Confirmation by Mass Spectrometry of a Trisulfide Variant in Methionyl

Anal. Chem. 1996, 68, 4044-4051

Confirmation by Mass Spectrometry of a Trisulfide Variant in Methionyl Human Growth Hormone Biosynthesized in Escherichia coli Eleanor Canova-Davis,*,† Ida P. Baldonado,† Rosanne C. Chloupek,† Victor T. Ling,† Richard Gehant,‡ Kenneth Olson,‡ and Beth L. Gillece-Castro§

Medicinal and Analytical Chemistry, Process Sciences, and Protein Chemistry Departments, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, California 94080

A sulfur-containing compound found in acid hydrolysates of proteins was identified 30 years ago as a trisulfide: bis(2-amino-2-carboxyethyl) trisulfide (cysteine2S3). At that time, studies concerning the chemistry of sulfur-transferring enzyme systems suggested that cysteine2S3 also existed in biological systems. Two decades later, a cystine trisulfide structure was postulated in the regulator protein molecule for the activation of δ-aminolevulinate synthetase. Recently, a trisulfide bond was reported to occur in the minor loop disulfide at Cys182-Cys189 in human growth hormone. We have detected a trisulfide structure in methionyl human growth hormone in the major loop disulfide Cys53-Cys165. The development of mass spectral analyses of high molecular weight molecules, such as proteins, led to the eventual identification of the modification. A tandem mass spectral analysis on a Sciex electrospray instrument localized an addition of 32 Da to the Cys53-Cys165 fragment. Elemental composition was determined by accurate mass measurement obtained by peak matching to a synthetic peptide and established that an extra sulfur atom was involved. Sulfur is widely distributed in nature as free and inorganic forms in mineral deposits, the atmosphere, and interstellar space. Organosulfur compounds are found in petroleum deposits and living systems. Sulfur commonly occurs in proteins as the sulfhydryl group of cysteine, the disulfide group of cystine, and the thioether group of methionine. In addition, acid-labile or “inorganic” sulfide is found in iron-sulfur proteins such as the ferredoxins. The first of the sulfur-containing amino acids to be discovered was cystine by Wollaston in 1810 in the stones of the bladder,1 from which it received its name (cystic, “pertaining to the bladder”). Many years later, cystine was found in proteins by Morner,2 and its structure was established at the beginning of the 20th century.3,4 In 1907, Heffter5 discovered the sulfhydryl group in proteins. Methionine was identified a few decades later by Mueller,6-8 who separated it from the products of the hydrolysis †

Medicinal and Analytical Chemistry Department. Process Sciences Department. Protein Chemistry Department. (1) Wollaston, W. H. Philos. Trans. R. Soc. London 1810, 21-33. (2) Morner, K. H. A. Hoppe-Seyler’s Z. Physiol. Chem. 1899, 28, 595-615. (3) Neuberg, C. Chem. Ber. 1902, 35, 3161-3164. (4) Friedmann, E. Beitr. Chem. Physiol. Pathol. 1903, 3, 1-47. (5) Heffter, A. Med. Naturwiss. Arch. 1907, 1, 81-103. (6) Mueller, J. H. Proc. Soc. Exper. Biol., N.Y. 1922, 19, 161-163. ‡ §

