Probing the Interaction of Cisplatin with Cytochrome c by Electrospray

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Probing the Interaction of Cisplatin with Cytochrome c by Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Ningbo Zhang,†,‡ Yonggang Du,†,‡ Meng Cui,*,† Junpeng Xing,† Zhiqiang Liu,† and Shuying Liu† †

Changchun Center of Mass Spectrometry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun 130022, People’s Republic of China ‡ Graduate School of the Chinese Academy of Sciences, Beijing 100039, People’s Republic of China S Supporting Information *

ABSTRACT: As one of the most important platinum drugs, the interactions of cisplatin with proteins play very important roles in its anticancer action and side effects. Here, the interaction of cisplatin with cytochrome c was investigated using Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). On the basis of the high-resolution data, cytochrome c−Pt(NH3)Cl was found to be the primary monoadduct. The platinated monoadducts were related to molar ratios of cytochrome c and cisplatin, and corresponding reaction pathways were proposed. Multiple binding sites of cisplatin on cytochrome c were directly determined by FTICR MS combined with trypsin digestion without liquid chromatography (LC) separation. Four binding sites (Met65, Met80, His18, and His33) for cisplatin on cytochrome c were identified. Moreover, hydrogen/deuterium exchange (H/DX) combined with FTICR MS provides the sensitive method to insight the small conformation change of cytochrome c induced by cisplatin. This strategy can be applied to comprehensive investigation of metallodrug/protein complexes.

A

studied the reactions of four platinum drugs and cytochrome c and observed their platinated adducts by ESI-MS.31 Zhao and King used a bottom-up method to assign one binding site (Met65) for cisplatin on cytochrome c by Fourier transform mass spectrometry (FT-MS).15 Recently, Moreno-Gordaliza et al. combined in-gel and in-solution digestion to characterize the multiple binding sites (Met80, Glu61/Glu62/Thr63, and Met65) for cisplatin on cytochrome c by liquid chromatography coupled with LTQ-MS.32 Until now, most of works on studies of reactions of platinum drugs with cytochrome c by mass spectrometry focused on the determination of drug−protein adducts and mapping platinum binding sites on cytochrome c. However, the conformation of a protein also plays a key role in its biological activity. The binding of platinum(II) to a protein likely changes the conformation of protein and leads it to malfunction. The conventional methods such as NMR,33,34 circular dichroism (CD),34 and IR35 are often used to monitor conformational changes of proteins induced by platination. In 1998, Neault and Tajmir-Riahi reported that, at high cisplatin concentration, cisplatin binding to HSA resulted in major alterations of the protein secondary structure from that of the α-helix to β-sheet conformation by Fourier transform infrared (FT-IR) spectroscopy.35 Recently, Williams et al. first applied

s one of the most important metal-antitumor drugs, cisplatin has been successfully used for the treatment of a variety of solid tumors, particularly testicular and ovarian cancers.1,2 Although the action mechanism has not been very clear, it is generally accepted that cisplatin can interact with DNA which is the key event to induce the cancer cell’s apoptosis.3,4 However, it also causes severe toxic side effects.5,6 It has been found that 65−98% of the platinum is bound to proteins in the blood within 24 h of intravenous injection of cisplatin, and the platination of proteins may contribute to the severe side effects of cisplatin, such as nephrotoxicity and ototoxicity.7 Therefore, more and more attention of researchers has been attracted to investigate the interactions between cisplatin with proteins. Cytochrome c is a small, well-characterized protein containing several potential binding sites such as methionine and histidine for Pt metallodrugs.8 Recently, researchers found that cisplatin may cause the release of cytochrome c from mitochondria into cytoplasm and cytochrome c plays an important role during the apoptosis of cancer cells.9−11 Since Gibson and Costello first reported the studies of ubiquitin− cisplatin interaction using electrospray ionization mass spectrometry (ESI-MS) in 1999,12 ESI-MS has played an important role in studies of the interactions of platinum metallodrugs with various proteins such as human serum albumin (HSA),13,14 cytochrome c,15,16 transferrin,17,18 ubiquitin,12,19−21 insulin,22,23 calmodulin,24,25 lysozyme,26 myoglobin,27 the human chaperone Atox1,28−30 and so on. Casini et al. © 2012 American Chemical Society

