The Reductive Desulfurization of Met and Cys Residues in Bovine

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The Reductive Desulfurization of Met and Cys Residues in Bovine RNase A Is Associated with trans Lipids Formation in a Mimetic Model of Biological Membranes Carla Ferreri,*,† Chryssostomos Chatgilialoglu,† Armida Torreggiani,† Anna Maria Salzano,‡ Giovanni Renzone,‡ and Andrea Scaloni*,‡ ISOF, Consiglio Nazionale delle Ricerche, via P. Gobetti 101, 40129 Bologna, Italy, and Proteomics & Mass Spectrometry Laboratory, ISPAAM, Consiglio Nazionale delle Ricerche, via Argine 1085, 80147 Napoli, Italy Received October 25, 2007

Damage to bovine pancreatic RNase A, due to the H• atom and/or solvated electron attack at protein sulfur-containing residues, was investigated by Raman spectroscopy and mass spectrometry techniques. To the already known desulfurization process affecting Met residues, novel reactivity was observed involving disulfide moieties, leading to the chemical transformation of Cys into Ala residues. Mapping experiments demonstrated that desulfurization selectively affected Met79, Cys110, Cys58 and Cys72 during first stages of reaction. While this reaction was performed on protein species added to large unilamellar vescicles, desulfurization yielded sulfur radicals able to induce a cis–trans isomerization of lipids at the onset of irradiation. These findings reveal new scenarios on reactions generated by radical stressing conditions, suggesting the need for specific assays and for future investigations to detect these modifications in proteins and lipids within challenged cells. Keywords: tandem damage • reductive radical stress • protein modification • trans lipids • sulfurcontaining amino acids

Introduction Tandem radical damage to biomolecules is the result of a consecutive process, where the attack to a first molecule generates a radical species able to carry out another insult, leading to a second molecular damage. An example is provided by the lipid-DNA damage derived from the reaction of malondialdehyde (MDA) with cytosine, adenine or guanine deoxynucleosides.1,2 Tandem damage processes are very harmful in biological systems and may generate highly diffusible radical species in a certain cell compartment, which are then able to reach other target sites, producing molecular species with potent biological effects.3 Recently, a tandem radical damage involving polypeptide and lipid molecules has been evidenced for Met-containing protein and peptide substrates in the presence of unsaturated membrane phospholipids.4–7 Damage is initiated by the attack of H• at the Met residues, which are then transformed to R-aminobutyric acid (Aba). This desulfurization process generates the diffusible methanethiyl radical (CH3S•), able to migrate from aqueous phase into membrane bilayer. At this site, this radical reacts with the cis double bond in phospholipid fatty * To whom correspondence should be addressed. Carla Ferreri: ISOF, Consiglio Nazionale delle Ricerche, via P. Gobetti 101, 40129 Bologna, Italy; tel., +39-051-6398289; fax, +39-051-6398349; e-mail, [email protected]. Andrea Scaloni: ISPAAM, Consiglio Nazionale delle Ricerche, via Argine 1085, 80147 Napoli, Italy; tel., +39-081-5966006; fax, +39-081-5965291; e-mail, [email protected]. † ISOF, Consiglio Nazionale delle Ricerche. ‡ Proteomics & Mass Spectrometry Laboratory, ISPAAM, Consiglio Nazionale delle Ricerche. 10.1021/pr700691x CCC: $40.75

 2008 American Chemical Society

acids, causing its transformation to the corresponding geometrical trans isomers.8,9 These studies evidenced the role of trans lipids as highly sensitive reporters of protein damage, since very low levels of protein modification can be amplified by the catalytic cycle of the radical-based isomerization of lipid cis double bond, corresponding to a consistent formation of trans isomers into membranes. The effects of trans lipids on membrane properties as well as their correlation to health problems have been revealed over the past decade.10,11 The reaction of H• atoms with sulfur-containing proteins is not limited only to the Met attack. Forty years ago, Stein and co-workers suggested that the reaction of H• atoms with disulfide moieties of RNase A may produce Ala and H2S, on the basis of a very preliminary characterization of the irradiation products.12–14 The reaction mechanism for such observations is still lacking. On the other hand, the interaction of H• atoms and solvated electrons with disulfides in aqueous solution is well-understood.15 The H• atom attack gives rise to the sulfuranyl adduct, which is in equilibrium with its deprotonated form, namely the disulfide radical anion RSSR•-. The latter species may also be derived from the direct electron attachment to the disulfide moiety. Both intermediates dissociate reversibly into two entities, RS• and RSH (or RS-). Recent EPR studies on the solid-state radiolysis of lysozyme demonstrated that the sulfuranyl radical can afford irreversibly perthiyl radical, this path becoming prevalent since RS•/RSH do not diffuse freely and since the reactions in Scheme 1 are in equilibrium.16 Journal of Proteome Research 2008, 7, 2007–2015 2007 Published on Web 04/02/2008

research articles Scheme 1

In the current study, a detailed characterization of RNase A damage by reductive reactive species was obtained by combined Raman spectroscopy and mass spectrometry experiments, which evidenced the modification of Tyr, Met and disulfide-forming Cys residues. In particular, conversion of specific Met and Cys residues to Aba and Ala derivative was observed, respectively. The resulting sulfur-containing radicals induced lipid damage, as evidenced by targeted experiments performed in RNase A-liposome suspensions. Our results provide new insights into the reactivity of disulfide bridges under radical attack, enlarging the number of chemical modifications detectable on proteins and lipids under specific reductive conditions.

