Article pubs.acs.org/molecularpharmaceutics
Oxidation of Human Growth Hormone by Oxygen-Centered Radicals: Formation of Leu-101 Hydroperoxide and Tyr-103 Oxidation Products Daniel Steinmann,† J. Andrea Ji,‡ Y. John Wang,‡ and Christian Schöneich*,† †
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66047, United States Late Stage Pharmaceutical Development, Genentech Inc., South San Francisco, California 94080, United States
‡
ABSTRACT: Human growth hormone (hGH) was exposed to oxygen-centered radicals generated through the thermolysis of AAPH in the presence of dioxygen. Such conditions mimic oxidative processes which protein pharmaceuticals can encounter during formulation in the presence of polysorbates. We detected the oxidation of Met to Met sulfoxide, the formation of protein carbonyls, the oxidation of Tyr to dityrosine and several additional Tyr oxidation products, the conformation-dependent oxidation of Trp, and the site-specific formation of protein hydroperoxides. The sensitivity of Met oxidation correlates with their solvent accessible surface, i.e. the yields of MetSO decreased in the order Met-14 > Met-125 > Met-170. Trp oxidation in native hGH was negligible, but was enhanced through denaturation. Dityrosine formed predominantly intramolecularly but did not contribute significantly to protein cross-linking. Hydroperoxides formed selectively on Leu-101 and were generated specifically by alkoxyl radicals, generated through the decomposition of peroxyl radicals. Tyr-103 was converted into a series of oxidation products characterized by mass shifts of Tyr + 14 Da and Tyr + 16 Da. KEYWORDS: human growth hormone, hGH, AAPH, oxidation, leucine hydroperoxide, dityrosine, tyrosine oxidation, ROS, reactive oxygen species, peroxyl radicals, alkoxy radicals, protein stability
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dityrosine was monitored for glutamine synthetase.14 The main objective of the current study was to localize and quantify protein hydroperoxide formation on a pharmaceutically relevant protein, hGH, exposed to oxygen centered radicals. In addition to oxygen centered radicals, singlet oxygen was shown to generate hydroperoxides on amino acid side chains, particularly on Tyr and Trp.16−20 Protein hydroperoxides must be considered important precursors for oxidative degradation as they can give rise to secondary oxidizing species in the presence of transition metals, heat and light, which, in turn, can induce further protein damage.21−23 Hence, protein hydroperoxides may be as important impurities in formulations as organic hydroperoxides of excipients. Gebicki and Gebicki showed that aliphatic amino acids are prone to form hydroperoxides during exposure to ionizing radiation.15 In fact, Leu and Val hydroxides have been detected in proteins subjected to ionizing radiation and subsequent treatment with NaBH4, suggesting the initial hydroperoxide formation on these amino acids.24,25 In addition, there is indirect evidence for the formation of Tyr hydroperoxides in myoglobin.26 However, to date hydroperoxides have not been located in a protein by MS/MS analysis. Such studies are described here for the exposure of hGH to oxygen-centered
INTRODUCTION The production, storage and delivery of protein pharmaceuticals presents many challenges due to their relatively fragile stability compared to small organic molecules.1 Exposure to oxygen leads to oxidative degradation of protein pharmaceuticals, which may result in the loss of potency.2,3 Protein formulations are often stabilized by polysorbates to prevent surface adsorption and protein aggregation. However, polysorbates are sensitive toward oxidative degradation, involving the formation of peroxyl radicals.4 Such peroxyl radicals can mediate the oxidative damage of proteins.5,6 Peroxyl radicals react via hydrogen abstraction from suitable hydrogen donors to form secondary radicals, which may subsequently react with dioxygen to form secondary peroxyl radicals, potentially propagating a chain reaction.7 Moreover, peroxyl radicals undergo fast combination reactions