Bioconjugate Chem. 1002, 3, 130-146
138
Site-Directed Conjugation of Nonpeptide Groups to Peptides and Proteins via Periodate Oxidation of a 2-Amino Alcohol. Application to Modification at N-Terminal Serine Kieran F. Geoghegan' and Justin G. Stroh Central Research Division, Pfizer Inc., Groton, Connecticut 06340. Received October 15, 1991 ~~
~~
The 2-amino alcohol structure -CH(NH&H(OH)- exists in proteins and peptides in N-terminal Ser or Thr and in hydroxylysine. Its very rapid oxidation by periodate at pH 7 generates an aldehyde in the peptide and is the first step in a method for site-directed labeling with biotin or a fluorescent reporter. The modifying group is a hydrazide, RCONHNH2, which reach with the new aldehyde to form a hydrazone-peptide conjugate, RCONHN=CH-peptide. Experimenh with two synthetic pepand Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly, and with recomtides, Ser-Ile-Gly-Ser-Leu-Ala-Lys binant murine interleukin-la (an 18-kDa cytokine with N-terminal Ser) demonstrated this method of peptide tagging. The use of a low molar ratio of periodate to peptide minimized the potential for side reactions during the oxidation, and the desired oxidation was rapid and highly specific. The hydrazones formed were stable at pH 6-8 for at least 12 h at 22 "C, but were labile at more acidic pH values. Potential uses of this method include the attachment of biotin, reporter groups, metal chelating groups, imaging agents, and cytotoxic drugs to peptides.
RCH(NH2)CH(OH)R'+ 1 0;
INTRODUCTION Covalent conjugates of polypeptides with nonpeptide "labels" or "tags" form a useful class of reagents in protein and peptide research. A conjugate is usually intended to retain the native properties of the peptide while gaining a new, non-native property due to the label. Biotinylation, for example, permits proteins to be separated, quantitated, or immobilized by mechanisms based on the strong interaction of biotin with avidin or streptavidin (1). I?luorescent or metal-chelating groups can also be introduced in order to generate newly bifunctional modified peptides. It would be convenient to be able to introduce a nonpeptide label as the peptide is produced, but this is only sometimes feasible in chemical synthesis (2, 3) and is impractical when the peptide is produced biologically. Generally, the conjugate must be formed by treating the peptide with a group-specific reagent that contains the label. Unless the peptide contains only one group attacked by the reagent, this procedure yields a mixture of products. This random form of labeling is sometimes adequate, but it is often preferable to modify a peptide at a single specified site and to employ the modified product in purified form. For such cases, it would be valuable to have a method of directing the modifying group into a single, preselected location. This precisely targeted chemical modification could be termed site-directed peptide tagging. Several schemes for N-terminal (4, 5) or C-terminal(6-8) site-directed tagging have already been described. This paper describes and illustrates a method which can potentially lead to site-directed modification at any locus within a synthetic peptide and at the N-terminus of proteins with N-terminal Ser or Thr. The method is based on the very fast oxidation by periodate of the 2-amino alcohol grouping
* Correspondence: Dr. K. F. Geoghegan, Pfizer Central Research, Eastern Point Road, Groton, CT 06340. 1043-1802/92/2903-0130~03.00/0
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RCHO + NH, + OHCR' + I O:
to create a single aldehyde function at a known site in the peptide (9-13). The aldehyde is then reacted with the tagging group in the form of a hydrazide, R"CONHNH2, to form a hydrazone, R"CONHN=CH-peptide. R" can be any of a variety of useful groups, and the hydrazones are adequately stable for many biochemical applications in the range pH 6-8. Site-selectivity is possible because the target site for the periodate reaction exists in peptides only when the N-terminus is Ser or Thr, or when hydroxylysine is present; hence the location at which an aldehyde is generated is known or can be selected in advance. (Hydroxylysine is only known to occur naturally in collagen, but in principle can be placed anywhere in a synthetic peptide.) Results presented here introduce the method by demonstrating site-directed modification at N-terminal Ser. EXPERIMENTAL PROCEDURES Reagents. Biotin-X-hydrazide,' Lucifer Yellow, and Cascade Blue hydrazide were purchased from Molecular Probes (P.O. Box 22010,4849Pitchford Ave., Eugene, OR 97402). The peptides SIGSLAK and SYSMEHFRWG were from Sigma (P.O. Box 14508, St. Louis, MO 63178). Recombinant mIL-la was produced as described (14).Avidin-HRP conjugate and biotinylated M, markers were from Pierce (P.O. Box 117, Rockford, IL 61105). Sodium m-periodate was from Aldrich (1001West Saint Paul Ave., Milwaukee,WI 53233). Periodate solutions were prepared on the day of use and kept in a foil-wrapped tube. 1 Abbreviations used: PDMS, plasma desorption mass spectrometry; ESMS, electrospray mass spectrometry; biotin-X-hydrazide, 6-(biotinoy1amino)caproic acid hydrazide; Lucifer Yellow, Lucifer Yellow CH; mIL-la, murine interleukin-la; IEF, isoelectric focusing; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; Tris, tris(hydroxymethy1)aminomethane:Mes, 2-morpholinoethanesulfonic acid; CAPS, 3-(cyclohexylamino)-l-propanesulfonicacid;the oneletter code for representing amino acid residues in peptide sequences may be found in most textbooks of biochemistry.