Experimental Procedures Irradiation of RNase A in Aqueous Solution. In a 4 mL vial equipped with an open-top screw cap and a Teflon faced septum, bovine RNase A (600 µg) was dissolved in 1 mL of aqueous 0.2 M t-BuOH or 10 mM phosphate, 0.2 M t-BuOH, flushed with N2O or Ar, respectively, and then irradiated. The t-BuOH concentration corresponded to a value of 2% (v/v); pH was set to 7. Continuous radiolysis was performed by using a 60 Co-Gammacell (Atomic Energy of Canada Ltd., Canada) at a dose rate of 10–12 Gy/min. The exact absorbed radiation dose was determined with the Fricke chemical dosimeter, by taking G(Fe3+) ) 1.61 µmol/J. The pH of the protein solution did not change significantly after irradiation. Samples were withdrawn at time 0 and after irradiation with a 84 and 650 Gy, were lyophilized and further characterized. Raman Spectroscopy. Raman spectroscopy experiments were carried out on a Bruker (Germany) IFS 66 spectrometer equipped with a FRA-106 Raman module and a cooled Gediode detector. The excitation source was a Nd3+-YAG laser (1064 nm), the spectral resolution was 4 cm-1 and the total scans for each spectrum were 8000. The laser power acting on each sample was almost 100 mW. All the spectra were corrected and processed as previously described.17,18 MS Analysis of Intact Protein Samples. Protein samples were freed of salts using ZipTipC4 pipet tips (Millipore). Mass spectra were recorded using an ESI Q-TOF Premier instrument equipped with a nanoelectrospray source (Waters, Milford, MA). Proteins were directly injected into the ionization source at a 0.5 µL/min flow rate using an external syringe pump. Best ionization parameters were sought to minimize in source fragmentation effects; capillary voltage was set at 2500 V, cone voltage at 40 V, and collision energy at 10 eV. Mass spectra were acquired in a range spanning m/z 800–3000, and a mass calibration over the entire range was performed by means of separate injections of horse heart myoglobin (Sigma) (average molecular mass 16 951.5 Da). Spectra were elaborated using the Masslynks software (Waters). SDS-PAGE. Protein samples (30 µg) subjected to different radiation doses were directly analyzed by SDS-PAGE according 2008

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Ferreri et al. 19

to Laemmli. Separated bands were visualized by colloidal Coomassie staining.20 Protein Digestion. Protein bands from SDS-PAGE were excised from the gel, triturated, S-alkylated and digested with trypsin.21,22 To reduce undesidered protein oxidation, all reactions were performed in NH4HCO3 buffer, pH 7, under N2 atmosphere. For the same reason, proteolysis was performed only for 3 h, under N2 atmosphere. The importance of using this experimental setup was verified by preliminary mass spectrometric experiments (data not shown). Gel particles were extracted with 25 mM NH4HCO3/acetonitrile (1:1 v/v) by sonication and peptide mixtures were concentrated. Samples were desalted using µZipTipC18 pipet tips (Millipore) before MALDI-TOF-MS analysis or directly analyzed by µLC-ESI-IT-MS/MS.21–23 Peptide Mapping Analysis. Peptide mixtures were loaded on the MALDI target together with CHCA as matrix, using the dried droplet technique. Samples were analyzed with a VoyagerDE PRO spectrometer (Applera).23 Mass spectra for PMF were acquired in reflectron mode; internal mass calibration was performed with peptides from trypsin autoproteolysis. Data were elaborated using the DataExplorer 5.1 software (Applera). All masses are reported as monoisotopic values. Peptide mixtures were also analyzed by µLC-ESI-IT-MS/MS using a LCQ Deca Xp Plus mass spectrometer (ThermoFinnigan) equipped with an electrospray source connected to a Phoenix 40 pump (ThermoFinnigan).21–23 Peptide mixtures were separated on a capillary ThermoHypersil-Keystone Aquasil C18 Kappa column (100 × 0.32 mm, 5 µm) (Hemel Hempstead, U.K.) using a linear gradient from 10% to 60% of acetonitrile in 0.1% formic acid, over 60 min, at flow rate of 5 µL/min. Spectra were acquired in the range m/z 200–2000. Acquisition was controlled by a data-dependent product ion scanning procedure over the three most abundant ions, enabling dynamic exclusion (repeat count 2 and exclusion duration 3 min). The mass isolation window and collision energy were set to m/z 3 and 35%, respectively. Data were elaborated using the BioWorks 3.1 software provided by the manufacturer. All masses are reported as monoisotopic values. A qualitative measurement of the modification extent during irradiation was obtained by extracting and integrating peak areas corresponding to m/z values of the modified and nonmodified peptides in the same total ion chromatogram. During experiments, RNase A modified peptides bearing Gln11 cyclization to pGlu and Asn67 deamidation to isoAsp/Asp were also detected,24 but were not reported in the text for simplicity. A semiquantitative evaluation of the modification extent was obtained by measuring peak areas corresponding to m/z values of the modified and unmodified peptides in the same total ion chromatogram that were extracted and integrated. Irradiation of RNase A present in Large Unilamellar Vescicles. Liposomes in the form of large unilamellar vesicles (LUVET) (diameter ) 100 nm) were prepared in bidistilled water or 10 mM phosphate buffer, pH 7, with 1-palmitoyl-2oleoyl phosphatidylcholine (POPC) or dioleoyl phosphatidylcholine (DOPC), by membrane extrusion using LiposoFast.4–7,25 The monounsaturated residue was oleic acid (cis-9-octadecenoic acid). To reach the same oleic acid content, 2.5 mM DOPC or 5.0 mM POPC LUVET suspensions were transferred into a 4 mL vial equipped with an open-top screw cap and a Teflon faced septum. RNase A dissolved in a minimal amount of aqueous medium was added to the LUVET suspension yielding a lipid/protein molar ratio of 57:1 or 116:1 (44 µM protein final concentration). When necessary, t-BuOH was also