followed by a variety of possible rearrangements, during which more reactive alkoxy radicals (and singlet oxygen) may be formed.7−9 Azocompounds, such as azobisisobutyronitrile (AIBN) and 2,2′azobis(2-methylpropionamidine) (AAPH), are able to simulate the oxidative degradation caused by peroxyl radicals resulting from polysorbate oxidation.4 They undergo facile thermolysis and, in the presence of oxygen, form peroxyl radicals.10 Specifically the decomposition of AAPH has recently been revisited, which, besides peroxyl radicals and alkoxy radicals (Scheme 1), leads also to disproportionation and cyclization products.11 Studies with model proteins revealed that AAPH-derived oxidants target the amino acids Cys, Met, Trp, Tyr and His.12−14 For the reaction of AAPH-derived oxidants with bovine serum albumin, iodometric analysis revealed the accumulation of protein hydroperoxides,15 while the accumulation of protein carbonyls and © 2012 American Chemical Society
Special Issue: Advances in Biophysical and Bioanalytical Protein Characterization Received: Revised: Accepted: Published: 803
February 20, 2012 March 2, 2012 March 7, 2012 March 7, 2012 dx.doi.org/10.1021/mp3001028 | Mol. Pharmaceutics 2012, 9, 803−814
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Scheme 1
If hydroperoxides were to be reduced, oxidized hGH was precipitated with 0.5 M perchloric acid, centrifuged at 15000g at 4 °C for 10 min, and washed twice with water. The resulting protein pellet was dissolved into 30 μL of a solution of 6 M guanidine hydrochloride, basified with KOH to pH 12. Then, 20 μL of a solution of 1 M NaBH4 were added, and after 15 min the reaction was quenched with 1 mL of a solution of 0.5 M HClO4. The protein was purified again by centrifugation and washed twice with water. Thiols that were formed during the reduction with NaBH4 were subsequently alkylated. Protein pellets obtained after the reduction with NaBH4 were dissolved into 6 M guanidine hydrochloride and treated with a 10-fold excess of N-ethylmaleimide at pH 7.8 for 15 min. Thereafter, the proteins were precipitated with 0.5 M HClO4, centrifuged and washed twice with water. Tryptic digestions of either reduced or nonreduced hGH were carried out in ACN/H2O (10/90, v/v), buffered at pH 7.8 with 0.1 M NH4HCO3. The initial trypsin/protein ratio was 50, and samples were incubated at 37 °C for 8−10 h. Digests were stored at −20 °C prior to analysis. Hydroperoxides. The overall content of protein hydroperoxides was determined with the G-PCA-Fox method.29 Prior to analysis, proteins were precipitated with 0.5 M perchloric acid, centrifuged at 15000g at 4 °C for 10 min and washed twice with water. The resulting protein pellets were dissolved in 0.2 mL of guanidine hydrochloride. The 1 cm light-path quartz cuvette from Hellma (Plainview, NY) used for the colorimetric assay was pretreated with the Fox reagents in 0.7 mL of guanidine hydrochloride for 15 min to control for iron contaminants in the absorbance background. Immediately after addition of the protein solution to the cuvette, the background absorbance was measured at 560 nm and final readings were taken after 40 min of incubation. The assay was calibrated with 10 μM Fe2(SO4)3 for each series of experiments. Protein Carbonyls. The content of carbonyl groups was measured coloriometrically with 2,4-dinitrophenylhydrazine (DNPH) as suggested by Hawkings et al.30 Dityrosine. For the analysis of dityrosine formation, hGH was precipitated with 0.5 M perchloric acid, centrifuged at 15000g at 4 °C for 10 min and washed once with water. Protein pellets were dissolved in a solution of 600 μL of guanidine hydrochloride (6 M) and 100 μL of NH4HCO3 (1 M, pH 7.8). Samples were analyzed by fluorescence spectroscopy with a Shimadzu RF-5000U fluorescence spectrophotometer in 0.5 mL fluorescence cuvettes with 1 cm light-pass from Hellma (Plainview, NY). The excitation and emission band widths were set at 5 nm. The excitation wavelength was 315 nm and the
radicals, which show that especially Leu-101 is a sensitive target for hydroperoxide formation on hGH.