0 1992 American Chemical Society
Site-Dlrected Modiflcatlon
Chromatography. For the experiments with SIGSLAK and SYSMEHFRWG, HPLC was performed using a gradient system from LDC Analytical or a Hewlett-Packard 1090 Series I1system. A Vydac C4 column (4.6 X 150 mm; type 214TP10415)(Separations Group, Hesperia, CA 92345) was used with linear gradients of CH3CN in 0.1 % TFA (1 mL/min flow rate). Peaks selected for PDMS were dried in a SpeedVac (Savant Instruments, 110-103 Bi-County Blvd., Farmingdale, NY 11735) to 1-5 pL. Peptide mapping was performed on the Hewlett-Packard system using a C18 Aquapore cartridge (2.1 X 100 mm) (Applied Biosystems, 850 Lincoln Centre Dr., Foster City, CA 94404) with a flow rate of 0.2 mL/min. The solvents were A, 0.1 % TFA in 5% (v/v) CH3CN; and B, 0.085 % TFA in 70% (v/v) CH3CN. A linear gradient from 0% to 80% solvent B was run from 2 to 66 min after injection. Peaks were collected by hand. Reversed-phase HPLC of mIL-la and its oxidized and biotinylated forms was performed on the Hewlett-Packard system, which was equipped with a diode-array detector to allow measurement of the UV-visible absorption spectrum of each component. A Vydac C4 column (2.1 X 50 mm; type 214TP5205) was used with solvents as follows: A, 0.1 5% TFA; and B, 0.085% TFA in 80% (v/v) CH3CN. The flow rate was 0.2 mL/min. A linear gradient from 10% to 82 % solvent B was run from 2 to 16 min after injection. Protein/Peptide Sequencing. Sequence analysis was performed using an Applied Biosystems Model 470A gasphase sequencer equipped with a Model 120A PTH analyzer. Electrophoresis. IEF, SDS-PAGE, and electroblotting to PVDF membrane (ProBlott; Applied Biosystems) were performed using a PhastSystem (Pharmacia LKB, 800 Centennial Ave., Piscataway, N J 08854) and gels were developed using a PhastGel silver kit (Pharmacia LKB). Western blots were tested for biotinylated proteins using avidin-HRP conjugate. Mass Spectrometry. PDMS was performed using a Bio-Ion 20 spectrometer (Applied Biosystems). Samples were dissolved in 15 pL of 20% CH3CN and applied to nitrocellulose targets (Applied Biosystems). The instrument was run in the positive ion mode with an acceleration potential of 15 kV. Masses were estimated by placing the twin cursors on each side of the peak at the bottom of the Gaussian-shaped area and calculating a centroid. This procedure generally produced a value of mass accuracy within 0.2 5%. ESMS data were obtained on a Finnigan TSQ-700 mass spectrometer operating in the positive-ion mode. The instrument was scanned over the m/z range 450-2200. Thirty-two spectra were averaged and the averaged data were smoothed using a seven-point smoothing routine. Spectra of mIL-la and its derivatives were collected on the HPLC column fractions with no further concentration. Samples were infused into the ES ion source at 2 pL/min with a sheath liquid flow of 2 pL/min of 2-methoxyethanol. Periodate Oxidations and Coupling Reactions. (1) Ser-Ile-Gly-Ser-Leu-Ala-Lys (SIGSLAK). SIGSLAK (2 X lo4 M) in 0.01 M sodium phosphate, pH 7.0, was treated with a 2-fold molar excess of NaI04 for 4 min at 22 OC. The reaction mixture (200 pL) was then injected into the HPLC and fractionated using a linear gradient from 0% to 42% CH&N between 5 and 20 min after injection. A small portion of the product peak was used for PDMS, and the remainder was dried. The oxidation product a-N-glyoxylyl-IGSLAK(1.5 mM)
Bioconjugate Chem., Vol. 3, No. 2, 1992
130
was reacted with biotin-X-hydrazide (10 mM) in 0.03 M sodium acetate (pH 4.5)/16% CH3CN for 70 min at 22 OC, after which the 30-pL reaction mixture was diluted to 220 p L with water and injected into the HPLC (see Results). Separately, a-N-glyoxylyl-IGSLAK (1.5 mM) was allowed to react with Lucifer Yellow (16 mM) in 0.03 M sodium acetate (pH 4.5) for 120 min at 22 OC, after which this reaction mixture was fractionated by HPLC (see Results). Products were characterized by PDMS. (2) Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly (SYSMEHFRWG). In a series of reactions at different pH values, SYSMEHFRWG (28 pM) was treated with NaI04 (44 pM) in a mixed buffer containing 7 mM each of sodium acetate, Mes, and sodium phosphate. The experiment was performed with the buffer set to pH 4.0, 5.0,6.0, 7.0, and 8.0. After 2 min at 22 "C, the reactions were terminated by HPLC injection. (In preliminary experiments, the major oxidation products were mass analyzed; mass data interpreted in terms of Met oxidation were confirmed by showing that components suspected of containing Met sulfoxide failed to undergo cleavage by CNBr). N-Terminally oxidized peptide a-N-glyoxylyl-YSMEHFRWG was coupled to biotin-X-hydrazide as described. The conjugate was then used in a study of the stability of peptidehydrazone conjugates. Biotin-X-YSMEHFRWG was separately incubated at 22 OC in a series of buffers with pH in the range pH 2-10, and the samples were analyzed by automated injection into the HPLC. Buffers used were 0.05 M sodium phosphate (pH 2.0), 0.05 M sodium acetate (pH 4.0), 0.05 M Mes (pH 6.01, 0.05 M Tris-HC1 (pH 8.01, 0.05 M CAPS (pH 10.0). (3) Murine Interleukin-la (mIL-la). In a 2.2-mL reaction, mIL-la (13 pM) was incubated at 0 "C for 30 min with NaI04 (40 pM) in 0.02 M sodium phosphate (pH 7.0). Periodate was removed by gel filtration using Sephadex G-25 equilibrated with 0.05 M sodium acetate (pH 4.5). Quantitative oxidation of the N-terminus was confirmed by IEF, and the N-terminally oxidized protein was concentrated using a Centricon 10 (Amicon,24 Cherry Hill Dr., Danvers, MA 01923) before reaction with biotinX-hydrazide. The coupling reaction contained 57 pM oxidized mIL-la with 8 mM biotin-X-hydrazide in 25% acetonitrile/0.038 M sodium acetate (pH 4.5). After 16 h a t 22 "C, the protein was gel filtered into 0.02 M Tris (pH 7.0) to remove biotin-X-hydrazide and subjected to