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Reductive Desulfurization of Met and Cys Residues added to reach a final concentration of 0.2 M, as value known to be compatible with vesicle stability.26 The total volume of the reaction sample was 1 mL. RNase A-free DOPC or POPC LUVET suspensions were prepared as control. All samples were flushed with N2O or Ar prior to γ-irradiation. The irradiation dose was in the range of 10–12 Gy/min. Sample aliquots were withdrawn before and after time intervals of the reaction. Workup of the irradiated reaction mixture was carried out as previously reported.4–7 To quantify the formation of the trans fatty acid isomer (elaidic acid), lipid isolation and derivatization to methyl ester for gas chromatographic (GC) analysis was performed as previously reported.4–7,17,18

Results and Discussion Generation of Hydrogen Atoms. Radiolysis of neutral water leads to eaq-, HO• and H•, as shown in eq 1.27,28 The values in parentheses represent the radiation chemical yields (G) in µmol/J. In an N2O-saturated solution (∼0.025 M at 25 °C), eaqare converted into HO• radicals with a k2 ) 9.1 × 109 M-1 s-1 (eq 2). Under these conditions, H• atoms and HO• radicals account for 10 and 90% of the reactive species, respectively. In the presence of 0.2 M t-BuOH, HO• radicals are scavenged efficiently (eq 3, k3 ) 6.0 × 108 M-1 s-1], whereas H• atoms react only slowly (eq 4, k4 ) 1.7 × 105 M-1 s-).27,28 Thus, the major reactive species expected under the first experimental conditions were H• atoms and C-centered radicals from t-BuOH. Alternatively, Ar-deoxygenated 10 mM phosphate buffer was used to generate H• atoms, since eaq- may be converted into H• atoms with a k5 ) 1.5 × 107 M-1 s-1 (eq 5), depending on the pH and other additives. γ

H2O 98 eaq- (0.27),

HO• (0.28),

H• (0.062)

H2O

(1)

eaq- + N2O 98 N2 + HO• + OH-

(2)

HO• + tBuOH f (CH3)2C(OH)CH2• + H2O

(3)

•

•

H + tBuOH f (CH3)2C(OH)CH2 + H2

(4)

eaq- + H2PO4- f H• + HPO42-

(5)

When RNase A was present, it can be calculated that about 90% of eaq- were trapped by the protein, based on known high reactivity of RNase A with electrons (k ) (1.5–2.1) × 1010 M-1 s-1).29 Under these conditions, the radiation chemical yield of H• atoms was considered equal in buffer or water as well as in all degassing conditions. Raman Spectroscopic Characterization of Irradiated RNase A. RNase A samples were treated under two experimental conditions generating H• atoms (see above) and were analyzed by Raman spectroscopy, a technique which provides valuable information on preferential sites of radical attack. The Raman spectral region corresponding to the vibrations of sulfurcontaining residues for untreated and irradiated protein samples in 0.2 M t-BuOH is provided in Figure 1A. The S-S and C-S stretching frequencies of the four C-S-S-C cross-links in untreated RNase A appeared respectively at 515 and 653 cm-1, respectively, whereas the Met residues corresponded to the band at 724 cm-1.30 Exposure of the protein to the 84 Gy irradiation dose was able to produce significant changes in the S-S bridges, as suggested by the higher wavenumber shifts of

Figure 1. The 900–450 cm-1 Raman region of native RNase A (a) and RNase A samples subjected to γ-irradiation with dose of 84 (b) and 650 Gy (c). Spectra from samples irradiated in 0.2 M t-BuOH (panel A) and 10 mM phosphate, 0.2 M t-BuOH (panel B) are reported.

the 518 and 657 cm-1 bands. Similarly, modifications in the C-S bond of Met were observed, as indicated by the splitting of the 724 cm-1 band in two components at 734 and 725 cm-1 (Figure 1A(b)). Modifications of the S-S bonds were further confirmed by the intensity increase of the bands at 748 and 674 cm-1 due to νC-S of cystines. By increasing the irradiation dose, additional spectral changes were visible in both the Met and Cys residue bands (i.e., wavenumber shift and broadening), indicating that the action of H•atoms may cause a profound change in the sulfur-containing residues (Figure 1A(c)). These findings confirmed previous reports on Met4–7 and preliminary data on cystine.12–15,31 Unfortunately, it was not possible to obtain unquestionable spectroscopic evidence for the occurrence of Met > Aba and Cys > Ala substitutions, due to the weakness of the bands corresponding to Aba and Ala, and to their overlapping with the backbone vibration bands of the protein. Some changes in the ≈1470, ≈1455 and ≈770 cm-1 bands, due to CH3 deformation modes of Ala, were, however, visible after γ-irradiation. Spectral modifications were also visible in the bands due to phenyl ring vibrations of Tyr and Phe residues (Figure 1A). For example, the intensity ratio of the high-frequency to the low-frequency component of the 850–830 cm-1 doublet, due to the six Tyr residues, slightly increased, indicating a small change in the binding network involving some of the phenolic hydroxyl side chains. Regarding the Phe residues, the splitting of the Phe band at 621 cm-1 into two components at 621 and 614 cm-1, Journal of Proteome Research • Vol. 7, No. 5, 2008 2009

