 V for the first half hour and 60 V for approximately 16-18 h until the dye was within 0.5 cm of the bottom of the gel. The separating gels were fixed with 10% acetic acid, 25% isopropanol overnight, dried under vacuum, and the protein bands located by autoradiography. Trypsinization andpeptide mapping. Protein bands were cut out, dried overnight under high vacuum, and hydrated with 0.5 ml of ammonium bicarbonate (0.2 M), pH 8.0, containing 25 hg of trypsin (chymotrypsinfree, Calbiochem). The heavy paper (Whatman 3MM) on which gels were dried down upon was scraped off of each dried-down, protein-containing gel slice to permit greater access of trypsin to the protein. Proteolysis of gel slices was carried out at 25 or 37°C overnight with gentle rocking of the tube rack. The supernatants were lyophilized and redigested to completion in 20 ~1 of 0.05 M sodium borate buffer, pH 8.5, with 25 pg trypsin. Finally, the peptides were spotted on 20 X 20-cm thin-layer cellulose plates [ (0.1 -mm cellulose on glass or 0.15-mm cellulose on polyethylene-terephthalate (Brinkman)]. About 1 ~1 of 0.3 mg/ml pyronine Y tracking dye (Eastman Organic Chemicals) was spotted just below the peptide sample. Immediately before electrophoresis, the plates were dampened with a spray of 0.8% acetic acid, 0.2% formic acid (Baker), pH 2, and placed inside a Plexiglas trough fitted with a cooling plate maintained at 15°C. The plates were connected to the electrode compartments containing 8% acetic acid, 2% formic acid with a double layer of Whatman 3MM paper, and then overlaid with 100 to 200 ml of n-heptane (Baker, analyzed reagent) to prevent water evaporation. Since n-heptane is flammable, the trough accommodating the n-heptane-overlaid, thin-layer
PEPTIDE
MAPPING
WITH
BOLTON-HUNTER
plates was covered with a glass plate to reduce evaporation of the organic solvent; therefore, electrophoresis should be performed in a well-ventilated room or in a fume hood. After electrophoresis for 2 h at 400 V and 4-16 mA, the plates were dried overnight at room temperature or for at least 1 h under a heat lamp followed by 30 min at room temperature. Thin-layer chromatography was ,then carried out in the second dimension at room temperature for approximately 4 h using n-butanol:water:ethyl acetate:acetic acid:pyridine at the ratio 120:60:20:4: 1 (v/v). After chromatography, the plates were dried, wrapped tightly in cellophane (Saran Wrap), and autoradiographed for 2 to 10 h at -70°C using Kodak RP/RZ Omat X-ray film and DuPont Cronex Xtra Life (lighting plus) intensifying screen (4). A.11 reagents used for chromatography were obtained from Baker. Chloramirw-T iodination and peptide mapping of model proteins. Two hundred fifty nanograms each of BSA, ovalbumin, chymotrypsinogen, and HEL dissolved in 10 ~1 of 0.05 M sodium phosphate buffer, pH 7.5, was added to 50 ~1 of 0.5 M sodium phosphate buffer, pH 7.5, and 0.5-1.0 mCi ‘25I in a I-ml screwcap vial, at room temperature, on a magnetic stirrer (5). Iodination was initiated by the addition of 100 pg chloramine-T and terminated after 1 min by the addition of 200 fig sodium metabisulfite (Sigma). Ten microliters of glycerol (Baker) and SDS to final concentrations of 10 and l%, respectively, were added and the proteins separated from free “‘1 on a P-4 column as described above. Reduction, alkylation, gel electrophoresis, and tryptic peptide mapping were carried out as described above, except that the chromatography solvent used was n-butanol:pyridine:acetic acid:water in the ratio 15:9: 3:9 (v/v). RESULTS
AND
DISCUSSION
BSA, ovalbumin, chymotrypsinogen, and HEL have lbeen labeled with the Bolton-
REAGENT
475
AB
FIG. I. SDS-electrophoresis profile of the four model proteins labeled by radioacylation or direct iodination. BSA (68K), ovalbumin (43K). chymotrypsinogen (25K), and HEL (14K) were prepared as described under Materiak and Methods. Approximately 250 ng (5-25 pmol) of each protein labeled by either procedure was analyzed by slab gel electrophoresis. Dried gels were exposed for autoradiography 30 min as outlined under Materials and Methods. (A) Bolton-Hunter-reagent-labeled proteins; (B) chloramine-T-iodinated proteins.