Received: May 4, 2012 Accepted: June 19, 2012 Published: June 29, 2012 6206

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Protein Digestion. The cytochrome c−platinum adducts were prepared by reacting 160 μM cytochrome c with cisplatin at molar ratio of 1:12 at pH 6.8, 37 °C for 9, 24, and 72 h and then were processed by the centrifugation procedure as mentioned above. Then the reaction products were diluted to 160 μM using 5 mM NH4OAc buffer. The cytochrome c− platinum adducts were diluted to 32 μM with 50 mM NH4HCO3 (pH 7.8) and then subjected to trypsin digestion at a protein to enzyme ratio of 40:1 (w/w) at 37 °C for 4 h. As a control, 32 μM free cytochrome c was digested under the same condition. The digestion solution was diluted to 5 μM with 50% methanol−0.1% formic acid buffer immediately before mass spectrometry analysis. Global Hydrogen/Deuterium Exchange Analysis. The cytochrome c−platinum adducts were prepared by reacting 160 μM cytochrome c with cisplatin at molar ratio of 1:12 at pH 6.8 for 24 h. The exchange reactions were initiated by diluting 30 μL solution of 160 μM cytochrome c−cisplatin adducts with 270 μL of D2O buffer (5 mM NH4OAc) and incubated at room temperature for various reaction times. The exchange reactions were quenched by reducing the pH to 2.5 with a 1:4 dilution with iced 0.5% HCOOH−20% CH3OH−H2O buffer. And after 3 min of back-exchange of the side hydrogen, the quenched samples were injected directly into the FTICR MS using a Harvard Apparatus syringe pump (Holliston, MA) at a flow rate of 3 μL/min. As a control, 160 μM free cytochrome c was exchanged under the same condition. The m/z values of cytochrome c and adducts after H/D exchange were computed from the centroid of the isotopic envelope. No adjustment was made for deuterium back-exchange during analysis, and therefore all results were reported as the relative deuterium level. Peptide-Level Hydrogen/Deuterium Exchange Analysis. Exchange was performed as mentioned above, and deuterated samples were quenched by reducing the concentration of the protein to 5 μM and the pH to 2.5 with iced 2% CH3COOH−20% CH3OH−H2O buffer. And then 4 mg/mL pepsin in 5 mM NH4OAc (pH 6.8) was added to a protein (protein/enzyme ratio of 1:2) to digest the protein for 5 min, and then the sample was injected directly into the FTICR MS using a Harvard Apparatus syringe pump (Holliston, MA) at a flow rate of 3 μL/min. The m/z values of peptides after H/D exchange were computed from the centroid of the isotopic envelope. FTICR Mass Spectrometry. All ESI-MS experiments were carried out on an IonSpec ESI-FTICR mass spectrometer (IonSpec, Irvine), equipped with a 7.0 T superconducting magnet (Cryomagnetics, Oak Ridge) and a Z-Spray ESI source (Micromass, Manchester). All of the solutions were introduced into the FTICR MS by a 100 μL gastight syringe (Hamilton, Las Vegas) and a Harvard Apparatus syringe pump (Holliston, MA) at a flow rate of 3 μL/min directly. For the sustained offresonance irradiation collision-induced dissociation (SORI CID) experiments, the parent ions were accumulated in a hexapole ion guide for 3−10 s and then were transferred to the ICR cell. The operating parameters were as follows: probe HV was set to 3300 V, sample cone and extractor cone were set to 45 and 5 V, respectively; high-purity nitrogen was used as desolation gas and cone gas; 45 Torr of nitrogen was used as the collision gas introduced into the ICR cell by a pulse valve, and the pulse duration was 524 ms.

ion mobility mass spectrometry to detect conformational changes in ubiquitin in the gas phase induced by reaction with cisplatin and found that the N-terminal platination of ubiquitin could make it to be more compact.36 The combination of hydrogen−deuterium exchange with mass spectrometry made it possible to characterize the influence of ligands on the conformation of proteins. Since Katta et al. combined hydrogen/deuterium exchange (H/DX) with ESI-MS to detect the conformational changes in proteins in 1991,37 it has been proved that H/DX-MS is a particularly useful tool to probe conformational dynamics and structural changes of proteins.38−40 Combined with enzymatic digestion, deuterium incorporation information of peptides can be localized in 3−10 residues to characterize the local alteration of protein conformation.41,42 With the development of Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS), it has become an extremely powerful tool to provide effective binding information of proteins and ligands due to its high resolution and high accuracy.43,44 Here, by using high-resolution mass spectrometry, the strategy was developed to comprehensive characterization of cisplatin−cytochrome c interaction. On the basis of high-resolution and high-accuracy MS data, cytochrome c−Pt(NH3)Cl was found to be the primary monoadduct, which was different from previous studies. The possible reaction pathways were proposed. The binding sites of cisplatin on cytochrome c were directly determined by FTICR MS combined with trypsin digestion without complex liquid chromatography (LC) separation. Met65 and Met80 were the primary and secondary binding sites, respectively. Besides, His18 and His33 also were observed to be binding sites for cisplatin in cytochrome c. Moreover, H/DX combined with FTICR MS provided the sensitive method to insight the small conformation change of cytochrome c by the binding of cisplatin. To our best knowledge, this is the first time to provide direct evidence for multiple binding sites for platinum complexes with cytochrome c without complex LC separation and observe the small conformation change of cytochrome c induced by cisplatin.