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Figure 2. (A) The 1720–1580 cm-1 Raman region and (B) curve fitting of the Amide I band of native RNase A (a) and RNase A samples subjected to γ-irradiation with dose of 84 (b) and 650 Gy (c) in 0.2 M t-BuOH.

indicated the tendency of the H• atoms to react with these aromatic amino acids as well. The use of phosphate buffer did not generate significant differences on protein modification, as just evidenced under similar experimental conditions but at different irradiation doses.5 In this case, the lowest dose caused significant changes in the sulfur moieties of RNase A (Figure 1B(b)). The spectral modifications visible in the bands due to the C-S stretching mode of Cys (from 742 to 750 cm-1) and Met (from 725 to 732–723 cm-1) residues were very similar to those observed for RNase A in aqueous solution, confirming these residues as preferential sites of a reductive radical attack. One of known potential advantages of Raman spectroscopy for the study of proteins resides in the correlation between the vibrational frequencies of the peptide backbone and the various protein conformations. In particular, the amide I bands, appearing in the 1620–1700 cm-1 region, may act as sensitive conformational markers. Unfortunately, the presence of the phosphate buffer in the samples rendered an in deep analysis of some spectral regions (such as 1640–1670 cm-1) impossible, since it gave rise to strong overlapping with other Raman bands, that is, the Amide I band. Thus, it was possible to carry out an in deep evaluation of the secondary structure modifications only in the aqueous samples. The exposure of the protein to both irradiation doses induced significant changes in the 2010

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spectral features of the amide I band (i.e., new shoulders at ≈1690 and ≈1648 cm-1), indicating modification in the R-helix and β-sheet content of RNase A (Figure 2A). To probe the extent of the conformational changes induced by the radical attack, the analysis of the amide I band was performed by curve fitting (Figure 2B). All the components were assigned to a particular secondary structure on the basis of previous reports,32–35 and the conformational contents were calculated from the integrated intensities of the individual assigned bands. Although this method does not yield the absolute content of the secondary structures (since the effective intensities of the bands corresponding to different structure elements are not completely equivalent), it may be used to demonstrate protein conformational changes induced by external factor interaction, such as radical attack. The curve fitting results indicated a strong decrease in the R-helix content (from 17% to 4% and 0% after 84 and 650 Gy, respectively), with parallel increase in the random coil percentage (from 30% to 37% and 46%, respectively). The β-sheet content resulted in an increase up to ≈60% after irradiation with 84 Gy, whereas further exposure also caused a slight decrease in this ordinate structure content (54%). Under the experimental conditions used, γ-irradiation was able to progressively destabilize RNase A structure, providing increased conformational freedom to the

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Reductive Desulfurization of Met and Cys Residues

Table 1. Modified Peptides Observed in RNase A Tryptic Digests As Revealed by µLC-ESI-IT-MS/MS Analysisa

Figure 3. ESI-Q-TOF-MS analysis of RNase A samples irradiated in 0.2 M t-BuOH with 0, 84 and 650 Gy (panels A, B and C, respectively) or in 10 mM phosphate, 0.2 M t-BuOH with 0, 84 and 650 Gy (panels D, E and F, respectively). Protein samples were freed from salts before analysis, as described in the Experimental Procedures. Components A, B, C, D, E, F, G and H corresponded to native RNase A and protein adducts showing a ∆m ) +16, +32, -17, - 46, -32, +48 and +72 Da, respectively, with respect to native RNase A.

amino acid side chains and rendering some of them more susceptible of reductive radical attack. Mass Spectrometric Characterization of Irradiated RNase A Products. To further investigate structural modifications induced by radical attack, RNase A samples were desalted and analyzed by ESI-Q-TOF-MS. The resulting mass spectra are shown in Figure 3. As expected, RNase A samples non irradiated in aqueous and buffer solutions showed almost identical MS profiles (Figure 3, panels A and D), demonstrating the occurrence of one major (component A) and three minor species (component B, C and F) within starting protein material. On the basis of the measured mass values, these components were tentatively associated with native RNase A (theoretical value 36 682.3 Da), mono-oxidized RNase A (theoretical value 36 698.3 Da), dioxidized RNase A (theoretical value 36 714.3 Da) and a succinimide-containing RNase A form (theoretical value 36 665.3 Da), respectively. It is worth mentioning that Met oxidation has been reported to result from protein manipulation.36 In contrast, clear differences were observed as result of irradiation progress. In addition to components reported above, four additional protein species were visible in the RNase A sample irradiated in 0.2 M t-BuOH with 84 Gy (Figure 3, panel B). These species corresponded to those protein components with a ∆m ) -46, -32, +48 and +72 Da (indicated as compounds H, G, D and E, respectively), with respect to native RNase A. A concomitant increase of the signals associated to mono- and dioxidized RNase A was also observed. The relative abundance of the eight protein derivatives appearing in the spectra changed in response to the radiation dose (Figure 3, panel C).

