Article pubs.acs.org/ac
Combined Use of Ion Mobility and Collision-Induced Dissociation To Investigate the Opening of Disulfide Bridges by Electron-Transfer Dissociation in Peptides Bearing Two Disulfide Bonds Philippe Massonnet,† Gregory Upert,‡ Nicolas Smargiasso,† Nicolas Gilles,‡ Loïc Quinton,† and Edwin De Pauw*,† †
Laboratory of Mass Spectrometry, Department of Chemistry, GIGA-R, University of Liege, Allée de la Chimie 3, B-4000 Liege, Belgium ‡ Commissariat à l’Energie Atomique, DSV/iBiTec-S/SIMOPRO, F91191 Gif-sur-Yvette, France S Supporting Information *
ABSTRACT: Disulfide bonds are post-translational modifications (PTMs) often found in peptides and proteins. They increase their stability toward enzymatic degradations and provide the structure and (consequently) the activity of such folded proteins. The characterization of disulfide patterns, i.e., the cysteine connectivity, is crucial to achieve a global picture of the active conformation of the protein of interest. Electrontransfer dissociation (ETD) constitutes a valuable tool to cleave the disulfide bonds in the gas phase, avoiding chemical reduction/alkylation in solution. To characterize the cysteine pairing, the present work proposes (i) to reduce by ETD one of the two disulfide bridges of model peptides, resulting in the opening of the cyclic structures, (ii) to separate the generated species by ion mobility, and (iii) to characterize the species using collision-induced dissociation (CID). Results of this strategy applied to several peptides show different behaviors depending on the connectivity. The loss of SH· radical species, observed for all the peptides, confirms the cleavage of the disulfides during the ETD process.
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different excitation methods such as UV photons,17,18 radical oxygen species (ROS), 19 electron-capture-dissociation (ECD),20 or electron-transfer dissociation (ETD).21 Electron-based techniques (ETD22 and ECD23) have been widely exploited due to the high electron affinity of the S−S bond24,25 and to their ability to maintain labile posttranslational modifications.26,27 The efficiency of the ETD is, however, limited by a side reaction that transfers one proton from the peptide to the ETD reagent. This reaction, that competes with the electron transfer,28−30 leads to a decrease of the ETD efficacy (Figure 1). In addition to what can be found in Figure 1, Cole et al.31 showed that backbone cleavages occur on peptides bearing a single disulfide bridge by a two-step process. ETD reagent transfers first an electron to the peptide, creating a backbone radical that reacts in a second step with the disulfide bond. The result of this reaction is the generation of specific losses from cand z-ion types (c − 33, c + 32, z, z − 32, z + 33).31 Tan et al. applied ETD to assign cysteine pairing in peptides bearing two disulfide bonds.32 They confirmed the occurrence of the mechanism described by Cole et al.
isulfide bonds are post-translational modifications (PTMs) ensuring the stability of the structure and the activity of peptides or proteins.1,2 Intrachain disulfide bonds are encountered in many natural peptides including defensines,3 cyclotides,4,5 or many animal toxins.6,7 The identification of cysteine pairing is still very challenging in the presence of multiple disulfide bridges. Therefore, different protocols have been developed based on a combination of enzymatic digestion, chromatographic separation, and mass spectrometry analysis.7−9 They suffer several drawbacks such as the amount of sample needed to perform the characterization or the lack of enzymatic cleavage sites between the cysteines. The cleavage sites are mandatory to identify the connectivity. Another problem often encountered in the study of the cysteine pairing is the occurrence of disulfide bonds scrambling. Scrambling has already been observed in solution,10,11 in MALDI12 (matrixassisted laser desorption ionization), as well as in ESI analysis (electrospray ionization).13 An elegant way to overcome part of these problems consists in performing the analysis of disulfide-bridged peptides directly in the gas phase. Previous studies focused on the reactivity of peptides containing one disulfide bond regarding to collisioninduced dissociation (CID).14−16 These studies pointed out the fact that symmetric16 cleavages (i.e., at the S−S bond) as well as asymmetric15,16 cleavages (at the C−S bond) can occurs. Disulfide bond gas-phase reactivity was also tested using © XXXX American Chemical Society
Received: January 19, 2015 Accepted: April 27, 2015
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DOI: 10.1021/acs.analchem.5b00245 Anal. Chem. XXXX, XXX, XXX−XXX
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Figure 1. Reaction pathways when ETD is performed on a peptide containing one S−S bond (ref 21).