4044 Analytical Chemistry, Vol. 68, No. 22, November 15, 1996

of casein. Its structure was established in 1928 by Barger and Coyne.9 This article describes the positive identification of a trisulfide structure in recombinant DNA-derived methionyl human growth hormone. Interestingly, an enyzyme-trisulfide mechanism for the action of β-mercaptopyruvate transsulfurase was proposed more than 30 years ago by Kun and Fanshier.10 Shortly afterward, Hylin and Wood11 demonstrated that the reactive form of sulfur produced by this enzyme was indeed a protein-bound polysulfide. These uncommon sulfur forms were reported to occur in a number of biological systems. Thiocysteine, a persulfide (-SSH group), was postulated as an intermediate in both the nonenzymatic and enzymatic degradations of cystine.12-15 The occurrence of trisulfide structures was also documented in the degradation of cystine,16 found in ponerine ants as a dimethyl trisulfide,17 and in commercial samples of glutathione.18 About this time, definite physical measurements using nuclear magnetic resonance spectroscopy determined a trisulfide structure in an allylic bicyclic polysulfide.19 The formation of trisulfide structures in proteins was postulated as a result of alkaline treatment20 and that in enzymes as reaction of the acid-labile sulfur in transsulfuration reactions.21-24 Sandy et al.25 isolated naturally occurring thiocystine, glutathione trisulfide, and the corresponding mixed trisulfide of glutathione from Rhodopseudomonas spheroides, which served as activators of (7) Mueller, J. H. J. Biol. Chem. 1923, 56, 157-169. (8) Mueller, J. H. J. Biol. Chem. 1923, 58, 373-375. (9) Barger, G.; Coyne, F. P. Biochem. J. 1928, 22, 1417-1425. (10) Kun, E.; Fanshier, D. W. Biochim. Biophys. Acta 1959, 32, 338-348. (11) Hylin, J. W.; Wood, J. L. J. Biol. Chem. 1959, 234, 2141-2144. (12) Dann, J. R.; Oliver, G. L.; Gates, J. W., Jr. J. Am. Chem. Soc. 1957, 79, 1644-1649. (13) Cavallini, D.; De Marco, C.; Mondovi, B. Arch. Biochem. Biophys. 1960, 87, 281-288. (14) Cavallini, D.; De Marco, C.; Mondovi, B.; Mori, B. G. Enzymologia 1960, 22, 161-173. (15) Flavin, M. J. Biol. Chem. 1962, 237, 768-777. (16) Fletcher, J. C.; Robson, A. Biochem. J. 1963, 87, 553-559. (17) Casnati, G.; Ricca, A.; Pavan, M. Chem. Ind. (Milan) 1967, 49, 57-58. (18) Massey, V.; Williams, C. H., Jr.; Palmer, G. Biochem. Biophys. Res. Commun. 1971, 42, 730-738. (19) Hofle, G.; Baldwin, J. E. J. Am. Chem. Soc. 1971, 93, 6307-6308. (20) Catsimpoolas, N.; Wood, J. L. J. Biol. Chem. 1964, 239, 4132-4137. (21) Sorbo, B. Acta Chem. Scand. 1963, 17, 2205-2208. (22) Szczepkowski, T. W.; Wood, J. L. Biochim. Biophys. Acta 1967, 139, 469478. (23) Wider De Xifra, E. A.; Sandy, J. D.; Davies, R. C.; Neuberger, A. Philos. Trans. R. Soc. London B. 1976, 273, 79-98. (24) Yamanishi, T.; Tuboi, S. J. Biochem. 1981, 89, 1913-1921. (25) Sandy, J. D.; Davies, R. C.; Neuberger, A. Biochem. J. 1975, 150, 245-257. S0003-2700(96)00591-4 CCC: $12.00

© 1996 American Chemical Society

δ-aminolevulinate synthetase. It was also reported that a high molecular weight regulator protein containing a trisulfide was required for full activation. The cystine trisulfide structure was confirmed by amino acid analysis following pronase digestion of the regulator protein.26,27 In 1983, Westley et al.28 postulated the involvement of persulfide structures in the mechanism of action of enzymes such as rhodanese and thiosulfate reductase. Additionally, they claimed that mercaptopyruvate sulfurtransferase may not be a trisulfide, as proposed by Kun and Fanshier,10 but that a persulfide structure may reside in the substrate. Subsequently, many investigators proposed the presence of a persulfide group essential for catalytic activity in iron-sulfur enzymes such as xanthine or aldehyde oxidase.29,30 Petering et al.31 proposed that oxidation of ferridoxin and several other iron-sulfur proteins can lead to protein-bound trisulfides. Likewise, Pagani et al.32 postulated that a trisulfide structure was formed from labile sulfur in deactivated molecules of succinate dehydrogenase. In many of the above investigations, distinction between a thiocysteine or trisulfide structure in proteins was not definitively demonstrated. Recently, a trisulfide structure was characterized in biosynthetic human growth hormone in the Cys182-Cys189 bridge by means of peptide mapping procedures coupled with detection of sulfur liberation.33 Another recent study34 identified this same trisulfide using mass spectrometry and two-dimensional nuclear magnetic resonance. The following study reports the positive identification of a trisulfide structure in recombinant DNA-derived methionyl human growth hormone in the Cys53-Cys165 bridge using tandem mass spectrometry and exact mass determinations. EXPERIMENTAL SECTION Isolation of a Methionyl Human Growth Hormone Variant Using Reversed-Phase HPLC. A 3 mL aliquot containing approximately 48 mg of a side fraction from a methionyl human growth hormone purification was loaded onto a 4.6 mm × 50 mm PLRP-S, 300 Å, 8 µm reversed-phase HPLC column (Polymer Labs, Inc., Amherst, MA). The separation was performed at a flow rate of 1 mL/min at ambient temperature on a HewlettPackard 1090M HPLC system fitted with a Rheodyne manual injector. Solvent A was 30% acetonitrile (J. T. Baker) in 50 mM sodium phosphate, pH 7.5, and solvent B was 60% acetonitrile in 50 mM sodium phosphate, pH 7.5. The run conditions were 100% A, 0-10 min, then 75% A/25% B, 10-20 min, followed by a gradient of 25-50% B until the peak of interest was fully eluted. A total of 168 mg of methionyl human growth hormone was processed by this method. Fractions of the latest eluting peak were pooled, and the solvent was removed by rotary evaporation. Estimated (26) Oyama, H.; Tuboi, S. J. Biochem. 1979, 86, 483-489. (27) Yamanishi, T.; Kubota, I.; Tuboi, S. J. Biochem. 1983, 94, 181-188. (28) Westley, J.; Adler, H.; Westley, L.; Nishida, C. Fundam. Appl. Toxicol. 1983, 3, 377-382. (29) Massey, V.; Edmondson, D. J. Biol. Chem. 1970, 245, 6595-6598. (30) Branzoli, U.; Massey, V. J. Biol. Chem. 1974, 249, 4346-4349. (31) Petering, D.; Fee, J. A.; Palmer, G. J. Biol. Chem. 1971, 246, 643-653. (32) Pagani, S.; Cannella, C.; Cerletti, P.; Pecci, L. FEBS Lett. 1975, 51, 112115. (33) Jespersen, A. M.; Christensen, T.; Klausen, N. K.; Nielsen, P. F.; Sorensen, H. H. Eur. J. Biochem. 1994, 219, 365-373. (34) Andersson, C.; Edlund, P. O.; Gellerfors, P.; Hansson, Y.; Holmberg, E.; Hult, C.; Johansson, S.; Kordel, J.; Lundin, R.; Mended-Hartvig, I.; Noren, B.; Wehler, T.; Widmalm, G.; Ohman, J. Int. J. Pept. Protein Res. 1996, 47, 311-321.