anionexchange chromatography (Mono& HR 5/5, Pharmacia LKB) using a flow rate of 1mL/min and a NaCl gradient of 0.0-0.1 M NaCl in 20 min in 0.02 M Tris (pH 7.0). The modified product was characterized by SDS-PAGE and by Western blotting followed by detection of the biotin label with avidin-HRP. Control samples were treated as described above with the omission of periodate from the oxidation stage or of biotin-X-hydrazide from the coupling stage. Tryptic Digestion of mIL-la. Unmodified and oxidized samples of mIL-la were treated in parallel with trypsin to prepare digests. In a first attempt, 0.2 mL of each protein sample at 0.14 mg/mL (28 pg of mIL-la) was incubated with 1 pg of sequencing-grade trypsin (Boehringer Mannheim, P.O. Box 50414, Indianapolis, IN 46250) for 18 h at 37 "C in 0.1 M Tris (pH 8.0). When it was found that this accomplished no digestion, the samples were made 1M in guanidinium chloride, a further 2 pg of trypsin was added, and the tubes were again incubated at 37 OC for 18 h. These conditions led to a substantial, though incomplete, digestion of the mIL-la samples which sufficed to permit isolation and characterization of the
140
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Bloconjugate Chem., Vol. 3, No. 2, 1992
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Figure 1. HPLC and PDMS analyses of periodate oxidation of SIGSLAK. See the methods for experimental details. (a) HPLC analysis of SIGSLAK before oxidation; the 13.7-min peak was collected for PDMS. (b) PDMS analysis of SIGSLAK peak; the calculated MH+for SIGSLAK is 675.8. (c) HPLC fractionation of periodate oxidation of SIGSLAK; the 14.9-minpeak was collected for PDMS. (d) PDMS analysis of peak from SIGSLAK oxidation reaction; the calculated MH+ for a-N-glyoxylyl-IGSLAK is 644.8 (unhydrated form of aldehyde) and 662.8 (hydrated form). unmodified and oxidized forms of the anticipated Nterminal undecapeptide (see Results). RESULTS Initial Test of Oxidation and Coupling. Oxidation and coupling experiments were first performed using SIGSLAK, a peptide in which the only periodate-sensitive residue was the N-terminal Ser. SIGSLAK gave a single major peak in HPLC (Figure la), and in PDMS (Figure lb) gave an MH+ of 675.6 (calculated MH+ 675.8). The reaction of SIGSLAK with a 2-fold molar excess of periodate (see Methods) gave a single product (Figure IC). PDMS of this material gave two species with MH+ values of 644.2 and 662.4, respectively (Figure Id), assigned to the unhydrated (calculated MH+ 644.8) and hydrated (calculated MH+ 662.8) forms of the expected product, a-N-glyoxylyl-IGSLAK. HzNCHCO-IGSLAK
I
HOCH, SIGSLAK
IO -
A
O=CHCO-IGSLAK
+
(HO)&HCO-IGSLAK
glyOXylyl-IGSLAK
glyOXylyl-IGSlAK
(643.8 Da, unhydrated)
(661.8 Da. hydrated)
(674.8 Da)
Prewetting the nitrocellulose target for PDMS with ethanol resulted in a third peak in addition to the two seen in Figure l d (spectrum not shown). The new peak, corresponding to an MH+ of 690.8, was assigned to a hemiacetal formed by ethanol with a-N-glyoxylyl-IGSLAK (calculated MH+ 690.8 Da). a-N-Glyoxylyl-IGSLAK was dried and then allowed to react with 10 mM biotin-X-hydrazide for 70 min or 16 mM Lucifer Yellow for 120 min (see the methods) in 0.03 M sodium acetate (pH 4.5) at 22 OC. Reactions were
terminated by injection into the HPLC, and the products were collected, concentrated, and characterized by PDMS. The reaction of a-N-glyoxylyl-IGSLAK with biotin-Xhydrazide gave a single major product which was the 18.9min peak in Figure 2a; the broad peak at 12-13 min (Figure 2a) was due to biotin-X-hydrazide. The calculated MH+ for the hydrazone conjugate of biotin-X-hydrazide with a-N-glyoxylyl-IGSLAKwas 998.3; in agreement with this, the product gave an MH+ of 997.9 (Figure 2b). Reaction with Lucifer Yellow also converted a-N-glyoxylyl-IGSLAK to a single product (Figure 2c). PDMS (Figure 2d) showed that the product had an MH+ of 1072.3, in agreement with the calculated MH+ of 1072.1 for the hydrazone formed by Lucifer Yellow and a-N-glyoxylylIGSLAK. The Lucifer Yellow adduct gave a weak mass spectrum lacking any dominant component unless the nitrocellulose target was first washed with 0.1 % TFA. Very similar oxidation and coupling experiments were successfully performed using the peptide TIGSLAK, in which the N-terminus is Thr rather than Ser. These results confirmed the expectation that N-terminal Ser and Thr are equally viable targets for site-directed modification. Selectivity of Periodate Oxidation. As periodate can oxidize several amino acid side chains in proteins (15),it was important to define optimum conditions for selective oxidation of the 2-amino alcohol. This was done by studying periodate oxidation of another synthetic peptide, SYSMEHFRWG, which in addition to N-terminal Ser contained one residue each of Tyr, Met, His, and Trp. Apart from Cys, which was excluded from consideration because of its presumed sensitivity to oxidation (101, these were the residues most likely to undergo side reactions with periodate (15). The effect of treating SYSMEHFRWG with low molar ratios of periodate was examined
Bloconjugate Chem., Voi. 3, No. 2, 1992
Site-Directed Modification
a
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Figure 2. HPLC and PDMS analyses of coupling reactions between a-N-glyoxylyl-IGSLAK and hydrazide reagents. See the methods for experimental details. (a) HPLC analysis of coupling reaction with biotin-X-hydrazide; the 18.9-min peak was collected for PDMS. (b) PDMS analysis of hydrazone adduct between biotin-X-hydrazide and a-N-glyoxylyl-IGSLAK; the calculated MH+ for the desired adduct is 998.3. (c) HPLC analysis of coupling reaction with Lucifer Yellow; the 16.2-min peak was collected for PDMS. (d) PDMS analysis of hydrazone adduct between Lucifer Yellow and a-N-glyoxylyl-IGSLAK; the calculated MH+ for the desired adduct is 1072.1.