Hunter reagent and analyzed by gel electrophoresis and tryptic peptide mapping. For comparison, these same proteins have been labeled by direct iodination using chloramine-T and then similarly analyzed. Results from SDS gels show that, with the exception of chymotrypsinogen, the mobilities of the proteins were reduced following acylation in comparison to direct iodination (Fig. 1). Although the reason for the anomalous behavior of chymotrypsin remains to be clarified, it is likely that the slower mobility of the other proteins is due to acylation, which
476
FEITELSON,
WETTSTEIN,
AND
STEVENS
A
c
FIG. 2. Tryptic peptide mapping of labeled proteins. Proteins labeled by either method were separated by gel electrophoresis (Fig. 1) and trypsinized as described under Materials and Methods. Approximately 50-759~ of the cpm in each protein band was recovered following trypsinization of each dried gel slice. Trypsinization of gel slices at 37’C resulted in slightly higher recoveries than at 25°C. After lyophilization and redigestion, approximately 10% of the recovered counts ( IO5 cpm) were used for peptide mapping. Plates were exposed for autoradiography for 10 h. Freshly prepared or commercially obtained Bolton-Hunter reagent gave identical results. Electrophoresis is from right to left and the direction of chromatography is up. (A,B) BSA; (C,D) ovalbumin; (E,F) bovine chymotrypsinogen; (G,H) HEL. (A,C,E,G) Chloramine-T-iodinated; (B,D,F,H) BoltonHunter-reagent-labeled.
could add as much as several thousand daltons of molecular weight in each case. This potential change in mobility in gel electrophoresis has to be kept in mind when comparing acylated to nonacylated proteins. Peptide maps of Bolton-Hunter-reagentacylated and chloramine-T-iodinated proteins are presented in Fig. 2. The peptide maps of the Bolton-Hunter-reagent-labeled proteins (Figs. 2B, D, F, H) exhibit more
spots than those of chloramine-T-iodinated proteins, thus providing more detailed information. This should be the case, since primary amino groups are more frequent in most proteins than tyrosine residues, resulting in a larger number of peptides being labeled by acylation than by direct iodination. The theoretical number of labeled tryptic peptides for the two smaller proteins, bovine chymotrypsinogen and HEL, has been de-
PEPTIDE
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H
FIG. 2 continued termined from their amino acid sequences (6) and is five for both when labeled by the method described here and four and three, respectively, when labeled by the ,chloramine-T method. The relatively large number of spots observed in the peptide maps of acylated protein digests could be accounted for by conditions dependent upon and independent from the radioacylation step. Some spots could have been generated as a consequence of the autocatalytic breakdown of trypsin, since during this process an enzymatically active species (pseudotrypsin) having chymotryptic-like activity is formed and may contribute up to several percent of
the total activity during proteolysis (7). Protein microheterogeneities resulting from the spontaneous deamination of asparagine and glutamine (8) and/or possible oxidation of cysteine to cysteic acid prior to reduction and alkylation (9) might also give rise to some of the faint spots on the autoradiograms of Fig. 2. The presence of these microheterogeneities and/or the action of pseudotrypsin is also compatible with the peptide maps of HEL and chymotrypsinogen labeled by direct iodination, where a number of minor spots are seen and the total number of spots on each map is slightly greater than the number predicted from the primary se-
478
FEITELSON.
WETTSTEIN.
quence of each protein. A major contributing factor to peptide heterogeneity in the Bolton-Hunter-reagent-labeled proteins is radioacylation itself. The large bulk and short half-life of the Bolton-Hunter reagent (halflife 9 min in aqueous media), combined with steric restrictions and/or differences in the intrinsic reactivities of epsilon amino groups, suggest that radioacylation does not go to completion. Residues neighboring epsilon amino groups may affect labeling, depending upon their charge and/or bulk. Since the extent of radioacylation versus hydrolysis is dependent upon the ratio of Bolton-Hunter reagent to protein as well as protein concentration (data not shown, lo), the reaction is driven as far to completion as possible by the addition of cold ester (TAGIT). The extent of chemical heterogeneity introduced into a protein as a result of radioacylation would vary depending upon these precise conditions of reaction. Further acylation with TAGIT serves to reproducibly limit this heterogeneity so that the presence and intensity of all the spots in a peptide map will remain uniform from experiment to experiment. In addition to causing small changes in the electrophoretic mobility of proteins on SDS gels and introducing chemical heterogeneity, acylation affects other properties of proteins and peptides. Acylation of epsilon and N-terminal amino groups removes the charge from those groups ( 11) which results in slightly retarded mobility of acylated peptides in relationship to unmodified peptides in the electrophoretic dimension. As pointed out under Materials and Methods, acylation changes the chromatographic properties of the tryptic peptides, since different chromatography solvents are employed for acylated and iodinated hydrolysates. Further, protein acylation results in derivatized lysyl residues which are resistant to trypsinization. This follows from studies in which replacement of the t amino hydrogens by methyl groups prevents trypsinization at lysyl peptide bonds (2,12). In our hands, a synthetic peptide with a single lysyl residue
AND
STEVENS