purity by HPLC analysis was 70%. This pool was then loaded onto a 4.6 mm × 50 mm PLRP-S, 1000 Å reversed-phase HPLC column (Polymer Labs). The separation was performed at a flow rate of 2 mL/min at ambient temperature. Solvent A was 30% acetonitrile in 50 mM sodium phosphate, pH 7.5, and solvent B was 60% acetonitrile in 50 mM sodium phosphate, pH 7.5. The run conditions were 100% A, 0-5 min, followed by a gradient of 2550% B in 25 min. Fractions of the latest eluting peak were pooled, and the solvent was removed by rotary evaporation. Estimated purity by HPLC analysis was 97%, and the yield was 1.7 mg. Analytical Neutral pH Reversed-Phase HPLC. This analysis was performed on a 4.6 mm × 250 mm Vydac C4, 300 Å, 5 µm reversed-phase column (The Separations Group, Hesperia, CA) at a flow rate of 0.5 mL/min, with the column temperature controlled at 30 °C. Solvent A was 70% 15 mM ammonium bicarbonate, pH 7.8, 30% acetonitrile, and solvent B was 40% 15 mM ammonium bicarbonate, pH 7.8, 60% acetonitrile. Samples (20 µL, 1 mg/mL) were loaded and eluted with a linear gradient of 25-50% B in 40 min. Trypsin Digestion. The protein samples (1 mg/mL) were exchanged into 100 mM ammonium bicarbonate using a Pharmacia PD-10 column at ambient temperature according to the manufacturer’s recommendations. Digestion was conducted with L-1-(tosylamino)-2-phenylethyl chloromethyl ketone-treated trypsin (Worthington) prepared fresh in 0.01 mM HCl. An aliquot of 1:100 enzyme/substrate by weight was added at zero time and another after 2 h of digestion at 37 °C. The reaction was terminated after a total of 4 h with 25 µL of 50% phosphoric acid. The resulting peptide mixture was separated by reversed-phase HPLC on a 4.6 mm × 150 mm Nucleosil C18, 5 µm, 100 Å column and monitored at 214 nm. The separation was performed at a flow rate of 0.5 mL/min and a column temperature of 40 °C. Solvent A was 0.05% trifluoroacetic acid (TFA, Pierce sequencing grade), and solvent B was 0.04% TFA in acetonitrile. Elution was effected with a linear gradient of 0-60% B in 2 h. Peptide peaks were collected manually for further analysis or subjected directly to mass spectral analysis. Reduction and Reoxidation of Methionyl Human Growth Hormone Samples. Approximately 100 µg of protein was dissolved in 100-500 µL of 0.1 M Tris, pH 7.5-8.5, containing 6 M guanidine-HCl. A 25-fold molar excess of dithiothreitol (DTT) over the total disulfide content was added, and the mixture was incubated at room temperature for 1 h. This solution was dialyzed against deoxygenated 0.1 M Tris, pH 7.5-8.5, containing 1 mM DTT at 4 °C for 2-3 h without stirring. Dialysis was continued against fresh buffer at 4 °C with stirring overnight. The sample was then autoxidized against 0.1 M Tris, pH 7.5-8.5, at room temperature for 12-24 h. Thermolysin Digest of Purified Peptides. The purified T6T16-linked peptide pairs were exchanged into 100 mM ammonium bicarbonate. Samples were digested with a single addition of thermolysin (Boehringer/Manheim) prepared fresh in 100 mM ammonium bicarbonate (1:10 enzyme/protein by weight) and incubated at 37 °C for 1 h. The peptide mixtures were loaded onto a 2 mm × 150 mm Zorbax SB-300, C8, 5 µm, stable bond reversed-phase HPLC column (MAC-MOD Analytical, Inc., Chadds Ford, PA). The separation was performed at a flow rate of 0.4 mL/min and a column temperature of 40 °C on a Hewlett-Packard 1090M HPLC system. Solvent A was 0.05% TFA, and solvent B was 0.04% TFA in acetonitrile. Elution was effected with a linear Analytical Chemistry, Vol. 68, No. 22, November 15, 1996