in the pH range 4-8,and reaction products were separated by HPLC and characterized by PDMS. Only three significant products were detected in addition to unreacted peptide. In order of increasing HPLC retention time (Figure 3a), the products were (I)the methionine sulfoxide form of the peptide, characterized by mass spectrometry and its inability to be cleaved by CNBr; (11) the methionine sulfoxide form, also N-terminally oxidized; (111)unmodified peptide; and (IV) the peptide modified only by the desired N-terminal oxidation. The effect of pH on the distribution of reaction products was then examined using an 104-:SYSMEHFRWG ratio of 1.6:l.O (Figure 3b). At pH 4.0, the principal reaction was oxidation of Met to its sulfoxide. The yield of this product diminished with increasing pH, and at pH 7.0 the amount of the desired N-terminal oxidation product, aN-glyoxylyl-YSMEHFRWG, was 40-fold higher and accounted for >90% of the total peptide (Figure 3a,b). Thus, at neutral pH, the desired oxidation of N-terminal Ser was rapid and highly favored. This was not true at pH 4-6 (Figure 3b), where oxidation of Met was favored. His, Tyr, and Trp were much less reactive to oxidative attack by periodate than either the N-terminus or the thioether group of Met. Very similar results were reported from an earlier study of periodate oxidation of corticotropin (161, of which SYSMEHFRWG is the N-terminal decapeptide. Stability of the HydrazoneAdduct. a-N-GlyoxylylYSMEHFRWG, the product of N-terminal oxidation of SYSMEHFRWG, was coupled to biotin-X-hydrazide as described above for a-N-glyoxylyl-IGSLAK. The product was isolated by HPLC (not shown) and gave a mass spectrum consistent with the expected hydrazone (calculated MH+ 1622.9,MH+ observed 1623.6). The stability of biotin-X-YSMEHFRWG at pH 2-10 at 22 "C was then analyzed by HPLC sampling over 12 h
I IV
a PEAK MASS MEASUREMENTS Peak I.M 8 U 1315 Met4-sulfoxideSYSMEHFRWG P08k II. Ma- 1302 Met'.SulIoxide and N.terminally oxidized SYSMEHFRWG (hydrate) Peak Ill. Mus 1209 unreacted SYSMEHFRWG Pe8k IV. Mass 1286 N-terminally oxidized SYSMEHFRWG (hydrate)
/I 18
20
22
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Figure 3. Effect of pH on periodate oxidation of SYSMEHFRWG. See the methods for experimental details. (a) HPLC analysis of oxidation of SYSMEHFRWG with a 1.6-fold molar excess of sodium periodate at pH 7. Peak identifications (inset text) were based on preliminary experiments from which peaks were collected and mass analyzed. (b) Percent of total SYSMEHFRWG undergoing (D) conversion to Met sulfoxide at the Met residue, and (A)N-terminal oxidation in experiments which were identical to that in panel a except for variation of the pH.
(Figure 4). The hydrazone broke down rapidly at pH 2, was less labile at pH 4, and remained intact through the course of the experiment at pH 6 and 8. Slow breakdown occurred at pH 10,but the hydrazone was still >95% intact after 12 h. Modification of mIL-la with Biotin-X-hydrazide.
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Bioconjugate Chem., Vol. 3, No. 2, 1992
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Figure 4. Stability of Biotin-X-YSMEHFRWG in the pH range 2-10. See the methods for experimental details. The hydrazone was incubated at 22 OC at (A)pH 2.0,).( pH 4.0, ( 0 )pH 6.0, ( 6 ) pH 8.0, and (x) pH 10.0.
mIL- l a , a 156-residuesingle-chain cytokine, was produced by expression of a synthetic gene in Escherichia coli (14). As it contained no thiol groups but had an N-terminal Ser, it was a good candidate for site-directed modification by the present method. mIL-la (1.4 X M) in 0.02 M sodium phosphate (pH 7.0) was treated with a 3-fold molar excess of sodium periodate at 0 "C for 30 min. Periodate was then removed by gel filtration, at the same time buffer-exchanging the protein into 0.05 M sodium acetate (pH 4.5) in preparation for the coupling reaction. Oxidation of N-terminal Ser would convert a positively charged site into a neutral one, lowering the isoelectric point of the protein and allowing the reaction to be monitored by IEF (Figure 5A). As anticipated, IEF showed that mIL-la was quantitatively converted to a modified form with a more acidic PI,consistent with oxidation of the N-terminus. A minor band in the preparation, originally with a more acidic p l than the major component, also shifted upon oxidation. This minor component has not been identified, but may be a deamidated form of mIL-la. A 500-pmol sample of periodate-oxidized mIL-la gave no sequence result in the gas-phase sequencer, with the quantity of protein submitted being verified by amino acid analysis. This loss of capacity to undergo automated E d " degradation confirmed that mIL-la had undergone modification of its N-terminus. To characterize the modification further, unmodified and oxidized mIL-la were digested with trypsin in the presence of 1M guanidinium chloride and the digests were compared by reversed-phase HPLC peptide mapping (Figure 6a,c). Selective modification of N-terminal Ser would result in the maps differing only by a shift in the position of the predicted N-terminal undecapeptide, SAPYTYQSDLR, due to its oxidation. With one additional discrepancy (see below),this was the result obtained. The peak near 19 min in the map of unmodified mIL-la was missing from the oxidized map, but the oxidized map