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EXPERIMENTAL SECTION Materials. Human growth hormone (somatropin, hGH) stock solutions were prepared from Nutropin AQ formulations provided by Genentech, Inc. (South San Francisco, CA). The formulations were dialyzed overnight against water with SlideA-Lyzer Dialysis Cassettes, 3.5K MWCO. Aliquots of ca. 0.1 mL were frozen at −20 °C for storage. The concentrations of hGH stock solutions were measured by means of UV spectro0.1% scopy at 277 nm, where A277nm = 0.82 cm−1.27 Additionally, a solution of pure hGH was supplied by Genentech, Inc. Small levels of aggregates therein were removed by centrifugation (15000g, 10 min) and clear solutions were stored at 4 °C. 2,2′Azobis(2-methylpropionamidine) dihydrochloride (purity >97%; AAPH), was obtained from Sigma-Aldrich (Saint Louis, MO). N-Ethylmaleimide (purity >99%; NEM) was supplied by Fluka (Saint Louis, MO). TPCK-treated sequence-grade trypsin was from Promega (Madison, WI). All other chemicals were of the highest commercial grade available and obtained from Sigma or Fisher (Pittsburgh, PA, USA). All solutions were prepared with MilliporeQ-water, and the chemicals were used as received. Synthesis of Dityrosine. For the preparation of dityrosine, a 10 mL solution of Tyr (1 mM), NaN3 (25 mM) and H2O2 (5 mM) in phosphate (25 mM) was prepared at pH 5 under Ar. Via a syringe, 3.5 mL of an Ar-saturated solution of 20 mM Fe2+ was slowly added while bubbling with Ar and stirring. Thereafter, Fe3+ precipitates were removed by centrifugation at 15000g for 5 min, and dityrosine was isolated by HPLC with a Discovery C18 5 μm column (250 × 4.6 mm, 110 Å pore size, Supelco, Bellefonte, PA). The mobile phase consisted of 0.1% (v/v) trifluoroacetic acid (TFA) and 9% acetonitrile (ACN) in water. Concentrations of dityrosine in the stock solutions were determined by absorbance spectroscopy, where A283nm = 5,680 M−1 cm−1 at pH 4.28 Reaction Mixtures. Reaction mixtures contained 50 μM hGH and were buffered at pH 7.4 with 0.4 M phosphate. The concentration of AAPH and the duration of incubation were varied to generate the desired amount of peroxyl radicals according to [ROO•] = 1.36 × 10−6 [AAPH] M s−1 at 37 °C.10 The relative ratios of concentration of peroxyl radicals and hGH are denoted as ROO•/hGH. AAPH concentrations ranged from 1 mM to 200 mM, and the incubation times from 1.5 to 22 h at 37 °C. After incubation the reaction was stopped by cooling to 4 °C. 804
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Table 1. Comparative Summary of Protein Modifications for ROO•/hGH = 30 oxidative modification
% modified
Met sulfoxide Trp + 16 Da Trp + 32 Da dityrosine hydroperoxides covalent aggregates protein carbonyls
Met-14, 100%; Met-125, 80%; Met-170, 70% 9%, 9% ≤1.2% 15% dimers, 21%; trimers and higher oligomers, 13% 25%
Figure 1. (A) LC−MS, total ion count chromatogram of a tryptic digest of an hGH control sample. (B) LC−MS of tryptic digest of a sample of hGH oxidized with AAPH (ROO•/hGH = 16). Insets: MS spectra at tR = 50−51 min.