protection except for cysteine residues, on a Prelude synthesizer (Protein Technologies) using ChemMatrix Rink amide resin (Sigma-Aldrich, St. Quentin en Fallavier). HCTU (Activotec, Cambridge, U.K.) was used as a coupling reagent in the presence of N-methylmorpholine (NMM, Sigma-Aldrich, St. Quentin en Fallavier). Each coupling step was carried out twice for 5 min using a mixture 10 equiv/10 equiv/20 equiv FmocAA/HCTU/NMM in N-methyl-2-pyrrolidinone (NMP, 0.1 M final) followed by a capping step of 5 min with a solution of Ac2O/NMM 1/1 v/v in NMP (2.5 mL). Fmoc deprotection was performed twice for 2 min using 2 mL of a 20% piperidine solution in NMP. Cysteine residues were protected in pairs with either S-trityl (Trt) or S-acetamidomethyl (Acm) groups, see Table 1. The peptides were cleaved from the resin and unprotected using the cocktail TFA/H2O/anisole/TIS 87.5/5/ 5/2.5 (8 mL per 50 mg of resin). The peptides were then precipitated in cold Et2O and centrifuged (three cycles). The linear peptide bearing free cysteine residue (Trt-protected during the synthesis) and Acm-protected was then purified by high-performance liquid chromatography (HPLC). A two-step oxidation protocol was used to selectively fold the peptides. The first disulfide bridge was formed by dilution of the peptide (final concentration of 20 μM) into 0.1 M ammonium carbonate buffer, pH 7.5, containing 10% DMSO for one night, and the cyclic peptide was purified by reversed-phase HPLC and lyophilized. Simultaneous deprotection and cyclization of the second disulfide bridge was carried out for 1 h using 10 equiv of I2 in acetic acid/water 4/1 v/v mixture (1 mg of peptide/mL). The solution was 3-fold diluted using water and washed three times with equal volume of CH2Cl2, and the final peptide was obtained after HPLC purification. Reagents. Formic Acid (FA) was purchased from SigmaAldrich (Saint Louis, MO, United States), and acetonitrile (ACN) was purchased from Biosolve (Dieuze, France). Peptide 1 was analyzed at a concentration of 1 μM in a mixture of FA 0.1%/ACN 50/50 (v/v), while peptides 2, 3, and 4 were analyzed at a concentration of 5 μM in the same mixture of solvent. Mass Spectrometry. Instrument and Ionization Settings. All the experiments were performed on a SYNAPT-G2 mass spectrometer (Waters, Manchester, United Kingdom) in the “resolution mode” equipped with an electrospray ion source. Capillary and sampling cones were set at 3 kV and 10 V, respectively.
To investigate more deeply the ETD-generated species, ion mobility seems useful. Ion mobility mass spectrometry has already been described as an efficient tool to achieve the separation of partially opened species.13 In this previous study, oxidation and partial reduction of the disulfides bonds were performed in solution. “In solution” incomplete reductions highlighted unfortunately disulfide scrambling, making the attribution of the cysteine pairings tricky. On the basis of these results, a workflow was designed to avoid the limitations stressed above. Our study aims at combining the opening one of the two disulfide bond using ETD, then isolating the species using ion mobility and characterizing finally each of them by CID. This workflow obviously assumes that disulfide opening in the gas phase limits/avoids disulfide scrambling, and this aspect was evaluated. The proof of concept of this strategy is first demonstrated using a synthetic peptide (named as P1) that was designed for this purpose. P1 bears two independent disulfide bonds, forming one small loop (8 amino acids) and a larger one (15 amino acids). With this design, the opening of the bigger loop should cause a larger collision cross section (CCS) variation than the one of the smaller loop. The cross section of the two partially opened structures should be different enough to allow their separation by ion mobility spectrometry (IMS) and their individual MS/MS using CID. On the basis of these expected results, other peptides displaying various cysteine connectivities have been analyzed as well.