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gradient of 0-35% B in 35 min. Peptide peaks were detected by the diode array detector at 214 nm and collected manually for further analysis or subjected directly to mass spectral analysis. Circular Dichroism Spectra. Fractions obtained from the reversed-phase HPLC purification were dialyzed against 0.1 M Tris, pH 8.0. Spectra were taken on a Cary 60/AVIV spectropolarimeter at 25 °C in 0.5 cm path length quartz cells from 360 to 240 nm. Each sample was scanned 10 times. During each scan, the ellipticity at 0.1 nm intervals was read 150 times and averaged, and then the 10 scans were averaged. The spectral bandwidth was 0.2 nm. Mass Spectrometry. A Sciex API III triple-quadrupole mass spectrometer (Perkin Elmer Sciex Instruments, Thornhill, Canada) was used to obtain the electrospray ionization mass spectra. Samples were dissolved in a 35% methanol solution with 0.5% acetic acid and infused into the mass spectrometer at 2-3 µL/min. The instrument was calibrated with a mixture of ammonium adducts of poly(propylene glycol)s. For collision-induced dissociation tandem mass spectra (CID MS/MS), Q1 was operated at unit m/z resolution (about 80% valley), and Q3 was operated at a lower resolution that gave ions of about 2-3 Da width at half-height. Ion spray voltage was at 5 kV, with the orifice potential set at 60 V. The collision cell (Q2) was offset at 65 V lower than Q0. The collision gas was argon at a collision gas thickness (CGT) of 6 × 1014 molecules/cm2, as calculated from a CGT setting of approximately 600 on the Sciex instrument. For the LC/MS analysis, the tryptic map effluent was split 1:20 with a Valco tee (Valco Instruments, Houston, TX) to give a flow rate of 25 µL/min into the ion spray nebulizer and the larger fraction flowing into the HP 1090 diode array detector, with monitoring at 220 nm. The ions generated in the ion spray nebulizer were analyzed in Q1. The quadrupole was scanned from 300 to 2000 Da in 4.3 s using a step size of 0.5 Da and a 1.2 ms dwell time per step. Further mass spectrometry experiments were performed on a tandem magnetic sector mass spectrometer (JEOL HX/110 HX/ 110) with fast atom bombardment (FAB) ionization. Samples were analyzed by loading 1 µL of peptide solution from reversed-phase HPLC onto the FAB target with 1 µL of matrix solution. Glycerol was used as the matrix solution for the elemental composition experiment. The exact molecular weight was determined by peak matching to a synthetic peptide which had a protonated molecular mass of 772.4739 Da. Instrument mass resolution for peak matching was set at 10 000. A mixture of thioglycerol/glycerol/ m-nitrobenzoic acid/acetic acid (9:4:6:1) was used to produce a strong and long-lived signal for the high-energy CID experiment. Mass resolution for the high-energy CID experiment was 1000, the accelerating voltage was 10 kV, and the collision cell voltage was 6 kV. The instrument was calibrated with Li, Na, and clusters of CsI. Helium gas pressure was adjusted to decrease the protonated molecule peak to 30% or its original intensity. The CID product ions were collected by array detection on a 3 in. microchannel plate held at 1.5 kV, while the associated phosphor was held at 4.0 kV. The magnet was stepped from 70 to 800 m/z in approximately 2 min. Activity Assays. Protein fractions were analyzed by a radioreceptor assay as described by Leung et al.35 and in an (35) Leung, D. W.; Spencer, S. A.; Cachianes, G.; Hammonds, G.; Collins, C.; Henzel, W. J.; Barnard, R.; Waters, M. J.; Wood, W. I. Nature 1987, 330, 537-543.