modified peptide

experimental (theoretical) mass (Da)

modification site

sample

(105–124)ButOH (105–124)Cys > Ala (40–61)Cys > Ala (67–85)ox (67–85)ButOH (67–85)ButOH (67–85)Met > Aba (67–85)Cys > Ala (62–85)ox (62–85)Met > Aba (11–31)ox (11–31)ox (11–31)ox2 (11–31)ox2

2296.14 (2296.63) 2135.08 (2135.41) 2428.22 (2428.75) 2301.91 (2302.47) 2357.97 (2358.57) 2357.97 (2358.57) 2239.93 (2240.38) 2196.92 (2197.35) 2874.18 (2875.16) 2812.20 (2813.06) 2380.92 (2381.59) 2380.92 (2381.59) 2396.91 (2397.59) 2396.91 (2397.59)

Tyr115 Cys110 Cys58 Met79 Tyr73 Tyr76 Met79 Cys72 Met79 Met79 Met13 Met29/30 Met13, Met29 Met13, Met30

B, C, F C, F C, F A, B, C, D, E, F B, C B, C C, F C, F A, B, C, D, E, F C, F A, B, C, D, E, F B, C, E, F B, C, F B, C, F

a Experiments were performed on RNase A samples irradiated in 0.2 M t-BuOH with 0, 84 and 650 Gy (samples A, B and C) or in 10 mM phosphate, 0.2 M t-BuOH with 0, 84 and 650 Gy (samples D, E and F), respectively. Samples where each modified peptide was observed are specifically reported. Modification was assigned on the basis of measured mass difference with respect to the observed nonmodified counterpart and fragmentation data. Ox, oxidation; t-ButOH, addition of C-centered radical of t-BuOH to Tyr; Met > Aba, desulfurization of Met to Aba; Cys > Ala, desulfurization of Cys to Ala. The site of modification is also shown.

These components were also observed in the RNase A samples irradiated in 10 mM phosphate, 0.2 M t-BuOH (Figure 3, panels E and F). A quantitative measurement of the total ion current produced by each species demonstrated different relative formation rates under the two conditions. To identify amino acids subjected to progressive RNase A modification during irradiation, all samples were resolved by SDS-PAGE, yielding the expected monomeric protein band migrating at 17 kDa (data not shown). No significant electrophoretic differences were observed between irradiated samples. Protein bands were alkylated in parallel, digested with trypsin and subjected to massive peptide mapping experiments by MALDI-TOF-MS and µLC-ESI-IT-MS/MS. In all cases, this combined analysis allowed an almost complete coverage (98.4%) of the RNase A sequence (see below). In particular, MALDI-TOF-MS analysis confirmed the covalent nature of some of the protein adducts already observed in ESI-Q-TOFMS analysis of intact proteins (Figure 4) and revealed the appearance of specific modified peptides resulting from the irradiation. In addition to some oxidized peptides already detectable in nonirradiated RNase A samples (Figure 4, panels A and C), namely (67–85)ox and (11–31)ox, a series of satellite signals with a ∆m ) -89, -46, +32, and +72 Da with respect to the expected peptides was observed in those protein samples irradiated with 650 Gy. In particular, three modified forms of the peptide (67–85) (∆m ) -89, -46 and +72 Da), a modified form of the peptide (11–31) (∆m ) +32 Da), a modified form of the peptide (40–61) (∆m ) -89 Da) and a modified form of the peptide (62–85) (∆m ) -46 Da) were detected for RNase A irradiated in 0.2 M t-BuOH (Figure 4, panel B). In contrast, two modified forms of the peptide (67–85) (∆m ) -89, -46 Da), a modified form of the peptide (40–61) (∆m ) -89 Da) and a modified form of the peptide (62–85) (∆m ) -46 Da) were detected for RNase A irradiated in 10 mM phosphate, 0.2 M t-BuOH (Figure 4, panel D). Newly detected satellite peaks at ∆m ) -89 Da always occurred in Cys-containing peptides. This analysis restricted Journal of Proteome Research • Vol. 7, No. 5, 2008 2011

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Figure 4. MALDI-TOF peptide mass mapping analysis of tryptic digests from RNase A samples irradiated in 0.2 M t-BuOH with 0 and 650 Gy (panels A and B, respectively) and in 10 mM phosphate, 0.2 M t-BuOH with 0 and 650 Gy (panel C and D, respectively). A specific mass range highlighting signal differences between samples is shown. Modified derivatives showing a ∆m ) +16, +32, - 46, -89 and +72 Da with respect to expected RNase A peptides are indicated with *, **, °, + and §, respectively.