contained a new component at about 20.1 min. In PDMS, the peak eliminated by periodate oxidation had an MH+ of 1302.5 (calculated MH+ for SAPYTYQSDLR, 1301.4) (Figure 6B). The 20.1-min fragment from oxidized MH+ gave components with MH+ values of 1271.4 and 1289.7 1270.4 [calculated MH+ for a-N-glyoxylyl-APYTYQSDLR, (unhydrated) and 1288.4 (hydrated)] (Figure 6d). Sequence analysis of the putative SAPYTYQSDLR derived from unmodified mIL-la confirmed its identity, also showing that the discrepancy between observed and calculated MH+ values was not due to deamidation of the Gln residue, but was due to normal error in the PDMS. The maps also differed by the presence of a minor peak at about 15 min in the map of unmodified mIL-la (Figure 6a) which was absent from the digest of oxidized protein (Figure 6c). Sequence and mass analyses showed that this was due to a mixture of three fragments of mIL-la. It was apparent from analysis of other peaks (not shown) that digestion of mIL-la in the presence of 1 M guanidinium chloride was incomplete and that several peaks in the maps were due to fragments containing a potentially trypsinlabile bond. Thus, the presence of this small extra peak in the unmodified map was attributed to inconsistency between two incomplete digestions. Following overnight coupling with biotin-X-hydrazide (see the methods), the samples were gel filtered into 0.02 M Tris (pH 7.0) to remove the hydrazide and prepare them for anion-exchange chromatography using a Pharmacia MonoQ column. All three protein samples gave sharp single peaks; as an example, Figure 7 shows chromatography of the product from the biotinylation reaction. The oxidized/nonbiotinylated and oxidizedtbiotinylated forms of the protein were eluted slightly later in the NaCl gradient (-0.07 M NaC1) than unmodified mIL-la (-0.06 M NaC1) (Figure 7). The samples were next examined by SDS-PAGE (Figure 5B). The unmodified and oxidizedtnonbiotinylated forms of mIL-la ran together in a position consistent with their respective masses of 18kDa. The oxidized/biotinylated form of mIL-la, however, gave a single band of slightly reduced mobility, consistent with covalent attachment of biotin-X-hydrazide (M, of 371.5). To confirm that this was due to modification with biotin-X-hydrazide,a similar gel was electroblotted to a PVDF membrane and the blot was developed using avidin-HRP conjugate (Figure 5C). A single positive band at 18 kDa was found in the lane loaded with oxidized/biotinylated mIL-la; no bands were present in the lanes loaded with control mIL-la samples. Thus, the small mass increase in the oxidized/biotinylated protein could be attributed to attachment of biotinX-hydrazide. Conversion of the oxidized mIL-la to the biotinylated form appeared quantitative (Figure 5B), and the adduct was stable to SDS-PAGE. For definitive mass analysis of the modified cytokine, unmodified and modified forms of mIL-la were desalted by reversed-phase HPLC and subjected to ESMS. In the HPLC step (Figure 8A), the unmodified, oxidized, and biotinylated forms of the protein were eluted in the same relative order as similar series prepared from much smaller peptides (e.g. Figures 1 and 2). The absorption spectra of the unmodified and oxidized forms of the protein were essentially equivalent, but the biotinylated protein had a strong new absorption band centered at about 264 nm (Figure 8B). This was assigned to the hydrazone formed by biotin-X-hydrazide at the oxidized N-terminus. In support of this assignment, the hydrazone adduct of the aromatic-free peptide oxidation product a-N-glyoxylylIGSLAK with biotin-X-hydrazide possessed a very similar
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Bioconjugafe Chem., Voi. 3, No. 2, 1992
Site-Directed Modification
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pl 6.5-
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-97 -66 -45
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Figure 5. Electrophoretic analysis of N-terminal oxidation and biotinylation of mIL-la. (A) IEF of periodate oxidation showing, lane 1,PI standards; lane 2, mIL-la before periodate oxidation; lane 3, mIL-la after periodate oxidation. The gel was a Phast IEF pH 4-6.5 premade gel, silver stained. (B) SDS-PAGE of biotinylation reaction, lane 1, Mr markers; lane 2, mIL-la which was not periodate oxidized or treated with biotin-X-hydrazide; lane 3, mIL-la which was periodate oxidized but not treated with biotin-Xhydrazide; lane 4, mIL-la which was periodate oxidized and allowed to react with biotin-X-hydrazide. The gel (PhastGel 12% homogeneous) was silver stained. (C) Western blot analysis of biotinylation of mIL-la. A gel similar to that in the preceding panel was used, except that biotinylated M , markers were used. Lane 1, mIL-la which was not periodate oxidized or treated with biotinX-hydrazide; lane 2, mIL-la which was periodate oxidized but not treated with biotin-X-hydrazide; lane 3, mIL-la periodate oxidized and reacted with biotin-X-hydrazide; lane 4, biotinylated Mr markers.
(c
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MR
Figure 6. Comparative reversed-phase HPLC peptide mapping of unmodified and periodate-oxidized mIL-la. (a) HPLC peptide map of unmodified mIL-la digested with trypsin in the presence of 1M guanidinium chloride (see Experimental Procedures). The peak marked with an asterisk was selected for PDMS. (b) PDMS analysis of selected peak from digest of unmodified mIL-la. The calculated MH+ for SAPYTYQSDLR, the predicted N-terminal tryptic peptide of mIL-la, was 1301.4. (c) HPLC peptide map of periodate-oxidized mIL-la digested with trypsin in the presence of 1 M guanidinium chloride. The peak marked with an asterisk was selected for PDMS. (d) PDMS analysis of selected peak from digest of oxidized mIL-la. The calculated MH+ for a-N-glyoxylylAPYTYQSDLR was 1270.4 (unhydrated) and 1288.4 (hydrated).
absorption band (not shown); hence, the new band was not due to chemical modification or changes in the environment of aromatic residues in mIL-la. ESMS gave a result of 17 989.6 f 7.2 for unmodified mIL-la (theoretical mass, 17 990.3 Da) (Figure 9a; see inset text for deconvolution of the m/z peak series to a mass result). The biotinylated product gave a result of 18 314.8 f 1.2 (theoretical mass, 18 312.8) (Figure 9b). As the standard deviation provided information about the precision of the estimate rather than its accuracy, these
observed and theoretical values were not significantly discrepant; in fact, the error of about 0.01 % was consistent with standard estimates of the accuracy of ESMS. Thus, the result confirmed other indications that mIL-la had been monobiotinylatedby the specific site-directed chemistry. Mass analysis of the oxidizedintermediate form of mILla gave no clear result, probably due to the difficulty of separating contributions from the hydrated and unhydrated forms of the a-N-glyoxylyl N-terminus.