emission wavelength 400 nm.31 To bypass matrix effects, the method of standard addition was applied. Readings were taken after 3 consecutive additions of 40 μL of a 0.63 μM dityrosine stock solution. Control samples were treated equally as the oxidized samples but lacked AAPH during incubation. Fluorescence intensity of the control samples was subtracted from that of the oxidized samples. Aggregates. For SDS−PAGE analysis, protein samples of 10 μL were mixed with 30 μL of tris−glycine−SDS sample buffer and loaded onto 1.5 mm thick 10-well Novex 4−12% Tris−glycine gradient gels (Invitrogen, Carlsbad, CA). After running gel electrophoresis at 200 V for 90 min, the gels were stained with 0.2% Coomassie R250 in 7.5% acetic acid, 30% methanol and 62.5% H2O for 2 h, followed by destaining in 7.5% acetic acid, 40% methanol and 52.5% H2O until the bands were visible and the background was clear. For size exclusion chromatography (SEC), hGH was precipitated (after oxidation) with HClO4, washed with water, and resolubilized with 2% SDS. SEC was performed as described.32 Fluorescence Spectroscopy. Spectra were measured with a Shimadzu RF-5000U fluorescence spectrophotometer in 0.5 mL fluorescence cuvettes with 1 cm light-pass from Hellma (Plainview, NY). The excitation and emission band widths were set at 5 nm. Mass Spectrometry. LC−ESI-MS experiments were carried out with an Acquity UPLC system (Waters Corporation, Milford, MA) coupled to either a SYNAPT-G2 (Waters Corporation, Milford, MA) or Q-TOF-2 (Micromass Ltd., Manchester, U.K.) hybrid mass spectrometer. For the chromatography a Vydac column (25 cm × 1 mm C18, 3.5 μm) was used. Tryptic digests were eluted with a linear gradient of 10− 60% (v/v) ACN in aqueous formic acid (0.06%, v/v) within 30 min, followed by a linear gradient to 80% (v/v) ACN in aqueous formic acid (0.06%, v/v) within 10 min. The flow rate was 20 μL/min. The SYNAPT-G2 instrument was operated for maximum resolution with all lenses optimized on the [M + 2H]2+ ion from the [Glu]1-fibrinopeptide B. The cone voltage was 45 V, and Ar was admitted to the collision cell. The spectra were acquired using a mass range of 50−2,000 amu (amu = atomic mass unit). The data were accumulated for 0.7 s per cycle.
Figure 2. (A) Extent of Met oxidized for various ROO•/hGH molecular ratios: ● Met-14; ■ Met-125; ◆ Met-170. Different gray shades indicate independent series of measurements. All mass spectrometric analysis was performed by the ESI-Q-Tof instrument, and results were derived from the signals of the molecular ions. (B) Losses of the Met-containing tryptic peptides: ● Met-14; ■ Met-125; ◆ Met-170. Different gray shades indicate independent series of measurements. The losses were calculated based on the oxidationresistant tryptic fragments T13 as internal standard. All mass spectrometric analysis was performed by the ESI-Q-Tof instrument, and results were derived from the signals of the molecular ions.
The Q-TOF-2 was optimized on the [M + 2H]2+ ion from the cyclic peptide gramicidin S. The cone voltage was 30 V, and Ar was admitted to the collision cell at a pressure that attenuated the beam to about 20%. The cell was operated at 5 eV for maximum transmission. Spectra were acquired at 11,364 Hz pusher frequency covering the mass range 100− 2,000 amu and accumulating data for 5 s per cycle. Time to mass calibration was made with CsI cluster ions acquired under the same conditions. LC−ESI-MS/MS measurements were performed with the SYNAPT-G2 and an LTQ-FT hybrid linear quadrupole ion trap Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometer (ThermoFinnigan, Bremen, Germany). On the Synapt-G2 instrument, CID spectra were acquired by setting the MS1 quadrupole to transmit a precursor mass window of ±0.1 m/z. FT-ICR measurements were obtained as described.33 The MS and MS/MS spectra obtained by the Synapt-G2 and Q-TOF-2 were analyzed with the MassLynx program v4.1 805
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Figure 3. Extent of Trp oxidized for various ROO•/hGH molecular ratios: ◆ Trp + 16 Da; ■ Trp + 32 Da. Different gray shades indicate independent series of measurements. All mass spectrometric analysis was performed by the ESI-Q-Tof instrument, and results were derived from the signals of the doubly charged ions.