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MATERIALS AND METHODS Materials. Peptide Synthesis. The peptides used in this study are listed in Table 1. Peptides were synthesized on a 25 μmol scale using Fmoc chemistry standard side chain Table 1. List of the Peptides Used in This Studya
A * indicates a C‑ter amidation. Cysteines are presented in bold and Acm-protected cysteines (during synthesis) are depicted in red.
a
B
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Figure 2. Workflow used in this study. This workflow has been performed on a SYNAPT-G2 mass spectrometer.
Figure 3. (I) ATD of the 3+ species of the peptide 1 before and after ETD; corresponding possible structures are depicted above the peaks. (II) Corresponding isotopic profiles and theoretical isotopic profiles of the “ETD opened” and “ETD not opened” species.
Electron-Transfer Dissociation, Ion Mobility, and CID Fragmentation. ETD reaction was performed before the ion mobility cell, in the so-called “trap cell”. The signal intensity of bicyanobenzene, used as the ETD reagent, was optimized according to the manufacturer’s protocol (to reach roughly 106 in the glow discharge mode) before each reaction. In each case, the ETD reaction considered was the one that reduces the peptide from the 4+ to the 3+ charge state. The ion mobility contribution of the 3+ species is always analyzed in the following work. For all peptides, the wave velocity and the wave height in the ion mobility cell were, respectively, set at 1250 m/ s and 40 V, while the wave height and the wave velocity in the transfer cell (just after the ion mobility cell) were, respectively, settled at 508 m/s and 0.1 V. The wave velocity in the trap was fixed at 300 m/s while, for each peptide, the trap wave height had to be optimized. For the peptide 1, the IMS cell pressure (N2) was 1.69 mbar while the trap and the transfer pressures were 0.756 mbar. For peptides 2, 3, and 4, IMS pressure (N2) was 1.52 mbar while the trap and transfer pressure were 0.756 mbar. For all experiments, the He cell pressure was of 0.16 mbar. CID fragmentation was performed in the transfer cell. The accelerating voltage in this cell was set between 35 and 45 V depending of the peptide. For all the experiments involving ETD and ion mobility, the trapping release time, the mobility trap height, and mobility extract height parameters were, respectively, of 100 μs and 0.5 and 40 V. A summary of what has been done in the SYNAPT-G2 is depicted in Figure 2.
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Figure 4. Arrival time distribution (ATD) of the 3+ species of the peptide 1. An isotopic deconvolution of the 3+ charge state has been performed at each drift time in order to have access, at each drift time, to the ratio proton transfer/electron transfer.
P3, and P4), aiming at studying the differences in drift times induced by different cysteine connectivities in a same peptide sequence. Part 1: Proof of Concept on a Designed Peptide. The ETD reaction was performed on the 4+ charge state, and the total arrival time distribution (ATD) of the resulting 3+ ions was recorded. To visualize the effect of ETD on disulfide-bonded peptides, the ATD of the 3+ species (after ETD reaction on the 4+) was compared to the 3+ of the native peptide (without ETD). This comparison, depicted in Figure 3, shows the appearance of species with higher drift times (i.e., larger collisional cross sections) when ETD is performed (peaks B
RESULTS AND DISCUSSION
Results. The first part of the results is devoted to the peptide 1 (P1, Table 1), this peptide being a model peptide used for the demonstration of the proof of concept. The second part of the discussion is focused on the peptides 2, 3, and 4 (P2, C
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Figure 5. CID spectra of the ion mobility-separated species (35 V CID) for the peptide 1. The ATD of the reduced species (3+ charge state) is presented in the upper part. The corresponding CID spectra are presented below. (Remark: the CID experiment has also been performed on the 3+ obtained without ETD. The corresponding spectrum is similar to the spectrum A). Structures that are presented are for the purpose of illustration.