EXPERIMENTAL METHODS Materials. Milli-Q water (18.2MΩ) (Millipore, Bedford, MA) was used in all the experiments. Cytochrome c from horse heart, cisplatin, and deuterium oxide were purchased from Sigma-Aldrich (St. Louis, MO). Trypsin (mass spectrometry grade) was purchased from Promega (Madison, WI). Methanol and acetic acid (HPLC grade) were obtained from Fisher Scientific (Pittsburgh, PA). Formic acid (HPLC grade) was bought from TEDIA Company (Fairfield, OH). All chemicals were used directly without further purification. Reaction of Cytochrome c with Cisplatin. A solution of 160 μM cytochrome c was incubated with cisplatin in 5 mM NH4OAc aqueous solution (pH 6.8) at different molar ratios of 1:2 and 1:12. The samples were incubated at 37 °C for different times and then were centrifuged using Amicon Ultra filters (MW cutoff = 3 kDa) at 18 000g for 30 min at room temperature to remove unbound platinum and washed twice with 400 μL of 5 mM NH4OAc aqueous solution (pH 6.8). Then the reaction products were diluted to 160 μM using 5 mM NH4OAc buffer. Samples were diluted to 3.2 μM with 20% methanol−0.1% formic acid buffer immediately before mass spectrometry analysis. 6207

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6H]8+, [cyt c + PtCl + 7H]8+, [cyt c + Pt(NH3)Cl + 7H]8+, and [cyt c + Pt(NH3)2Cl + 7H]8+. Among them, [cyt c + Pt(NH3) Cl + 7H]8+ was the primary monoadduct during the reaction and the others’ relative intensities to it were very low. The predominant monoplatinated species suggested that there is a highly preferential platinum binding site to cytochrome c. Although the observation of cyt c−Pt(NH3)Cl species as the primary monoadduct is different from the previous studies,15,31 it is in agreement with the trans labilization.45−47 When the reaction time was very short such as 0.5 h (shown in Figure 1a), the monoadducts [cyt c + Pt(NH3)Cl + 7H]8+ and [cyt c + Pt(NH3)2Cl + 7H]8+ were easily recognized, while the peaks of [cyt c + Pt(NH3) + 6H]8+ and [cyt c + PtCl + 7H]8+ were too weak to be recognized. The peak of [cyt c + Pt(NH3)2Cl2 + 8H]8+ may be produced due to the noncovalent interaction. As the reaction time extended (shown in Supporting Information Figure S-1), the peaks of [cyt c + Pt(NH3) + 6H]8+ and [cyt c + PtCl + 7H]8+ increased gradually to a relatively stable percentage to [cyt c + Pt(NH3)Cl + 7H]8+. The possible reaction pathways for molar ratio of cisplatin/cytochrome c at 1:12 were proposed and shown in Supporting Information Scheme S-1. First, the reaction between cytochrome c and monohydrated cisplatin to form cyt c−Pt(NH3)2Cl adduct maybe the first step for the reaction.48,49 And then one NH3 was liberated from cyt c−Pt(NH3)2Cl to form cyt c−Pt(NH3) Cl because of the trans labilization effect.50 The Cl− and the other NH3 being liberated from cyt c−Pt(NH3)Cl were the two possible reactions for the next step. On the basis of the relative intensities of these monoadducts, the loss of amine ligand is a reactively fast process after the trans chlorine ligand replaced by sulfur of the Met residue. As the reaction time extended, multiple-platinated species coordinated to cytochrome c. Taking MS spectra at reaction time of 168 h as examples, up to five platinated complexes bound to cytochrome c were detected (Figure 1c). According to monoadducts and mass increases, the most abundant diadduct was speculated as [cyt c + 2Pt + 3(NH3) + Cl + 5H]8+. For the other diadducts, it is hard to confidently assign them. Molar Ratio of Cytochrome c/Cisplatin at 1:2. When the molar ratio of cytochrome c/cisplatin is decreased to 1:2, [cyt c + Pt(NH3)Cl + 7H]8+ is still observed as the main monoadduct. Its neighbor peaks are highly likely to be [cyt c + Pt(NH3) + 6H]8+ and [cyt c + Pt(NH3)(H2O) + 6H]8+ (Figure 1d and Supporting Information Figure S-2). It was different from the reactions of molar ratio of cytochrome c/ cisplatin at 1:12, which indicated that their reaction pathways may be different (shown in Supporting Information Scheme S1). The first and second step of the reactions are in the same manner to generate cyt c−Pt(NH3)Cl. And then, for the reactions of molar ratio of cytochrome c/cisplatin at 1:2, the Cl− will be hydrated to form cyt c−Pt(NH3)(H2O), which might be followed by the liberation of the H2O. On the basis of the above data, the peak of [cyt c + Pt(NH3)2Cl + 7H]8+ can be clearly observed only at high molar ratio (1:12). The [cyt c + Pt(NH3)Cl + 7H]8+ ion is the primary monoadduct at both high and low molar ratios; the other monoadducts’ relative intensities decrease as the molar ratio increases. The peak of [cyt c + Pt(NH3)(H2O) + 6H]8+ was observed at low molar ratio (1:2) of cytochrome c and cisplatin, and [cyt c + PtCl + 7H]8+ was observed at high molar ratio (1:12) of cytochrome c and cisplatin. The possible reason may be that the hydrolysis of Cl in cyt c−Pt(NH3)Cl species