modified by acylation failed to cleave in the presence of trypsin (data not shown). This result is consistent with the presence of t amino hydrogens for recognition by the active site of trypsin (12). Since not all of the lysyl residues may become fully acylated, reductive methylation is employed to modify any remaining free amino groups in the protein, thereby ensuring that all of the lysyl residues become resistant to trypsinization. Reductive methylation, then, helps to prevent ambiguities in the number of spots seen on peptide maps by eliminating lysyl residues as substrates for trypsin. Proteolysis, then, occurs only at arginyl and alkylated cysteinyl residues. Since cysteinyl residues alkylated by iodoacetamide, but not by iodoacetic acid, are susceptible substrates for trypsin (12), use of the former reagent is advantageous in creating another site for trypsinization in the absence of susceptible lysyl residues. The presence of this additional cleavage site increases the number of labeled peptides and thus provides more information. Alkylation is also important in preventing reformation of disulfide bonds, thereby facilitating complete tryptic cleavage. In addition to reductive methylation and alkylation, P-4 chromatography prior to gel electrophoresis improved the quality and reproducibility of this method. This step was found useful to separate labeled material which moved in a broad peak in electrophoresis with the mobility of proteins in the molecular weight range of 10,000 on 15% gels. This material is not completely washed out during fixation with acetic acid and isopropanol. Finally, the quality of peptide mapping was increased during thinlayer electrophoresis by the addition of a heptane overlay, which prevented evaporation of water from the plate, helped maintain a cool, constant temperature over the plate, and gave better separation of peptides in the first dimension. The method herein is capable of labeling proteins to high specific activities and pos-
PEPTIDE
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BOLTON-HUNTER
sesses many advantages over the direct iodination of proteins with chloramine-T. IJpon labeling the ester with 0.5 mCi of ‘251, approximately 10% of the label (0.05 mCi) becomes incorporated. Approximately 90% of the Bolton-Hunter reagent hydrolyses during the protein-labeling step. Even so, the specific activities of the model proteins herein are l-2 X lo4 cpm/ng. When the same proteins were labeled by chloramine-T, the specific activities were five- to sixfold higher. The use of greater quantities or higher specific activities of Bolton-Hunter reagent could be employed if the quantities of protein available warrant it. It is possible that in some systems radioacylation may result in extremely complicated patterns and that direct iodination may be preferable. However, in virus protein systems analyzed thus far, up to 80 spots in peptide maps have been clearly discerned using this approach (13; Feitelson et al., unpublished work).. The more uniform distribution of label in radioacylated proteins permits evaluation of differences in sequence or posttranslational modification which may well be missed by direct iodination by virtue of the smaller subset of labeled peptides. Furthermore, radioacylation can be used to generate maps of proteins completely lacking tyrosinle. The chemical conditions for labeling are milder; they do not include the oxidizing and reducing agents used in direct iodination, which can lead to artifactual aggregation of proteins ( 14). Direct iodination of proteins after their separation by gel electrophoresis (15) circumvents this problem, but larger quantities of protein are needed to permit the detection of proteins by staining. The method herein, then, provides a procedure for the molecular characterization of picomole quantities of protein which cannot be labeled by biosynthetic incorporation of amino acids. This situation may be encountered in the characterization of cell surface
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receptors or antigens, for virus- and/or parasite-associated proteins in systems lacking tissue culture, and other proteins of high biological activity when available only from whole animals. ACKNOWLEDGMENTS This research was supported by grant No. CA- I8 15 1 from the National Cancer Institute, National Institutes of Health, the California Institute for Cancer Research, and the Cancer Research Coordinating Committee of the University of California, M.A.F. was a recipient of a National Research Service Award CA-9030.
REFERENCES I. Bolton, A. E., and Hunter, W. M. (1973) Biochem. J. 133, 529-538. 2. Means, G. E., and Feeney, R. E. (1968) Biochemistry 7, 2192-2201. 3. Laemmli, U. K. (I 970) Nature (London] 227,680685. 4. Swanstrom, R.. and Shank, P. R. (1978) Anal. Biochem. 86, 184- 192. 5. Hunter, W. M. (1978) in Handbook of Experimental Immunology (Weir, D. M., ed.), 3rd ed., p. 14, Blackwell, Oxford. 6. Dickerson, R. E., and Geis, 1. (1969) The Structure and Action of Proteins, Harper & Row, New York. 1. Keil-Dlouha. V.. Zylber, N., Imhoff, J. M., and Keil, B. (1971) FEBS Letters 16, 291-295. 8. Robinson, A. B. (I 974). Proc. Nat. Acad. Sci. USA 71, 885-888. 9. O’Farrell, P. H. (1975). J. Biol. Chem. 250,40074021. IO. Bolton, A. E., Bennie, J. G., and Hunter, W. M. ( 1976) in Proteins and Related Subjects; Protides of Biological Fluids (Peeters, H., ed.), Vol. 24, p. 687, Pergamon, Elmsford, New York. I I. Wood, F. T., Wu, M. M., and Gerhart. J. C. (1975) Anal. Biochem. 69, 339-349. 12. Gorecki, M., and Shalatin. Y. (1967) Biochem. Biophys. Res. Commun. 29, 189-193. M. A., Marion, P. L., and Robinson, 13. Feitelson. W. S. (1981) J. Viral. 39, 447-454. 14. Spira. G., Estes, M. K., Dreesman, G. R., Butel, J. S.. and Rawls, W. E. (1974) Intervirology 3, 220-231. 15. Elder, J. H., Pickett II, R. A., Hampton, J., and Lerner, R. A. ( 1977) J. Biol. Chem. 252,6510-6515.