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Figure 1. Analytical neutral pH reversed-phase chromatography of methionyl recombinant human growth hormone. See Experimental Section for details. The peaks were previously identified as follows: (1) Met14(O)-containing, (2) Asp149-containing, and (3) methionyl recombinant human growth hormone. Peak 4 is the unknown variant.

enzyme-ligand immunosorbent assay (ELISA) with eight different monoclonal antibodies as the coat antibody. The epitopes of these monoclonals were mapped to the following sequences in human growth hormone: Mab 1, 88-104; Mab 2, 11-33 and 97-104; Mab 3, 12-33 and 109-129; Mab 4, 12-33 and 111-129; Mab 5, 57-73 and 164-190; Mab 6, 57-74; Mab 7, 46-52; and Mab 8, 32-46.36 RESULTS AND DISCUSSION Neutral pH Reversed-Phase High-Performance Chromatography. Protein preparations are routinely screened by reversedphase HPLC in acidic mobile phases to determine their purity. Such an analysis of methionyl human growth hormone biosynthesized and purified from Escherichia coli was essentially uninformative. To obtain a more discriminating chromatographic procedure, a neutral pH reversed-phase mobile phase was developed. A typical profile generated using sodium phosphate, pH 7.5, containing mobile phases is shown in Figure 1. The early eluting peaks were identified as the deamidated and methionine sulfoxide-containing species commonly seen in protein pharmaceuticals (data not shown). The fraction eluting at approximately 56 min was collected for identification. A likely candidate for a species of greater hydrophobicity is a norleucine-containing variant. It is known that the amino acid norleucine can be charged onto Met-t-RNA and become incorporated into proteins biosynthesized in E. coli.37-39 Since the norleucine-containing tryptic fragments can be easily detected by peptide mapping procedures, a tryptic map of this fraction was generated. Trypsin Digestion. A tryptic map of the late eluting material was obtained and compared to the map generated from the main peak (Figure 2). The predicted tryptic fragments are listed in Table 1. The methionine-containing tryptic peptides T1, T2, T11, and T18-19 were present in both maps in similar ratios, negating a possible norleucine involvement. However, it was readily apparent that the T6-T16 disulfide-containing peptide pair was modified (Figure 2B). This peptide pair is often nonspecifically digested by trypsin at the Ser62-Asn63 site, yielding the fragment designated as T6c-T16 in Figure 2A. Both of these peptide pairs are visible in the map of the unknown variant (Figure 2B) and exist as doublets, indicating the presence of the native pair in addition to modified fragments. Treatment of the digest with 5 (36) Cunningham, B. C.; Jhurani, P.; Ng, P.; Wells, J. A. Science 1989, 243, 1330-1336. (37) Bogosian, G.; Violand, B. N.; Jung, P. E.; Kane, J. F. Biochimie 1991, 73, 546-558. (38) Lu, H. S.; Tsai, L. B.; Kenney, W. C.; Lai, P.-H. Biochem. Biophys. Res. Commun. 1988, 156, 807-813. (39) Munier, R.; Cohen, G. N. Biochim. Biophys. Acta 1959, 31, 378-391.

Figure 2. Tryptic maps of fractions collected from neutral pH reversed-phase chromatography (Figure 1). Panel A, peak 3; Panel B, peak 4. See Experimental Section for details. Table 1. Predicted Tryptic Fragments of Methionyl Human Growth Hormone

a

tryptic peptides

sequence

T1 (0-8) T2 (9-16) T3 (17-19) T4 (20-38) T5 (39-41) T6 (42-64)T16 (159-167)a T7 (65-70) T8 (71-77) T9 (78-94) T10 (95-115) T11 (116-127) T12 (128-134) T13 (135-140) T14 (141-145) T15 (146-158) T17 (168) T18-19 (169-178) T20 (179-183)T21 (184-191)a