144
Qeoghegan and Stroh
Bloconlugete Chem., Vol. 3, No. 2, 1992
............................................................. Smhs
Mass Estimat.
ME
.............................................................
20:
ML?
Smhs
Mass Estimata
............................................................. IM+llHll'* lM+12HI"+
Figure 7. Anion-exchange chromatography of product from reaction of periodate-oxidized mIL-la with biotin-X-hydrazide. See the text for conditions. The arrow shows the position at which unmodified mIL-la was eluted in a similar run.
1668.0 1 527.4
18315.0 18 316.8
IM+15HI16+ 1 2 2 1 . 9
18313.6
1832.6
1527,4
A
1666.0
2e
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o
0.3: 1.2
< g0.6-
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4
'
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Figure 8. Reversed-phase HPLC of unmodified, periodateoxidized and biotinylated mIL-la. See the text for conditions. (A) Portion of chromatograms showing peaks due to (- -) unmodified, (-) periodate-oxidized, and (-) biotinylated mIL-la. (B) Absorption spectra corresponding to 280-nm peaks observed in panel A recorded by diode-arraydetector in HPLC (linestyles as in panel A). The receptor binding and other biologically relevant activities of periodate-oxidized and biotinylated mIL-la were indistinguishable from those of control unmodified samples. A detailed account of the biological activities of biotinylated mIL-la will be presented elsewhere (Otterness, I. G., Geoghegan, K. F., et al., in preparation). Numerous hydrazide tagging reagents are available. Those tested successfully in the present work have been biotin hydrazide (in three forms differing by the length of spacer group), Lucifer Yellow, and Cascade Blue hydrazide (not shown). One practical problem was the presence of hydrazine in the Cascade Blue hydrazide as supplied (probably a residue from synthesis); as hydrazine competed avidly for aldehyde, it had to be removed by passing the reagent through Dowex resin in the sulfonic acid form before use. As a further practical note, it was important to avoid introducing periodate-quenching substances such as glycerol into the protein sample before oxidation. Glycerol is present in some commercial membrane devices, such as the Centricon concentrator; use of these units was avoided until after the periodate reaction.
r
500
.
.
,
.
,
'Oo0
.
!
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1500
201
Figure 9. Electrospray mass spectrometry of unmodified and biotinylated mIL-la. See the text for conditions; text inset to each panel of the figure shows the basis for the mass estimate in each experiment,in which the mean of estimates provided by a series of multiply-charged ions is computed as the final result. (a) ESMS analysis of unmodified mIL-la (theoretical mass 17 990.3). (b) ESMS analysis of mIL-la monobiotinylated by hydrazone linkage at the N-terminus (theoreticalmass 18 312.8).
DISCUSSION Broadly applicable methods for site-directed tagging-the ability to deliver a label or reporter group to a single locus in a polypeptide-would reduce the tedium and complexity associated with the preparation of peptide conjugates. They would also promote the ability to apply knowledge of structure-function relationships in peptides to the selection of target sites for chemical modification. Modification of regions relevant to the biological function of a peptide could be avoided, maximizing the chances of producing conjugates with full biological activity. The need for such technology is widely appreciated (48,13), and three main approaches to the problem have been proposed: (i) to alter the peptide sequence so that only one group of the type to be tagged is present; examples are introduction of a single surface-accessible thiol into interleukin-lfl by site-directed mutagenesis (I7,18) and elimination of all but one Lys from calcitonin to leave a single t-amino group for modification (19);(ii) to use an enzyme to confer specificity, as in C-terminal biotinylation of proteins (6) or C-terminal incorporation of a hydrazide or other group as the locus for secondary modification (7,8); and (iii) to synthesize peptides with sitedirected modification in mind, so that a uniquely reactive group is present or can be generated. An example is incorporation of a protected thiol group at the N-terminus of synthetic products which may be exposed to provide a locus for site-directed coupling (4). The present strategy most closely resembles the third of these approaches. This paper has implemented a
SlteDlrected Modlflcatlon
scheme for N-terminal modification of peptides equipped with an N-terminal Ser or Thr (13),but the approach has wider potential for site-directed modification at Nterminal, internal, or C-terminal locations in synthetic peptides. For proteins produced biosynthetically, it will remain a means of N-terminal modification alone for the foreseeable future. Site-directed mutagenesis can be used to introduce N-terminal Ser or Thr when needed. In this procedure, a 2-amino alcohol group in a protein or peptide (N-terminal Ser or Thr, or any hydroxylysine side chain) is first oxidized with periodate to generate an aldehyde, and this is derivatized with a hydrazide reagent to form a hydrazone adduct. Both chemical steps are classical, direct, and reliable; thus, it was readily possible to demonstrate selective N-terminal modification both of small peptides (Figures 1-3) and a larger protein (Figures 5,6, and 9). The major issues requiring study have been the selectivity with which the desired oxidation can be achieved, and the stability of the hydrazone adducts once formed. The method could not be effective if periodate invariably caused widespread oxidation at different amino acid residues, nor would it be satisfactory if it generated conjugates that lacked the requisite stability for biochemical experiments such as receptor binding assays and