Figure 5. Extracted ion chromatogram (XIC; chromatogram created by plotting the intensity of the signal observed at a chosen m/z value as a function of retention time) for the doubly charged ion corresponding to the mass of T10 + 32 Da, m/z 1147.57. Black: hGH oxidized with AAPH, ROO•/hGH = 30. Gray: After reduction with NaBH4, peak a disappears completely. Inset: Spectrum at the retention time of peak a. According to the MS/MS spectrum (Figure 7) peak a can be ascribed to a T10 derivate that carries an additional mass of +32 Da on Leu-101.
Figure 4. Size exclusion chromatography (SEC) analysis of hGH that was oxidized with a ROO•/hGH ratio of 30. From the relative peak areas, we estimate that ca. 66% of hGH is present as monomer, 21% as dimer and 13% as trimer and higher weight oligomer.
Figure 6. XIC for doubly charged peptide T10 + 16 Da, m/z 1139.57. Black: hGH exposed to AAPH, ROO•/hGH = 30. Gray: After reduction with NaBH4. The abundance of T10/Leu-101 + 16 Da is increased after reduction of oxidized hGH by NaBH4, peak b. Peak c corresponds to a mixture of oxidized tryptic fragments T10 that carry an additional mass of +14 Da and +16 Da on Tyr-103 (denoted here as T10/Tyr-103 + 14 Da and T10/Tyr-103 + 16 Da). This mixture has a respective ratio of 48/52. At the expense of T10/Tyr-103 + 14 Da and T10/Tyr-103 + 16 Da, a new species T10/Tyr-103 + 18 Da is formed during the reduction with NaBH4, peak d. The MS/MS spectra are shown in Figures 9−12.
from Waters Corporation, and the raw files obtained from the FT-ICR measurements were read by the Xcalibur 2.0 software package (Thermo Scientific). We calculated specific oxidation yields (oxidized fragment/(oxidized + native fragments)) through the peak areas in the extracted ion chromatograms of abundant ions of the respective tryptic peptides. We assume that electrospray-ionized peptides containing the oxidative modifications have similar mass spectrometric properties as the respective ions of the native peptides due to the small mass differences. Furthermore, the MassMatrix program34 was used to screen MS/MS fragments for hydroperoxides and hydroxides.
representatively at a retention time tR = 50 min (insets in Figure 1), displays signals mainly between m/z 800 and 1800. We did not succeed to characterize any of these products in sufficient detail to assign a structure. At this point we conclude that the increase of these background signals is caused by a collection of degradation products present in low yields, which can presently not be characterized. However, a number of specific oxidation products have been identified, which are summarized in Table 1. More specific details on their analytical characterization are given below. Methionine Oxidation. The time course of AAPHdependent Met oxidation was investigated by means of LC−MS. Figure 2A shows the oxidation yields of Met at different positions in hGH for the exposure of hGH to various ROO•/ hGH ratios. Met-14 is the primary target for oxidation, followed by Met-125 and Met-170. The variability of the method was
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RESULTS Degradation Products Present in Low Yields. A first screen of the chemical stability of hGH against AAPH-induced oxidation was obtained by monitoring the tryptic fragments by chromatography connected to mass spectrometry. As shown in the total ion counts (TIC) in Figure 1, hGH is significantly degraded when subjected to a ROO•/hGH ratio of 16; however, we detected not only some changes in the peak areas of individual tryptic fragments but also a general, broad increase in the background between retention times of 40 and 60 min. The mass spectrometric analysis of these background signals, 806