covalent modification of RNase A to specific polypeptide regions and confirmed the variable modification extent of the protein as result of the buffer used. To precisely elucidate the nature of the modification products generated in the irradiated RNase A samples, peptide mixtures were also resolved by LC and automatically characterized by tandem mass spectrometry. These experiments definitively localized all covalent adducts present in the protein sequence as a function of radiation dose. In fact, µLC-ESI-ITMS/MS analysis confirmed the occurrence of modified peptides already revealed by MALDI-TOF-MS and allowed for the detection of additional trace peptide derivatives, not visualized by MALDI-TOF mapping experiments due to competitive ionization phenomena. The modified peptides detected in all RNase A digests are reported in Table 1. Small amounts of Met13 and Met79 sulfoxide derivatives were also present in nonirradiated samples, in agreement with ESI-Q-TOF-MS data (Figure 3) and previous literature data on protein oxidation propensity.30 Approximately 14 peptide adducts were detected in the RNase A sample irradiated in 0.2 M t-BuOH with 650 Gy. As representative examples, the MS/MS characterization of some modified peptides detected in this case is presented in Figure 5. On the basis of the mass difference measured for the covalent adducts with respect to nonmodified peptides and the good fragmentation data obtained for these 2012

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species, four main classes of modification reactions were revealed: (i) oxidation of Met to sulfoxide and sulfone derivative (∆m ) +16, +32 and +48 Da for intact proteins and ∆m ) +16 and +32 Da for modified peptides); (ii) addition of t-BuOH-derived radical to Tyr (∆m ) +72 Da for intact proteins and modified peptides); (iii) H•-mediated reduction of Met to Aba (∆m ) -46 Da for intact proteins and modified peptides); (iv) H•-mediated reduction of Cys to Ala (∆m ) -32 Da for intact proteins and ∆m ) -89 Da for modified carboxamidomethyl peptides). In all cases, modification was not widely distributed on the 4 Met, 6 Tyr and 8 disulfide-forming Cys residues present within protein sequence, but occurred at specific residues. In fact, the selective transformation of Met79 to Aba, desulfurization of Cys58, Cys72 and Cys110 to Ala, aromatic substitution at the phenolic moiety of Tyr73, Tyr76 and Tyr115, and oxidation at Met13, Met29, Met30 and Met79 were observed. In contrast, fewer modified peptides were detected in the RNase A samples irradiated with identical γ-ray doses in 10 mM phosphate, 0.2 M t-BuOH (Table 1). These findings further confirmed the variable modification extent of the protein as result of the solvent used and demonstrated the quenching effect of phosphate buffer on the addition of t-BuOH-derived radical to Tyr residues (Table 1). Apart from the buffer used,

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Reductive Desulfurization of Met and Cys Residues

Table 2. Dose-Dependent Formation of Elaidic Acid (trans-9-Octadecenoic Acid) by Isomerization of Oleic Acid (cis-9-Octadecenoic Acid) Present in Phospholipid (DOPC or POPC) Vesicle Suspensions Containing RNase A Prepared in Water or 10 mM Phosphate, with/without 0.2 M t-BuOHa

dose (Gy)

2.5 mM DOPC N2O, water (% trans)

2.5 mM DOPC 0.2 M t-BuOH N2O, water (% trans)

5 mM POPC 0.2 M t-BuOH N2O, water (% trans)

5 mM POPC 0.2 M t-BuOH Ar, 10 mM phosphate (% trans)

84 138 166 328 653 1000

0.5 0.9 1.5 3.2 4.7 5.5

5.9 9.9 11.9 23.4 40.1 49.9

8.3 12.5 17.2 28.6 44.3 52.3

12.3 14.5 16.4 26.7 38.8 45.0

a LUVET were prepared as described in Experimental Procedures. Quantitative GC analysis of lipid derivatives was performed as described in refs4-7.

Figure 5. Tandem mass spectrometry analysis of modified peptides present in the tryptic digest of RNase A irradiated in 0.2 M t-BuOH with 650 Gy. Newly formed amino acids are shown with a three-letter abbreviation. The fragmentation mass spectrum of the triply charged ion at m/z 938.7 associated with the peptide (62–85)Met79 > Aba is shown in panel A. The fragmentation mass spectrum of the doubly charged ion at m/z 1099.5 associated with the peptide (67–85)Cys72 > Ala is shown in panel B. The fragmentation mass spectrum of the doubly charged ion at m/z 1068.5 associated with the peptide (105–124)Cys110 > Ala, is shown in panel C. The fragmentation mass spectrum of the doubly charged ion at m/z 1149.4 associated with the peptide (105–124)Tyr115-CH2C(CH3)2OH, is shown in panel D.