and C on Figure 3). The analysis of isotopic profiles reveals a shift of one mass unit compared to the nonreduced peptide. These observations, regarding Figure 1, may be linked to a partial opening of the peptide structure (opening of one of the two disulfide bridges). The B peak could be associated with the opening of the small loop, while the C peak could be associated with the opening of the large loop (Figure 3). To study the nature of the species building the ATD distribution, each isotopic profile has been deconvoluted in order to determine the relative contribution of the protontransfer reaction (close species) compared to disulfide bond opening (partially open species) (Figure 4). This figure shows that the peak A (around 4.2 ms) is mainly composed of a proton-transfer species (about 90%) while B and C peaks are mainly composed of disulfide-bonded partially opened species. Isotopic analysis at ATD contribution shows that the first peak around 4.2 ms (Figure 3, peak A) is mainly constituted of a proton-transfer product (in blue). This species is identified, with a high confidence, to the fully folded peptide, displaying a reduced charge state. On the other hand, the peaks at 4.4 ms
(Figure 3, peak B) and 4.8 ms (Figure 3, peak C) are mainly due to partially disulfide-opened species (in green). According to this observation and to the structure of the peptide 1, several hypotheses are proposed: (i) the species A should correspond to the folded species, the most compact structure; (ii) the species B should be linked to the peptide with the smaller loop opened (C3−C4), creating a small increase of CCS and the corresponding arrival time; (iii) the species C should represent the peptide with the larger loop opened (C1−C2), displaying a larger variation of CCS, visualized by an important increase in drift time (∼0.6 ms). To verify these hypotheses, CID experiments were performed after the ion mobility separation on each of the three contributions A, B, and C setting the collision voltage at 35 V (Figure 5). According to the resulting tandem mass spectrum (MS/MS spectrum), the fragmentation pattern of the peak A is in agreement with a species that still has the two disulfide bonds intact (Figure 5A). Indeed, the two disulfides strongly stabilize the peptide resulting in the occurrence of only few fragments as D
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formation. The MS/MS spectrum extracted from the peak C (Figure 5C) is in agreement with the opening of the disulfide bond between C1 and C2. Indeed, the masses of the different fragments perfectly fit with the supposed sequence, i.e., classical y- and b-ions (and y-1 and b-1) and atypical a-2, b-2, and y-2 fragments for the sequence still embedded in the remaining disulfide. These observations clearly reinforce the initial assumptions. The variation of CCS observed by the opening of only one disulfide bridge is sufficient enough to isolate the partially opened and the proton-transfer species. The online MS/MS experiments provide enough information to confirm the identity of the different ETD-generated species. It is important to note that a loss of 33 mass units is observed from the [M + 4H]3+• species in the fragmentation spectra B and C. The mobility profiles of this species, as well as other selected fragment ions, are presented in Figure 6. This loss is associated with the generation of a dehydroalanine residue after the electron-transfer pathway presented in Figure 1.31 In this case, the loss of 33 Da confirms the presence of sulfur radicals in the peaks B and C and then the opening of the S−S bond by ETD. Figure 6 also presents the contributions of other fragment ions to the ATD. The results show that the b13 and y11 ions are mainly present in the peak C, confirming the opening of the disulfide bond between C1 and C2. This figure also shows that the b20 ion is mainly identified in the peak B, confirming the opening of the disulfide bond between C3 and C4 for this species. The presence of the specific [a6 − 2]+ species in the B peak additionally indicates that the remaining disulfide bond after ETD reaction (C3−C4) has been cleaved by CID (generation of thioaldehyde).33 The pathway that leads to the generation of