RESULTS AND DISCUSSION FTICR MS Analyses of the Interaction of Cisplatin with Cytochrome c. Molar Ratio of Cytochrome c/Cisplatin at 1:12. The reactions between cytochrome c and cisplatin over the time were studied by FTICR MS. Figure 1 shows the mass

Figure 1. ESI-FTICR MS mass spectra of reaction mixtures of cisplatin with cyt c at molar ratio 1:12 of cyt c/cisplatin for different times: (a) 0.5, (b) 24, and (c) 168 h and at molar ratio 1:2 of cyt c/cisplatin for reaction time (d) 168 h. (Symbols represent experimental data for (∗) monoadducts, (▲) diadducts, (⧫) triadducts, and (■) tetra-adducts.)

spectra of reactions of cytochrome c with cisplatin at molar ratios 1:12 and 1:2 for different time intervals, respectively. The platinum-modified protein ions were recognized based on their accurate m/z values and the characteristic isotopic patterns. The mean absolute deviation was within 3 ppm range. Besides, the charge states of the platinum compounds are considered in this paper. In Figure 1, the ions of cytochrome c showed charge states between 7+ and 9+, and the most abundant ions were those with charge state 8+. At a high molar ratio (1:12) of cytochrome c/cisplatin, four monoadduct peaks were detected (Figure 1a−c). Taking the complexes at the 8+ charge state as examples, monoadducts were assigned as [cyt c + Pt(NH3) + 6208

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Figure 2. ESI-FTICR MS mass spectrum of trypic peptides of platinated cyt c.

was suppressed and led to the different monoadducts at the high molar ratio of cisplatin and cytochrome c. Determination of the Binding Sites of Cisplatin on Cytochrome c. In order to determine the binding sites, the cytochrome c−cisplatin adducts were digested by trypsin. The digested peptides were analyzed by ESI-FTICR MS and SORI CID. Primary and Second Binding Sites. Figure 2 shows mass spectra of digested peptides of cytochrome c/cisplatin 1:12 at reaction time of 24 h by trypsin. The peptide coverage identified is shown in Supporting Information Scheme S-2. The platinated peptides can be recognized by their characteristic isotopic patterns and accurate masses. The three platinated peptides were clearly recognized as [(61−73) + Pt(NH3)]2+ (m/z 917.8895), [(61−72) + Pt(NH3)]2+ (m/z 853.8414), and [(80−86) + Pt(NH3) − H]+ (m/z 989.4243), respectively. Besides, the [(61−73) + Pt]2+ (m/z 909.3763), [(61−73) + PtCl +H]2+ (m/z 927.3643), [(61−73) + Pt(NH3)Cl +H]2+ (m/z 935.8774), [(56−73) + Pt(NH3)Cl + H]2+ (m/z 1228.5418), [(56−73) + Pt(NH3)]2+ (m/z 1210.5529), and [(56−73) + Pt]2+ (m/z 1202.0362) were also observed clearly. This is consistent with the monoadducts observed in the 1:12 reaction. In order to determine the binding sites in these peptides, SORI CID was performed on [(61−73) + Pt(NH3)] 2+ ion at m/z 917.9, [(80−86) + Pt(NH3) − H]2+ ion at m/z 989.4, and [(61−72) + Pt(NH3)]2+ at m/z 853.8. For the [(61−73) + Pt(NH3)]2+ ion, the observation of y8+ and [y9 + Pt(NH3) − H]2+ ions indicated that platinum coordinates to Met65 (Figure 3a). For the [(61−72) + Pt(NH3)]2+ ion, y7+ and [b5 + Pt − H2O − 2H]+ ions also further confirmed it (Supporting Information Figure S-3). However, for the [(80−86) + Pt(NH3)]+ ion, the fragment ions [b6 + Pt − 2H]+, [b4 + Pt − 2H]+, [b3 + Pt − 2H]+, and [a3 + Pt − 2H]+ (Figure 3b) indicated that platinum coordinates to MIF (80−82), in which Met80 has the highest affinity for the Pt(II) compounds. Therefore, Met65 and Met80 are the primary two binding sites for cisplatin on cytochrome c. Moreover, Met65 should be the primary binding site and Met80 should be the second binding site, based on two reasons as follows. First, at reaction time 9 h, the peaks of platinated peptide (61−73) were observed and no