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digestion may result in some of the observed variability. The rate of AAPH thermolysis, i.e. the rate of formation of oxygencentered radicals, did not influence the extent of Met oxidation, which provides evidence that oxygen concentration was not a limiting factor under our experimental condition. Tryptophan Oxidation. LC−MS/MS analysis of oxidized hGH shows comparably low yields of Trp oxidation relative to that of Met (Figure 3). LC−MS/MS analysis indicates that two equally abundant oxidation products are formed from Trp, characterized by a mass increase of +16 Da, possibly hydroxytryptophan and oxindolealanine. An additional oxidation product, characterized by a mass increase of +32 Da, was generated, probably N-formylkynurenine, which was as abundant as the combined derivatives showing a mass increase of +16 Da. No significant yields of kynurenine (Trp + 4 Da) were observed (99%.37 The remaining hydrogen peroxide was eliminated by the addition of 0.01 mg/mL catalase. Subsequently, the Met sulfoxide-containing hGH (hGH-MetSO) was exposed to ROO•/hGH-MetSO = 30, i.e. reaction conditions identical to those employed for the oxidation of native hGH. The yieldz of Leu-101 hydroperoxide detected for hGH-MetSO was similar to that for native hGH exposed to AAPH, indicating that Leu-101 hydroperoxide does not significantly react with either Met-14 or Met-125. Consistent with this result, also the yield of Leu-101 + 16 Da, immediately present after exposure of hGH-MetSO to AAPH, was similar to that obtained by exposure of native hGH to AAPH. Hence, the initial yields of T10/Leu-101 + 16 Da in the absence of NaBH4 must be due to alternative reactions. To probe mechanistic pathways leading to Leu-101 hydroperoxide formation, the exposure of hGH to AAPH was carried out in the presence of 10 mM sodium azide. According to the electrode potentials (E°′(N3•/N3−) = 1.33 V)38 azide will scavenge alkoxy radicals (E°′ (RO•,H/ROH) = 1.55−1.77 V)39,40 but not peroxyl radicals (E°′ (ROO•,H/ROOH) = 1.0−1.1 V).39,40 Azide completely suppressed the formation of hydroperoxides quantified by the G-PCA-Fox method. Moreover, no formation 809
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Figure 11. MS/MS spectrum of T10/Tyr-103 + 14 Da obtained from the doubly charged parent ion with m/z 1138.57; hGH was oxidized with a ROO•/hGH ratio of 30, and the reaction products were analyzed by an LTQ-FTICR mass spectrometer.
radical41 plays a role in the product formation of Tyr-103, the oxidation of hGH was carried out in the presence of 1 μM SOD at pH 7.4. No change in product formation was observed, excluding any involvement of O2•−. Several mechanistic possibilities exist for the formation of T10/Tyr-103 + 14/16 Da, which are summarized in Scheme 2 in the Discussion. In addition to the oxidation of Leu-101 and Tyr-103 we detected low yields of oxidative modification at the amino acids Tyr-28, Glu-32, Ile-36, Ile-78, Leu-80, Gln-84, Leu-87, Val-96, Asn-99, and Lys-167 by analyzing MS/MS survey scans with the MassMatrix program. In all these cases, however, overlap with coeluting isobaric species and/or low abundance did not allow unambiguous assignment such as presented for the oxidation products of Leu-101 and Tyr-103.