detection of modified peptides at low radiation doses highlighted the most reactive amino acids within RNase A sequence. Fatty Acid Isomerization in RNase A-Containing Phospholipids Vesicles. The formation of diffusible isomerising species, that is, thiyl radicals derived from the reductive desulfurization of the sulfur residues of RNase A, was assayed by monitoring the cis to trans double bond conversion of liposomes. This biomimetic model has found to be very useful in the initial identification of lipid–protein damage.4,5 Both DOPC or POPC, having in their structure oleic acid (cis9-octadecenoic acid) as monounsaturated fatty acid, were used to form large unilamellar vesicles in aqueous or buffer medium containing 0.2 M t-BuOH. RNase A was then added in order to mantain a high lipid/protein molar ratio (57:1 or 116:1), minimizing protein interaction with lipid bilayer.37,38 Samples were degassed according to the desired conditions (N2O or Ar) and irradiated, withdrawing aliquots of the suspension at different doses for GC determination of the trans isomer formation. The progressive isomerization in parallel with irradiation dose is shown in Table 2, starting from low radiation doses and progressing up to 1 kGy. Representative GC traces (Figure 6) show the selective elaidic acid production during irradiation in the presence of RNase A; isomerization was practically absent in LUVET not containing the protein (cf., trace B). The percentage of trans lipids found in the vesicles under different experimental conditions is provided in Table 2. In the first column, previously reported data4,17 are shown for comparative purpose; in the presence of t-BuOH (second column), isomerization proceeded to a greater extent, due to the trapping of most of the HO• radicals by the alcohol (cf., eqs 3 and 4). Irradiation using 2.5 mM DOPC or 5 mM POPC gave similar results (second and third columns, respectively) because of the comparable oleic acid content. These processes gave results similar to those obtained for reaction in phosphate buffer, indicating that the reductive transformations are not greatly influenced by protein conformational variations in different environments. Argon as the degassing agent (fourth column) did not affect the efficiency of the isomerization compared to N2O, thus, confirming the role of RNase A as the best electronscavenger in all systems. Taking into account all competitive reactions (cf., eqs 1–5), hydrogen atom reactivity emerges and cannot be excluded as Journal of Proteome Research • Vol. 7, No. 5, 2008 2013

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Ferreri et al. Scheme 2

Figure 6. GC analysis of the trans-esterification products from POPC vesicles not irradiated (panel A), after irradiation with a dose of 1 kGy in absence of RNase A (panel B) and after irradiation with a dose of 1 kGy in presence of RNase A (panel C). Fatty acid methyl esters were identified by chromatographic comparison with commercially available reference standards; peak 1, 16:0 (methyl palmitate); peak 2, 9t-18:1 (methyl elaidate); peak 3, 9c-18:1 (methyl oleate).

a contributing factor in the generation of the observed species under radical conditions, whereas until now much more attention has been devoted to electrons and HO• radicals. The fact that hydrogen atoms are highly specific for sulfur-containing moieties explains why tandem protein–lipid damage was also detectable in the presence of oxygen.5 These findings suggest that isomerization should be interesting in the context of living systems, where hypoxic conditions are present.39 Although we did not characterize the damaged proteins in vivo, we previously demonstrated that (i) trans lipids are indeed formed in eukaryotic cells, (ii) thiyl radicals are harmful to the integrity of cis lipid geometry and (iii) cis–trans isomerization occurs in vivo following redox insult.40,41 Our results therefore motivate future investigations for a more comprehensive description of the modifications affecting proteins and lipids within cells challenged by radical stress conditions. It should also be noted that double bond isomerization of the membrane fatty acid residues is a specific reaction for diffusible thiyl radicals generated from desulfurization of proteins in an aqueous environment.4–7 A very small quantity of thiyl radicals can effect the efficient lipid isomerization process, resulting in damage amplification. A connection between lipid isomerization and desulfurization was previously reported for some Met-containing polypeptides subjected to reductive H• atom attack,4–7 and was associated to the reactivity of CH3S• radicals resulting from transformation of Met to Aba residues. In the present study, reductive conditions associated to H• atoms production also strongly affected other sulfurcontaining amino acids, that is, disulfide-forming cysteines. Both reductive modifications are involved in the formation of diffusible radical species able to isomerize lipid double bonds, as summarized in Scheme 2. 2014

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Detection of the Cys > Ala modification products in RNase A-irradiated samples was highly indicative of the formation of perthiyl radicals as intermediate products of reaction (Scheme 1), confirming previous preliminary observations.12–14,16 However, the possibility that perthiyl radicals, themselves, contribute to this reaction in phospholipids is remote, since these species remain protein-bound and do not meet the requirement of diffusibility toward cellular membranes. The fate of these intermediates to yield the corresponding thiols, as reported in case of irradiated oxidized glutathione at solid state,42 is the subject of forthcoming studies on the possible occurrence of other radical species deriving from perthiyl radicals, that is, sulfhydryl radicals (HS•/S•-), able to contribute to cis–trans lipid isomerization phenomena in membranes. The potential of HS•/S•- radicals as isomerising agents toward lipid vesicles has been very recently described.43 Moreover, the fate of the perthiyl radicals in proteins should be further investigated also in view of its reactivity with nucleophilic centers or other thiol functions. 44

Conclusions The biomimetic model of unsaturated lipid vesicle suspensions containing polypeptide substrates proves to be a very interesting system for the study of the cis–trans isomerization of phospholipids present in biological membranes and associated to protein damage.4–11 The reaction is catalyzed by sulfur radical species generated from desulfurization of sulfurcontaining residues in proteins.4–7 In this contest, H• atoms emerge as the appropriate reactive species for the study of these processes and, in general, of protein damage, affording selective reductive radical conditions for specific amino acid targets. Various examples are reported in literature describing how strong reductive stresses may alter cellular redox environment leading to odd events such as radiation-induced cellular apoptosis during radiosensitizer use,45,46 abnormal protein aggregation associated to cardiomyopathy,47 accumulation of misfolded proteins in ER,48–51 atypical reduction/increase of protein activities52,53 and proliferation of some tumor cells.54–56 To the already known desulfurization process affecting Met residues and yielding Aba derivatives and methanethiyl radicals,4–7 a novel reactivity involving protein disulfide moieties was evidenced in this work. This process was associated with the chemical transformation of disulfide-linked cysteines into Ala residues and to the formation of perthiyl radicals. Mapping experiments on RNase A samples demonstrated that desulfurization affected, to a significant extent, specific Cys residues from the onset of the reaction. Although preliminary evidence of this