Figure 3. SORI CID mass spectra of the platinated peptides [(61−73) + Pt(NH3)]2+ ions (a), [(80−86) + Pt(NH3) − H]2+ ions (b), [(14− 22) + Pt(NH3)Cl + H]2+ ions (c), and [(28−38) + Pt(NH3)2]2+ ions (d).

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Figure 4. Time courses of deuterium incorporation into intact cyt c (■) and cyt c−Pt(NH3)Cl (▲) (a), and into peptide 83−94 (b), peptide 95− 104 (c), and peptide 1−36 (d) before (■) and after (▲) the reaction of cyt c with cisplatin.

very useful for detecting conformational and structural changes of proteins and was often used in the interaction of small ligand and protein.51−53 For the system of cytochrome c and cisplatin, there was only one predominant monoadduct with the strongest relative abundance while the others’ were relatively weak. Therefore, the method of H/D exchange was used to estimate the change of the conformation of cytochrome c induced by the platination of primary binding site Met65. For the primary binding site Met65, platinum coordinates to the side chain and the hydrogen in the platinum(II) compound is similar to the hydrogen located on functional groups of the side chain and will back-exchange quickly during the quenched step.54,55 H/DX-MS of Intact Free and Platinated Cytochrome c. Deuterium incorporation was assessed for free and platinated cytochrome c by incubating the protein in D2O for various amounts of time and measuring the mass increase. Three independent experiments were performed; each included an undeuterated sample and 14 deuterated samples. The mass error associated with each time point ranged from ±0.5 to ±1.8 Da. None of the data presented here were adjusted for backexchange, so all results were reported at the relative deuterium level. Note that mass increases were reported only on backbone amide deuteration because side chain deuterium was backexchanged within 3 min of the quenched step. The results of global exchange on cytochrome c (Figure 4a) showed that approximately 40 residues exchanged within cytochrome c within 1 min and 51 residues exchanged after 150 min. For the platinated cytochrome c, although there were about 41 residues exchanged within 1 min, there were about 60 residues exchanged after 150 min. It needs about 30 min before the exchange to approach equilibrium gradually. Upon platination, the increase of the exchange rate of the platinated cytochrome c species implied that the flexibility of cytochrome c after platination may be increased. According to previous results, horse heart cytochrome c is “composed of

platinated peptide (80−86) was observed. Second, with reaction time extended to 24 h, compared with digested cytochrome c, the relative ion abundance of peptide 61−73 greatly decreased while that of peptide 80−86 only reduced a little. Other Binding Sites. Apart from the primary and second binding sites of cisplatin on cytochrome c, other platinated peptides such as cyt c (14−22) and (28−38) were also observed in Figure 2. [Pt(NH3)2]2+, [Pt(NH3)]2+, and Pt2+ were bound to peptide (28−38), while [Pt(NH3)Cl]+ and [Pt(NH3)]2+ were bound to peptide (14−22). However, the sensitivity of platinated peptide (14−22) was too low to be used for SORI CID. In order to improve the intensity of platinated peptide (14−22), platinated peptide (14−22) before the ultrafilter was selected for SORI CID. Figure 3c shows the SORI CID spectrum of the m/z 940.8 ion [(14−22) + Pt(NH3)Cl + H]2+ at reaction time 72 h for cisplatin and cytochrome c at molar ratio 1:12. The fragments of [(14−22) − heme + Pt − H]+ and oxidative heme were observed. Besides these ions, the ions y4+, [b5 − NH3 + Pt − 2H]+, and [b6 − H2O + Pt −2H]+ indicated that platinum coordinates to cyt c (14−18). Because of the existence of the heme, it is very difficult for the cysteine 14 and 17 to coordinate to Pt. Therefore, the most likely binding site in the peptide (14−22) is His18. Figure 3d shows the SORI CID spectrum of the peptide [(28−38) + Pt(NH3)2]2+ at m/z 698.3. Fragments of y4+, y5+, and [b6 + Pt − 2H]+ suggested that platinum coordinates to cyt c (28−33), in which His33 has the strongest affinity to the Pt(II) compounds. Therefore, the most likely binding site in the peptide (28−38) should be His33. On the basis of the above data, Met65 and Met80 in cytochrome c are the primary and second platinated binding sites, respectively. His18 and His33 are also binding sites for cisplatin on cytochrome c if the reaction time is long enough. Conformation Change of Cytochrome c. Hydrogen/ deuterium exchange mass spectrometry has been shown to be 6210