of Leu-101 + 16/32 Da could be detected by MS analysis when azide had been present during the exposure of AAPH. In a separate control experiment we verified that azide did not react with Leu-101 hydroperoxide. For this purpose azide was added after the formation of Leu-101 hydroperoxides by exposure of hGH to AAPH, and the reaction mixture was analyzed by LC−MS. Tyr Oxidation. Besides the coupling product dityrosine (see above), we detected additional oxidative modifications of Tyr103, corresponding to T10/Tyr-103 + 14 Da and T10/Tyr-103 + 16 Da (MS/MS spectra are shown in Figures 10 and 11, respectively). These reaction products coeluted with a retention time of tR = 19.7 min (Figure 6, peak c). Spectral deconvolution suggests a mixture of T10 + 14 and T10 + 16 Da with a ratio of ca. 48:52. Peak c is reduced in area by treatment with NaBH4. A scan of the remaining peak (referred to as peak d) shows a minor contribution of Tyr-104 + 16 Da but a major presence of a new signal of Tyr-103 + 18 Da. A chromatogram for m/z 1140.56 yields Figure 7, where peaks e and f indicate the formation of the product Tyr-103 + 18 Da. Both peaks show an abundance of ca. 3% relative to unmodified T10. By means of MS/MS the incorporation of the additional mass of 18 Da was localized to Tyr-103 (Figure 12). Analogous to the formation of Leu-101 hydroperoxide, the preoxidation of Met-14 and Met-125 had no effect on the formation of T10/Tyr-103 + 14/16 Da. Hence, any reaction of hypothetical intermediary Tyr hydroperoxides with Met must be negligible. To test whether the reaction of superoxide (O2•−) with an intermediary tyrosyl
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DISCUSSION Mechanisms of Product Formation. The exposure of hGH to oxygen-centered radicals generated by AAPH leads to several oxidation products predominantly from Met, Trp, Tyr, and Leu. Oxygen-centered radicals generated through AAPH thermolysis have been demonstrated to be an appropriate mimic for oxidants generated through the degradation of polysorbates.4 Hence, the exposure to AAPH represents a convenient way for probing the stability of protein pharmaceuticals toward oxidative degradation by polysorbate oxidation.4 The oxidation of Met to Met sulfoxide is expected based on the known reactions of peroxyl radicals with aliphatic sulfides. For example, pulse radiolysis studies have revealed the potential of aliphatic peroxyl 810
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Figure 12. MS/MS spectrum of T10/Tyr-103 + 18 Da obtained from the doubly charged parent ion with m/z 1140.57; hGH was oxidized with a ROO•/hGH ratio of 30, and the reaction products were analyzed by an LTQ-FTICR mass spectrometer.
(HO•) generated through ionizing radiation.15,24,25 Alkoxy radicals are less powerful oxidants as compared to hydroxyl radicals, which may rationalize the observed selectivity toward Leu-101 in hGH exposed to AAPH, compounded by the solvent accessibility of Leu-101 in hGH. Protein hydroperoxides are prone to degrade during digestion conditions.15,26 The most likely degradation pathways are hydrolysis or oxidation of Met residues. Since the level of hydroxylated Leu-101 before reduction with NaBH4 is small, the recovery and stability of the Leu-101 hydroperoxide must be high. We found no clear evidence for the specific formation of other hydroxylated amino acids besides Tyr-103 and Trp-86 before reduction, and, therefore, specific formation of instable hydroperoxides on other aliphatic amino acids is unlikely. Thus, the Leu-101 hydroperoxide is quantitatively the most abundantly formed aliphatic amino acid according to our experiments. By comparing the total yield of hydroperoxides formed (15%) according to the G-PCA-Fox method and the estimated yield of the Leu-101 hydrperoxide (3%) we calculate that ca. 20% of the hydroperoxides are located on Leu-101. Importantly, the localization of hydroperoxide formation to Leu-101 represents one of the first examples for the site-specific formation of a hydroperoxide on a protein detected by mass spectrometry. Our data demonstrate that hydroperoxide formation not only is a problem for pharmaceutical excipients but can affect proteins as well. Hence, stability problems can arise when protein pharmaceuticals are contaminated by protein hydroperoxides rather than excipient hydroperoxides, and it
radicals to oxidize sulfides to sulfoxides under formation of alkoxyl radicals (reaction 1).42 ROO• + S< → RO• + OS