MFPTIPLSR LFDNAMLR AHR LHQLAFDTYQEFEEAYIPK EQK YSFLQNPQTSLCFSESIPTPSNR NYGLLYCFR EETQQK SNLELLR ISLLLIQSWLEPVQFLR SVFANSLVYGASDSNVYDLLK DLEEGIQTLMGR LEDGSPR TSQIFK QTYSK FDTNSHNDDALLK K DMDKVETFLR IVQCR SVEGSCGF

Disulfide-bonded tryptic peptides.

mM DTT prior to reversed-phase HPLC revealed that the cysteinecontaining peptides T6 (42-64), T16 (159-167), T6c (42-62), T20 (179-183), and T21 (184-191) were present and eluting in their respective expected positions (data not shown). This result

suggested that the modification was destroyed upon reduction, eliminating deamidation at any of the three asparagine residues as a possible explanation for the difference in elution behavior before reduction. It has previously been observed that deamidation at Asn149 in the T15 peptide results in a later eluting species.40 Investigation of Cysteine Linkages. To eliminate the possibility that this more hydrophobic variant was not simply an aggregate, an SDS-PAGE analysis was performed and established its monomeric nature (data not shown). Since reduction of the cysteine-containing peptides T6-T16 removed the modification, it was possible that this late eluting species may be a disulfide conformer. For example, such conformers have been detected in the X-ray diffraction analyses of carboxypeptidase A.41 Hence, the growth hormone variant was treated with urea and subsequently analyzed by the neutral pH reversed-phase HPLC procedure. The variant still eluted at about 56 min. Perhaps urea was not a sufficiently strong denaturant. Hence, the variant was treated with guanidine hydrochloride to unfold the molecule, reduced with DTT, and then allowed to refold as described in the Experimental Section. The main peak material was treated similarly as a control. The neutral pH reversed-phase HPLC analyses showed that the reduced and renatured variant material now behaved similarly to the reduced and renatured main peak (40) Johnson, B. A.; Shirokawa, J. M.; Hancock, W. S.; Spellman, M. W.; Basa, L. J.; Aswad, D. W. J. Biol. Chem. 1989, 264, 14262-14271. (41) Wang, D.; Bode, W.; Huber, R. J. Mol. Biol. 1985, 185, 595-624.

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Figure 3. Reversed-phase chromatography of T6-T16 fragments collected from the tryptic maps of peaks 3 and 4 (Figure 2). (1) T6T16 fragment from peak 4, (2) T6-T16 fragment from peak 3, and (3) T6-T16 fragment from peak 4 after treatment at 60 °C, pH 7.3, for 60 min.

material, lending credence to the hypothesis for a disulfide conformer. Studies to confirm this conclusion were performed on the isolated T6-T16-modified peptide pair. If this modification is truly conformational only, it should be possible to convert the T6-T16-modified peptide pair to the normally obtained conformation by increasing the temperature. However, even treatment at 60 °C for 60 min did not convert the modified peptide pair to the unmodified version when analyzed under tryptic mapping reversedphase HPLC conditions (Figure 3). Therefore, only treatment with DTT effected this change. The spectroscopic properties of an organosulfur compound reflect the electronic structure of the molecule, although generally in an indirect manner. Nevertheless, the ultraviolet absorption (UV) spectra of main peak and peak 4 material were obtained. No gross differences in the appearance of the two spectra were observed. To more accurately access the wavelength positions of the Trp, Tyr, and Phe chromophores in these molecules, the second-derivative spectra were computed. Again, no significant differences were seen in the major second-derivative minima, indicating that the average microenvironments of the Trp, Tyr, and Phe residues are essentially equivalent in these molecules. It should be noted that the weak absorbance of the two disulfide bonds in methionyl human growth hormone would be extremely difficult to assess from the zero-order spectra and would be impossible to assess from second-order spectra due to their extremely weak, broad, featureless nature. In an attempt to at least indirectly estimate the disulfide absorption, the circular dichroism (CD) spectra were obtained. It is well known42 that, although the absorbance of disulfide bonds is relatively weak, their contribution to the near-UV CD spectrum is often proportionately much stronger. Furthermore, the approximate strength and sign of the CD of the two disulfides in human growth hormone have been previously published.42 The CD spectra of main peak and peak 4 material are shown in Figure 4. The spectrum of main peak material is essentially identical to that previously published for human growth hormone in the same buffer. In sharp contrast, the CD spectrum of the peak 4 variant material shows exactly the kind of difference (Figure 4, lower trace) from main peak material expected for a perturbation in one or more of the disulfide bonds as described by Bewley.42 This reasonably smooth, broad (42) Bewley, T. Biochemistry 1977, 16, 209-215.