cell sorting. Considerations relating to the oxidative step should also apply to the method described by Rose et al. (8). To effect C-terminal site-directed tagging, they proposed the use of carboxypeptidase-catalyzed couplingof 1,3-diamino-2-propan01 to peptides followed by periodate oxidation of the resulting 2-amino alcohol as a means to generate an aldehyde for subsequent modification. Periodate oxidation of 2-amino alcohols -CH(NHdCH(OH)- is a very fast reaction, reportedly 1000-fold faster As than the periodate oxidation of 1,2-diols (9-13,20,21). a result, selective oxidation of a 2-amino alcohol structure in a protein is possible even though other side chains are potentially susceptible to oxidation. This was used to convert N-terminal Ser to Gly in corticotropin (16)and to reverse reductive hydroxyisopropylation (22)or reductive dihydroxypropylation (23)of amino groups. Periodate effects quantitative oxidation of N-terminal Ser when present in only a low molar excess over peptide, as demonstrated by the oxidations of SYSMEHFRWG (Figure 3a) and mIL-la (Figure 5A). The use of mass spectrometry has recently simplified the analysis of oxidative reactions in peptides, so that anomalous instances of a competing reaction that matches or exceeds the rate of the desired process can readily be detected. Periodate oxidation of SYSMEHFRWG in the pH range 4-8 occurred at the N-terminus and at Met. The Tyr, Trp, and His residues were not appreciably oxidized under the conditions of the experiment (see the methods). The conversion of Met to its sulfoxide was favored by acidification, while the desired N-terminal reaction was strongly favored at pH 7-8 (Figure 3) (12). Met residues in peptides vary in their susceptibility to oxidation (24,251, and there may be cases where oxidation of a particular Met will be accelerated to a rate closer to that of the reaction with N-terminal Ser; in synthetic peptides, a possible countermove will be to replace the sensitive Met with norleucine. Peptides containing a free thiol probably should not be used because of their extreme susceptibility to oxidation (IO),but the possibility of protecting thiols by reversible blocking remains to be explored. We have not studied the periodate susceptibility of either disulfide bonds or protein-linked carbohydrates in relation to the rapid oxidation of 2-amino alcohols. It seems quite
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145
possible, given the reported preferential oxidation of 2amino alcohols relative to 1,Zdiols at pH 7-8, that the present method can be applied successfully to glycoproteins. Conversely,however, O’Shannessy and Wilchek (26) noted that periodate oxidation of glycoproteins with Nterminal Ser or Thr probably cannot be directed selectively to the carbohydrates without also generating an aldehyde at the N-terminus. The conjugation of a-N-glyoxylyl peptides with hydrazides proceeds under mild conditions, generally going to near completion in 2-16 h at room temperature. Aqueous buffers of pH 4.5 have been used, sometimes with the addition of 8%-25% acetonitrile to maintain solubility of a hydrophobic reagent such as biotin-Xhydrazide. Progress of the reactions can be monitored by reversed-phase HPLC, although when hydrazides containing a highly electronegative substituent (such as Cascade Blue hydrazide) are used, the solvent pH should be pH 4-7; the hydrazone formed between glyoxylyl-YSMEHFRWG and Cascade Blue hydrazide decomposed with increasing time of residence when adsorbed on a column in the presence of solvents containing 0.1 % TFA (not shown). While hydrazones are acid-labile, they are relatively stable at pH 6-8 (Figure 4) and form the basis of several schemes for conjugating biomolecules (26,27).The ability of N-terminally biotinylated mIL-la to remain stable through SDS-PAGE (including sample preparation with heating in the presence of 0-mercaptoethanol and SDS) (Figure 5B) is a good indication of the stability of such conjugates. In addition, our many successful recoveries of hydrazone conjugates from HPLC experiments performed using solvents containing 0.1% TFA (pH -2.1) show that at least a selection of them, although perhaps not all (see Results), can endure this valuable procedure. In all such cases, however, samples have been kept cold when possible and the acidic solvent has been removed at the first opportunity. Labile hydrazones require chromatography at pH 4-7. Application of the method described here to site-directed modification at internal and C-terminal loci will require peptides synthesized with a strategically located residue of hydroxylysine. A barrier has been the commercial unavailability of L-hydroxylysine, which (in suitably protected form) is required for the synthesis of peptides that contain hydroxylysine and have an all-L peptide backbone. Using such peptides, it will be possible to produce peptide conjugates which are tagged at the site of the hydroxylysine. Hydroxylysine in peptides has been shown to undergo rapid periodate oxidation to create an aldehyde (28). The experiments presented here document the feasibility of an approach to site-directed chemical modification of peptides and certain proteins. The use of periodate does create a need for careful analysis of products for potential side reactions. On the other hand, the desired reaction of periodate with 2-amino alcohols is very fast and selective, and the potential for side reactions can be limited by using very low periodate:protein molar ratios. Also, the routine application of mass spectrometry to peptide and protein chemistry has greatly increased the precision with which peptide modifications can be monitored. This simple and direct procedure may prove to be a useful option among strategies for site-directed protein and peptide tagging. ACKNOWLEDGMENT We thank many colleagues for helpful advice and support, including G . C. Andrews, L. J. Contillo, G. 0.