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data revealed that cyt c−Pt(NH3)Cl is the primary monoadduct and different molar ratios of cytochrome c/cisplatin may lead to different monoadducts. A bottom-up approach using FTICR MS provides the direct determination of binding sites of cisplatin on cytochrome c without complex liquid chromatography. Met65 and Met80 were identified as the primary and secondary binding sites, respectively. Besides, His18 and His33 were also identified to be binding sites. Global H/DX-MS showed that the platination of Met65 disturbed the conformation of cytochrome c and made it to be locally unfolded, while peptide-level H/DX-MS provided the region influenced by platination. This work also highlights the ability of H/DX-FTICR MS to provide insight into the minor conformation change of cytochrome c induced by cisplatin. This strategy can be applied to comprehensive investigation of metallodrug/protein complexes or adducts.

three major (6−14, 60−69, 87−102) and two minor (49−54, 70−75) helix elements interconnected by strands of polypeptide chain and folded into a roughly globular shape within which a heme pocket is formed”.8 On the basis of our results, the primary binding site of the Pt(NH3)Cl group in cytochrome c is Met65 which is in the center of the helix (60−69), and it may result in disturbing the intramolecular hydrogen bonds of the helix. Meanwhile, because of the covalent interaction of platinum species with cytochrome c, the platination may disturb other secondary structural elements whose space length is very short to the platinum coordinated to Met65 in space structure. Consequently, the platination of Met65 disturbs the conformation of cytochrome c to be locally unfolded and increases the H/D exchange rate of the monoadduct. Peptide-Level Hydrogen/Deuterium Exchange Analysis. In order to further confirm the above results, pepsin digestion methodology was employed to investigate the regions on cytochrome c influenced by the platination. Briefly, the free and platinated cytochrome c were exchanged at various time (1, 15, 30, 60 min) points in 5 mM deuterated NH4OAc buffer, respectively. Labeling was quenched by iced 2% CH3COOH− 20% CH3OH−H2O buffer, and the protein was digested by adding 4 mg/mL pepsin to a protein/enzyme ratio of 1:2 for 5 min, and then the samples were directly injected into the FTICR MS. As a control, free and unlabeled platinated cytochrome c were digested by pepsin under the same condition. For free and unlabeled platinated cytochrome c, the peptides were identified according to their accurate m/z values. The peptide coverage identified for free cytochrome c is 100% while that of platinated cytochrome c is 92%. The MS measurements are reproducible within 0.5 Da. The deuterium exchange dynamics of free and platinated cytochrome c at Met65 were examined to gain insight into the structural changes. After deuterium exchanged, the mass spectra became very complicated and some peptides could not be identified. The difference of H/D level was found in peptide (83−94) which showed about one more hydrogen was deuterated after being platinated (Figure 4b). Although this peptide is far from Met65, the side chain of Met65 is in close proximity to the amino acids Arg91 and Glu92 in the space, and maybe the platination disturbs the hydrogen bond of the helix of (87−102) and leads to the increase of H/D exchange rate in these peptides. Besides, the deuterated level of peptides (1−36) and (95−104) showed a very little difference which is in the error range after platination. However, no peptide that contained the primary binding site Met65 could be observed, and the change of the number of deuteriums incorporated of peptides detected is far less than those of the intact protein. Therefore, it is very possible that the platination of Met65 disturbed the hydrogen bond of the helix (60−69) and led to the increase of deuterated rate of intact protein after platination. In the previous studies,16,34 it was reported that cytochrome c did not unfold by interaction of cisplatin. According to our results, H/D exchange combined with FTICR MS provides the sensitive method for insight of the small conformation change of cytochrome c induced by cisplatin.



ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (No. 21103160) and the Science and Technology Development Project of Jilin Province (No. 201105032).