4048 Analytical Chemistry, Vol. 68, No. 22, November 15, 1996

Figure 4. Circular dichroism spectra of peak 3 and 4 material (see Figure 1). The lower trace is the difference spectrum.

negative band has been attributed to disulfide dichroism.42-44 Such a difference would most probably include a large alteration in the dihedral angle of the bond.42-44 Additionally, an amino acid composition analysis revealed that the variant species contained 25% less cystine than the properly folded species, suggesting a possible disulfide difference. However, if the composition is determined after conversion of cystine to cysteic acid, no difference is detected, again implicating a transient sulfhydryl modification. Mass Spectral Analyses. The development of mass spectral analyses of high molecular weight molecules, such as proteins, led to the eventual identification of the modification. A preliminary analysis on an electrospray triple-quadrupole instrument of the intact variant indicated a mass that was approximately 32 Da greater than expected. Perhaps two extra oxygen atoms were associated with the cystine moiety. However, this postulate was not consistent with the behavior of the variant upon reversedphase HPLC, which suggested that the variant was more, not less, hydrophobic than human growth hormone itself. In addition, the observation that DTT reduces the variant to a normal disulfide suggests that the structures shown in Figure 7A,B are not probable. It has been reported that iodide, a stronger reducing agent than DTT, can easily reduce the disulfoxide of cystine, but not the thiosulfonate or monosulfone.45 An LC/MS analysis of the tryptic digest indicated that there was an ion with the mass of T6-T16 plus 32 Da. Hence, it was prudent to confirm that the 32 Da was, indeed, confined to the T6-T16 peptide pair, after which an exact mass measurement was needed to determine if the 32 Da was due to two oxygen atoms or one sulfur atom. A mass spectral analysis of the isolated T6-T16-modified peptide pair established that the 32 Da increase resided in this linked peptide pair. Since the mass of this tryptic fragment is 3762 Da, it was necessary to perform a second digestion to obtain a smaller fragment which would be amenable to tandem mass spectral analysis. This was accomplished with thermolysin to yield the LC/LLYC-linked peptide pair from both the native (calculated m/z 743.3) and modified tryptic fragments. Electrospray ionization mass spectrometry identified the protonated molecular masses of the normal and modified versions as 743.3 and 775.3 Da, respectively. The 32 Da increase was thus found on the disulfidecontaining portion of the T6-T16 peptide pair. (43) Beychok, S. Science 1966, 154, 1288-1299. (44) Yamashiro, D.; Rigbi, M.; Bewley, T.; Li, C. H. Int. J. Pept. Protein Res. 1975, 7, 389-393. (45) Toennies, G.; Lavine, T. F. J. Biol. Chem. 1936, 113, 571-582.

Figure 5. Low-energy electrospray tandem mass spectra of thermolysin fragments Leu52Cys53/Leu162Leu163Tyr164Cys165 of T6-T16 peptides from peak 3 and 4 material (see Figure 1). Upper panel, peak 3 fragment; lower panel, peak 4 fragment. See Experimental Section for details.

To further elucidate the structure of these peptides, both highand low-energy CID spectra were taken. The low-energy tandem mass spectra are shown in Figure 5. They confirm the structure LC/LLYC (Figure 5, upper panel); the ions at m/z 630, 517, 354, and 241 result from the sequential losses of Leu, Leu, Tyr, and Leu. These sequential losses were also observed in the modified peptide fragmentation spectrum, placing the plus 32 Da on the residual Cys53-Cys165 sulfur-linked fragment (Figure 5, lower panel; m/z 273.1) derived from the modified T6-T16 peptide pair. Since no cleavages occurred within the cystine residue in the low-energy regime, high-energy tandem mass spectra were obtained and are shown in Figure 6. Many of the same fragment ions were produced by the high-energy collisions, as shown in Figure 5, but important cystine cleavages were also seen.46,47 Four (46) Stults, J. T.; Bourell, J. H.; Canova-Davis, E.; Ling, V. T.; Laramee, G. R.; Winslow, J. W.; Griffin, P. R.; Rinderknecht, E.; Vandlen, R. L. Biomed. Environ. Mass Spectrom. 1990, 19, 655-664.