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Reductive Desulfurization of Met and Cys Residues 12–14,16

process was reported previously, this is, to our knowledge, the first definitive demonstration of its occurrence. The results reported here disclose new scenarios for the reactions generated by radical stressing conditions, highlighting the role of reducing radicals in selectively damaging sulfurcontaining residues and suggesting the need for specific assays to detect these modifications in proteins and lipids present within challenged cells. The concomitant occurrence of Met > Aba and Cys > Ala substitutions cannot be ascribed to other modification processes in aqueous media other than to radical-based desulfurization reactions, and can therefore be attributable to reductive radical stress conditions. Cys > Ala desulfurization has been already described to occur enzymatically in the production of elemental sulfur from Cys-containing polypeptides assisted by pyridoxal phosphate-dependent desulfurases NifSs,57 and by Raney nickel-catalyzed reactions for the t-RNA amino acid triplet modification.58,59 A comprehensive approach, including reductive stress modifications applied to model living systems, would positively contribute to a more complete understanding of puzzling biological questions regarding cellular degeneration associated with aging and pathologies.

Acknowledgment. This work was partially supported by grants from the European Community, Human Potential Programme contract HPRN-CT-2002–00184 (SULFRAD), from the Italian National Research Council (AG-PO4-PO14) and MiUR (FIRB2001, RBAU01PRLA). The support and sponsorship concerted by COST Action CM0603 on “Free Radicals in Chemical Biology (CHEMBIORADICAL)” are kindly acknowledged. Authors thank Dr. Fabrizio Dal Piaz, University of Salerno, for the assistance during MS analysis of intact proteins. References (1) Seto, H.; Okuda, T.; Takesue, T.; Ikemura, T. Bull. Chem. Soc. Jpn. 1983, 56, 1799–1802. (2) Marnett, L. J.; Basu, A. K.; O’Hara, S. M.; Weller, P. E.; Rahman, A. F. M. M.; Oliver, J. P. J. Am. Chem. Soc. 1986, 108, 1348–1350. (3) Niedernhofer, L. J.; Daniels, J. S.; Rouzer, C. A.; Greene, R. E.; Marnett, L. J. J. Biol. Chem. 2003, 33, 31426–31433. (4) Ferreri, C.; Manco, I.; Faraone-Mennella, M. R.; Torreggiani, A.; Tamba, M.; Chatgilialoglu, C. ChemBioChem 2004, 5, 1710–1712. (5) Ferreri, C.; Manco, I.; Faraone-Mennella, M. R.; Torreggiani, A.; Tamba, M.; Manara, S.; Chatgilialoglu, C. ChemBioChem 2006, 7, 1738–1744. (6) Kadlcik, V.; Sicard-Roselli, C.; Houée-Levin, C.; Kodicek, M.; Ferreri, C.; Chatgilialoglu, C. Angew. Chem., Int. Ed. 2006, 45, 2595–2598. (7) Mozziconacci, O.; Bobrowski, K.; Ferreri, C.; Chatgilialoglu, C. Chem.-Eur. J. 2007, 13, 2029–2033. (8) Chatgilialoglu, C.; Ferreri, C. Acc. Chem. Res. 2005, 36, 441–448. (9) Ferreri, C.; Chatgilialoglu, C. ChemBioChem 2005, 6, 1722–1734. (10) Wood, R. D. Biological effects of geometrical and positional isomers of monounsaturated fatty acids in humans. In Fatty Acids in Foods and Their Health Implications, 2nd ed.; Chow, C. K., Ed.; Dekker: New York, 2000; pp 637–665. (11) Mozaffarian, D.; Katan, M. B.; Ascherio, A.; Stampfer, M. J.; Willett, W. C. N. Engl. J. Med. 2006, 354, 1601–1613. (12) Stein, G. In Energetic and Mechanisms in Radiation Biology; Phillips, G. O., Ed.: Academic Press: London, 1968; pp 467–477. (13) Shapira, R.; Stein, G. Science 1968, 162, 1489–1491. (14) Holmes, B. E.; Navon, G.; Stein, G. Nature 1967, 213, 1087–1091. (15) Asmus, K. D.; Binifacic, M. In S-Centered Radicals; Alfassi, Z. B., Ed.; Wiley: Chichester, U.K., 1999; pp 141–191. (16) Faucitano, A.; Buttafava, A.; Mariani, M.; Chatgilialoglu, C. ChemPhysChem 2005, 6, 1100–1107. N.B.: Schemes 4 and 5 of this article are mixed. The drawings of Scheme 5 are related to the legend of Scheme 4 and the drawings of Scheme 4 are related to the legend of Scheme 5. (17) Torreggiani, A.; Tamba, M.; Manco, I.; Faraone-Mennella, M. R.; Ferreri, C.; Chatgilialoglu, C. Biopolymers 2006, 81, 39–50. (18) Torreggiani, A.; Bottura, G.; Fini, G. J. Raman Spectrosc. 2000, 31, 445–450.

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