REFERENCES

(1) Jamieson, E. R.; Lippard, S. J. Chem. Rev. 1999, 99 (9), 2467− 2498. (2) Wang, D.; Lippard, S. J. Nat. Rev. Drug Discovery 2005, 4 (4), 307−320. (3) Cepeda, V.; Fuertes, M. A.; Castilla, J.; Alonso, C.; Quevedo, C.; Perez, J. M. Anti-Cancer Agents Med. Chem. 2007, 7 (1), 3−18. (4) Reedijk, J. Proc. Natl. Acad. Sci. U.S.A. 2003, 100 (7), 3611−3616. (5) Borch, R. F.; Pleasants, M. E. Proc. Natl. Acad. Sci. U.S.A. 1979, 76 (12), 6611−6614. (6) Timerbaev, A. R.; Hartinger, C. G.; Aleksenko, S. S.; Keppler, B. K. Chem. Rev. 2006, 106 (6), 2224−2248. (7) Bischin, C.; Lupan, A.; Taciuc, V.; Silaghi-Dumitrescu, R. Mini Rev. Med. Chem. 2011, 11 (3), 214. (8) Bushnell, G. W.; Louie, G. V.; Brayer, G. D. J. Mol. Biol. 1990, 214 (2), 585−595. (9) Kojima, H.; Endo, K.; Moriyama, H.; Tanaka, Y.; Alnemri, E. S.; Slapak, C. A.; Teicher, B.; Kufe, D.; Datta, R. J. Biol. Chem. 1998, 273 (27), 16647−16650. (10) Gonzalez, V. M.; Fuertes, M. A.; Alonso, C.; Perez, J. M. Mol. Pharmacol. 2001, 59 (4), 657−663. (11) Park, M. S.; De Leon, M.; Devarajan, P. J. Am. Soc. Nephrol. 2002, 13 (4), 858−865. (12) Gibson, D.; Costello, C. E. Eur. Mass Spectrom. 1999, 5 (6), 501−510. (13) Hu, W.; Luo, Q.; Wu, K.; Li, X.; Wang, F.; Chen, Y.; Ma, X.; Wang, J.; Liu, J.; Xiong, S.; Sadler, P. J. Chem. Commun. 2011, 47, 6006−6008. (14) Esteban-Fernández, D.; Montes-Bayón, M.; Blanco González, E.; Gómez-Gómez, M. M.; Palacios, M. A.; Sanz-Medel, A. J. Anal. At. Spectrom. 2008, 23 (3), 378−384.



CONCLUSIONS It has been demonstrated that FTICR MS provides a powerful tool to permit accurate characterization of cisplatin−cytochrome c interaction at a molecular level. High-resolution MS 6211