possible structures can be proposed with the addition of either one sulfur atom or two oxygen atoms. If two oxygen atoms were each on separate sulfur atoms, ions 16 Da higher than the single sulfur-containing ions such as the 283/285 Da pair would be predicted. These ions were never found. Figure 7 illustrates the ions expected for the three remaining possible structures. Strong ions are seen at m/z 266 and 316 (Figure 6, lower panel), eliminating two oxygens on the LLYC sulfur (Figure 7A) and two oxygens on the LC sulfur (Figure 7B), respectively. The ions for the disulfide structures are, indeed, seen in the tandem mass spectrum of the peptide pair isolated from the main peak material (Figuer 6, upper panel). Similarly, the ions for the trisulfide structures at m/z 266, 316, and especially 348 (Figure 7C) are observed in the trisulfide tandem mass spectrum of the peptide pair isolated from peak 4 material (Figure 6, lower panel). (47) Bean, M. F.; Carr, S. A. Anal. Biochem. 1992, 201, 216-226.

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Figure 6. High-energy fast atom bombardment tandem mass spectra of LC/LLYC fragments of T6-T16 peptides from peak 3 and 4 material (see Figure 1). Upper panel, peak 3 fragment; lower panel, peak 4 fragment. See Experimental Section for details.

However, these data do not eliminate the possibility that a mixture of oxygenated structures (Figure 7A,B) are present. The elemental composition of the normal peptide was confirmed by accurate mass measurement as follows: m/z 743.3475 was found for the protonated normal peptide, and m/z 743.3472 was calculated for C33H55O9S2N6 (LC/LLYC). The elemental composition of the modified peptide was identified as follows: m/z 775.3220 was found, m/z 775.3192 was calculated for an additional sulfur atom, and m/z 775.3370 was calculated for two additional oxygen atoms. Therefore, the closest match, -0.0028 vs +0.0150 Da, suggests the presence of an additional sulfur atom, not two oxygen atoms. Activity Assays. The trisulfide-containing human growth hormone preparation was analyzed for biological activity by using a radioreceptor assay as a surrogate assay. It had a Ka of 5.3 × 109 M-1, as opposed to a value of 7.6 × 109 M-1 for human growth hormone. This difference is not statistically significant. 4050 Analytical Chemistry, Vol. 68, No. 22, November 15, 1996

The variant was also tested in an ELISA format using eight different monoclonal antibodies directed against various surfaces of human growth hormone, as illustrated in ref 35 and cataloged in the Experimental Section. All of these antibodies were effective in binding the trisulfide variant. CONCLUSIONS Recently, a trisulfide bond was reported to occur in the minor loop disulfide at Cys182-Cys189 in human growth hormone biosynthesized in E. coli.33,34 This variant was isolated using hydrophobic interaction chromatography. We have detected a trisulfide structure in methionyl human growth hormone in the major loop disulfide Cys52-Cys165. This variant was isolated using neutral pH reversed-phase chromatography, a technique which disrupts the three-dimensional structure to a much greater degree than hydrophobic interaction chromatography. The trisulfide structure was localized to the Cys52-Cys165 linkage by consecutive

(Figure 7C) as opposed to a cystine thiosulfonate (Figure 7A,B) by accurate mass measurement. It is possible that a trisulfide in the minor loop disulfide was undetected in this study due to its lability, as reported by Jespersen et al.33 under neutral pH conditions. As can be seen in Figure 2, the tryptic peptides containing the disulfide/trisulfide appear as doublets. The neutral pH of the trypsin digestion buffer seems to have led to a conversion of the trisulfide to the naturally occurring disulfide. The methionyl hGH was biosynthesized in E. coli and found in inclusion bodies within the cell. Extraction from the cells is effected with 8 M thiocyanate and may explain the formation of some trisulfide structures with the concomitant release of cyanide.16 Alternatively, an enzymatic mechanism involving perhaps cystathionase or a transsulfurase may be operative.

Figure 7. Structures for unknown variant: A, two oxygen atoms on Cys165 sulfur; B, two oxygen atoms on Cys53 sulfur; C, trisulfide structure. Expected daughter ions are indicated. Sulfur-sulfur bonds may cleave with hydrogen rearrangements which add or subtract 1 Da to the product ion.45,46 See Experimental Section for details.

ACKNOWLEDGMENT The authors thank Dr. Tom Bewley for the circular dichroism spectra and Ms. Kathy O’Connell for the accurate mass measurements.

Received for review June 14, 1996. Accepted August 20, 1996.X AC9605915

proteolysis using trypsin and thermolysin with mass spectral analyses. The structure was definitively identified as a trisulfide

X

Abstract published in Advance ACS Abstracts, October 1, 1996.

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