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Daumy, D. E. Danley, H. B. F. Dixon, M. E. Kelly, A. J. Lanzetti, I. G. Otterness, A. R. Proctor, K. J. Rosnack, N. A. Saccomano, A. D. Vodenlich, R. A. Volkmann, and P. T. Wingfield. LITERATURE CITED (1) Bayer, E. A., and Wilchek, M. (1990) Protein Biotinylation. Methods Enzymol. 184, 138-160. (2) Laveille, S., Chassaing, G., Beaujouan, J. C., Torrens, Y., and Marquet, A. (1984) Binding affinities t o r a t brain synaptosomes-synthesis of biotinylated analogues of Substance P. Znt. J. Pept. Protein Res. 24, 480-487. (3) Arya, R., and Garibpy, J. (1991) Rapid Synthesis and Introduction of a Protected EDTA-like Group during the SolidPhase Assembly of Peptides. Bioconjugate Chem. 2, 323326. (4) Drijfhout, J. W., Bloemhoff, W., Poolman, J. T., and Hoogerhout, P. (1990) Solid-Phase Synthesis and Applications of N(S-Acetylmercaptoacetyl)Peptides. Anal. Biochem.187,349354. (5) Wetzel, R., Halualani, R., Stults, J. T., and Quan, C. (1990) A General Method for Highly Selective Cross-linking of Unprotected Polypeptides via pH-controlled Modification of N-terminal a-Amino Groups. Bioconjugate Chem. 1, 114122. (6) Schwarz, A., Wandrey, C., Bayer, E. A., and Wilchek, M. (1990) Enzymatic C-Terminal Biotinylation of Proteins. Methods Enzymol. 184, 160-162. (7) Rose, K., Vilaseca, L. A,, Werlen, R., Meunier, A., Fisch, I., Jones, R. M. L., and Offord, R. E. (1991) Preparation of WellDefined Protein Conjugates Using Enzyme-Assisted Reverse Proteolysis. Bioconjugate Chem. 2, 154-159. ( 8 ) Rose, K., Jones, R. M. L., Sundaram, G., and Offord, R. E. (1989) Attachment of Linker Groups to Carboxyl Termini Using Enzyme-assisted Reverse Proteolysis. Peptides 1988 (G. Jung, 2nd E. Bayer, Eds.) pp 274-276, Walter de Gruyter & Co., New York. (9) Nicolet, B. H., and Shinn, L. A. (1939) The Action of Periodic Acid on a-Amino Alcohols. J. Am. Chem. SOC.61, 1615. (10) Dixon, H. B. F., and Fields, R. (1972) Specific Modification of NH2-Terminal Residues by Transamination. Methods Enzymol. 25, 409-419. (11) Dixon, H. B. F. (1984) N-Terminal Modification of Proteins-A Review. J. Protein Chem. 3, 99-108. (12) Sklarz, B. (1967) Organic Chemistry of Periodates. Q.Rev., Chem. SOC.21, 3-28. (13) Offord, R. E. (1990) Chemical Approaches to Protein Engineering. Protein Design and the Development of New Therapeutics and Vaccines (J. B. Hook and G. Poste, Eds.) pp 253-282, Plenum Publishing Corporation, New York. (14) Daumy, G. O., Merenda, J. M., McColl, A. S., Andrews, G. C., Franke, A. E., Geoghegan, K. F., and Otterness, I. G. (1989) Isolation and Characterization of Biologically Active Murine Interleukin-la Derived from Expression of a Synthetic Gene in Escherichia coli. Biochim. Biophys. Acta 998, 32-42.
Geoghegan and Stroh
(15) Clamp, J. R., and Hough, L. (1965) The Periodate Oxidation of Amino Acids with Reference to Studies on Glycoproteins. Biochem. J . 94, 17-24. (16) Dixon, H. B. F., and Weitkamp, L. R. (1962) Conversion of the N-Terminal Serine Residue of Corticotrophin into Glycine. Biochem. J. 84, 462-468. (17) Wingfield, P., Graber, P., Shaw, A. R., Gronenborn, A. M., Clore, G. M., and MacDonald, H. R. (1989) Preparation, characterization, and application of interleukin-18 mutant proteins with surface-accessible cysteine residues. Eur. J. Biochem. 179, 565-571. (18) Chollet, A., Bonnefoy, J.-Y., and Odermatt, N. (1990) Preparation, application and biological characterization of interleukin-18 mutant protein biotinylated at a single site. J. Immunol. Methods 127, 179-185. (19) D’Santos, C. S., Nicholson, G. C., Mosely, J. M., Evans, T., Martin, T. J., and Kemp, B. E. (1988) Biologically Active, Derivatizable Salmon Calcitonin Analog: Design, Synthesis and Applications. Endocrinology 123, 1483-1488. (20) Barlow, C. B., Guthrie, R. Do,and Prior, A. M. (1966) Periodate Oxidation of Amino Sugars. Chem. Commun. pp 268269. (21) Geoghegan, K. F., and Dixon, H. B. F. (1989) Synthesis of 2-Aminoethylarsonic acid. A new synthesis of primary amines. Biochem. J. 260, 295-297. (22) Geoghegan, K. F., Ybarra, D. M., and Feeney, R. E. (1979) Reversible Reductive Alkylation of Amino Groups in Proteins. Biochemistry 18, 5392-5399. (23) Acharya, A. S., and Manjula, B. N. (1987) Dihydroxypropylation of Amino Groups of Proteins: Use of Glyceraldehyde a s a Reversible Reagent for Reductive Alkylation. Biochemistry 26, 3524-3530. (24) Knowles, J. R. (1965) The Role of Methionine in a-Chymotrypsin-Catalyzed Reactions. Biochem. J. 95, 180-190. (25) Penner,M. H., Yamasaki,R. B.,Osuga, D. T., Babin,D.R., Meares, C. F., and Feeney, R. E. (1983)Comparative Oxidations of Tyrosines and Methionines in Transferrins: Human Serum Transferrin, Human Lactotransferrin, and Chicken Ovotransferrin. Arch. Biochem. Biophys. 225, 740-747. (26) O’Shannessy, D. J.,and Wilchek, M. (1990) Immobilization of Glycoconjugates by Their Oligosaccharides: Use of Hydrazido-Derivatized Matrices. Anal. Biochem. 191, 1-8. (27) Kaneko, T.,Willner, D., Monkovic, I., Knipe, J. O., Braslawsky, G. R., Greenfield, R. S., and Vyas, D. M. (1991) New Hydrazone Derivatives of Adriamycin a n d Their Immunoconjugates-A Correlation between Acid Stability and Cytotoxicity. Bioconjugate Chem. 2, 133-141. (28) Van Slyke, D. D., Hiller, A., and MacFadyen, D. A. (1941) The Determination of Hydroxylysine in Proteins. J. Biol. Chem. 141,681-705. Registry No. SIGSLAK, 115918-58-6; SYSMEHFRWG, 2791-05-1; TIGSLAK, 138354-10-6; a-N-glyoxylyl-(IGSLAK), 138354-05-9; a-N-glyoxylyl-(YSMEHFRWG), 138354-06-0; biotin-X-(YSMEHFRWG), 138354-09-3;biotin-X-hydrazidela-Nglyoxylyl-(IGSLAK) hydrazone conjugate, 138354-07-1; lucifer yellowla-N-glyoxylyl-(IGSLAK) hydrazone, 138354-08-2;biotinX-hydrazide lucifer yellow CH, 67769-47-5.