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Analytical Chemistry

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(15) Zhao, T.; King, F. L. J. Am. Soc. Mass Spectrom. 2009, 20 (6), 1141−1147. (16) Casini, A.; Gabbiani, C.; Mastrobuoni, G.; Messori, L.; Moneti, G.; Pieraccini, G. ChemMedChem 2006, 1 (4), 413−417. (17) Khalaila, I.; Allardyce, C. S.; Verma, C. S.; Dyson, P. J. ChemBioChem 2005, 6 (10), 1788−1795. (18) Zhao, Y. Y.; Mandal, R.; Li, X. F. Rapid Commun. Mass Spectrom. 2005, 19 (14), 1956−1962. (19) Zhao, T.; King, F. J. Biol. Inorg. Chem. 2011, 16 (4), 633−639. (20) Hartinger, C. G.; Tsybin, Y. O.; Fuchser, J.; Dyson, P. J. Inorg. Chem. 2008, 47 (1), 17−19. (21) Hartinger, C. G.; Ang, W. H.; Casini, A.; Messori, L.; Keppler, B. K.; Dyson, P. J. J. Anal. At. Spectrom. 2007, 22 (8), 960−967. (22) Moreno-Gordaliza, E.; Cañas, B.; Palacios, M. A.; GómezGómez, M. M. Anal. Chem. 2009, 81 (9), 3507−3516. (23) Moreno-Gordaliza, E.; Cañas, B.; Palacios, M. A.; GómezGómez, M. M. Analyst 2010, 135 (6), 1288−1298. (24) Li, H.; Lin, T. Y.; Van Orden, S. L.; Zhao, Y.; Barrow, M. P.; Pizarro, A. M.; Qi, Y.; Sadler, P. J.; O’Connor, P. B. Anal. Chem. 2011, 83 (24), 9507−9515. (25) Li, H.; Zhao, Y.; Phillips, H. I. A.; Qi, Y.; Lin, T. Y.; Sadler, P. J.; O’Connor, P. B. Anal. Chem. 2011, 83 (13), 5369−5376. (26) Casini, A.; Mastrobuoni, G.; Temperini, C.; Gabbiani, C.; Francese, S.; Moneti, G.; Supuran, C. T.; Scozzafava, A.; Messori, L. Chem. Commun. 2007, 2, 156−158. (27) Zhao, T.; King, F. L. J. Inorg. Biochem. 2010, 104 (2), 186−192. (28) Arnesano, F.; Banci, L.; Bertini, I.; Felli, I. C.; Losacco, M.; Natile, G. J. Am. Chem. Soc. 2011, 133 (45), 18361−18369. (29) Sze, C.; Khairallah, G.; Xiao, Z.; Donnelly, P.; O’Hair, R.; Wedd, A. J. Biol. Inorg. Chem. 2009, 14 (2), 163−165. (30) Crider, S. E.; Holbrook, R. J.; Franz, K. J. Metallomics 2010, 2 (1), 74−83. (31) Casini, A.; Gabbiani, C.; Mastrobuoni, G.; Pellicani, R. Z.; Intini, F. P.; Arnesano, F.; Natile, G.; Moneti, G.; Francese, S.; Messori, L. Biochemistry 2007, 46 (43), 12220−12230. (32) Moreno-Gordaliza, E.; Cañas, B.; Palacios, M. A.; GómezGómez, M. Talanta 2012, 88, 599−608. (33) Ivanov, A.; Christodoulou, J.; Parkinson, J.; Barnham, K.; Tucker, A.; Woodrow, J.; Sadler, P. J. Biol. Chem. 1998, 273 (24), 14721. (34) Palm, M. E.; Weise, C. F.; Lundin, C.; Wingsle, G.; Nygren, Y.; Björn, E.; Naredi, P.; Wolf-Watz, M.; Wittung-Stafshede, P. Proc. Natl. Acad. Sci. U.S.A. 2011, 108 (17), 6951−6956. (35) Neault, J. F.; Tajmir-Riahi, H. A. Biochim. Biophys. Acta 1998, 1384 (1), 153−159. (36) Williams, J.; Phillips, H.; Campuzano, I.; Sadler, P. J. Am. Soc. Mass Spectrom. 2010, 21 (7), 1097−1106. (37) Katta, V.; Chait, B. T.; Carr, S. Rapid Commun. Mass Spectrom. 1991, 5 (4), 214−217. (38) Engen, J. R. Anal. Chem. 2009, 81 (19), 7870−7875. (39) Kaltashov, I. A.; Eyles, S. J. Mass Spectrom. Rev. 2002, 21 (1), 37−71. (40) Kaltashov, I. A.; Bobst, C. E.; Abzalimov, R. R. Anal. Chem. 2009, 81 (19), 7892−7899. (41) Wales, T. E.; Engen, J. R. Mass Spectrom. Rev. 2006, 25 (1), 158−170. (42) Garcia, R. A.; Pantazatos, D.; Villarreal, F. J. Assay Drug Dev. Technol. 2004, 2 (1), 81−91. (43) Gajiwala, K. S.; Wu, J. C.; Christensen, J.; Deshmukh, G. D.; Diehl, W.; DiNitto, J. P.; English, J. M.; Greig, M. J.; He, Y. A.; Jacques, S. L.; Lunney, E. A.; McTigue, M.; Molina, D.; Quenzer, T.; Wells, P. A.; Yu, X.; Zhang, Y.; Zou, A. H.; Emmett, M. R.; Marshall, A. G.; Zhang, H. M.; Demetri, G. D. Proc. Natl. Acad. Sci. U.S.A. 2009, 106 (5), 1542−1547. (44) Seyfried, N. T.; Atwood, J. A.; Yongye, A.; Almond, A.; Day, A. J.; Orlando, R.; Woods, R. J. Rapid Commun. Mass Spectrom. 2007, 21 (2), 121−131. (45) Kasherman, Y.; Sturup, S.; Gibson, D. J. Biol. Inorg. Chem. 2009, 14 (3), 387−399.

(46) Lau, J. K.-C.; Deubel, D. V. Chem.Eur. J. 2005, 11 (9), 2849− 2855. (47) Gibson, D.; Kasherman, Y.; Kowarski, D.; Freikman, I. J. Biol. Inorg. Chem. 2006, 11 (2), 179−188. (48) Cui, M.; Ding, L.; Mester, Z. Anal. Chem. 2003, 75 (21), 5847− 5853. (49) Cui, M.; Mester, Z. Rapid Commun. Mass Spectrom. 2003, 17 (14), 1517−1527. (50) Chval, Z.; Sip, M.; Burda, J. V. J. Comput. Chem. 2008, 29 (14), 2370−2381. (51) Wang, F.; Li, W. Q.; Emmett, M. R.; Marshall, A. G.; Corson, D.; Sykes, B. D. J. Am. Soc. Mass Spectrom. 1999, 10 (8), 703−710. (52) Houde, D.; Arndt, J.; Domeier, W.; Berkowitz, S.; Engen, J. R. Anal. Chem. 2009, 81 (7), 2644−2651. (53) Zhu, M. M.; Rempel, D. L.; Du, Z.; Gross, M. L. J. Am. Chem. Soc. 2003, 125 (18), 5252−5253. (54) Zhang, Z.; Smith, D. L. Protein Sci. 1993, 2 (4), 522−531. (55) Esswein, S. T.; Florance, H. V.; Baillie, L.; Lippens, J.; Barran, P. E. J. Chromatogr., A 2010, 1217 (43), 6709−6717.

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dx.doi.org/10.1021/ac301122w | Anal. Chem. 2012, 84, 6206−6212