this species is explained in the Supporting Information Figure SI 1. Figure 6 also reveals that the [M + 4H + 33]3+ species can be found in the peaks B and C, which confirms that they are both partially opened species. Influence of Disulfide Bond Connectivity on ETD-IMS Experiments. To investigate the influence of disulfide bond connectivity on ETD-IMS experiments, three peptides displaying the same amino acid sequence but three different cysteine connectivities were synthesized (Table 1). Each isoform was analyzed according to the same ETD-IMS-CID workflow previously described (Figure 7). From a general point of view, the three ATDs present roughly the same profile. They show a large and intense peak (Figure 7). However, the isotopic distribution analysis of the ATD clearly highlights different behaviors of the three species (Figure 7b). Case of Peptide 2 (C1−C2/C3−C4). For the peptide 2, isotopic deconvolution at each drift time shows that the first peak around 3.4 ms (in blue) is composed of a mixture of proton-transfer (in red) and electron-transfer products (see Figure 7). The CID spectra of each ATDs (Supporting Information Figure SI 2) confirms and demonstrates also that the peak around 3.8 ms is mainly composed of an opened species (in green). This open species is associated with the opening of the C1−C2 disulfide bridge, because (i) this bridge is the biggest of the two and is supposed to cause a significant drift time (DT) variation after reduction (ΔDT ≈ 0.4 ms) and (ii) several characteristic fragment ions such as y14+ are mainly found in the peak at 3.8 ms (see Supporting Information Figure SI 3). However, for the y15+ ion, the situation is less clear and seems to show a more complex situation in which the resolution of the separation is not sufficient to distinguish
Figure 6. ATD contributions of selected peaks depicted in Figure 5. All the contributions are compared to the total ATD contribution of the ETD charge-reduced species.
expected for such peptides fragmented under low-energy CID. The spectrum of the peak B and the peak C display many more fragment ions (b20, b21, y5 for peak B and y8, y10, y11, y13, y14, y17, y21, y22, b4, b10, b11, b13 for the C peak), indicating the probable opening of one disulfide bridge, as deduced from the ATD changes (Figure 4). The MS/MS spectrum extracted from the peak B contains less fragment ions than the one from the peak C. In this peptide, the opening of the larger loop will create a free chain of 15 amino acids that would probably lead to a longer sequence tag by CID. Moreover, for the peak B, few fragmentations are detected originating from the remaining disulfide bond (y6-2, b6-2). The pathways that leads to these ions are depicted in the Supporting Information (Figure SI 1).33 These ions are generated by the homolytic cleavage of the S−S bond followed by a backbone cleavage and a thioaldehyde E
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Figure 7. (a) ATD of the 3+ species of the peptides 2, 3, and 4 without ETD. (b) Deconvoluted ATD for the same peptide after charge reduction (4+ to 3+) using ETD. The black line represent the total ATD contribution, the blue line represent the proton-transfer contribution, and the green line represent the partially disulfide-opened species.
is quite weak; proton transfer and disulfide bond opened species are superimposed in the ATD. This result is supported by the CID fragmentation spectrum of this peptide (see Supporting Information Figure SI 6) in which the opening of the C1−C4 connectivity is detected. This property can also be seen as a signature of C1−C4/C2−C3 connectivity, also referred as to the “hairpin fold”. In all cases, presence of 33 Da neutral loss confirms the opening of the disulfides, and the intensity of this ion can be used to monitor this reaction.31 No scrambling phenomenon was detected during this study. This could suggest that the reaction time scale is not sufficient to visualize or to generate scrambling. Indeed, using this experimental setup, the reaction time is only around 100 μs while ETD reactions in ion trap can go to 100 ms long. One other explanation could come from the fact that the energy given to the system is not high enough to perform scrambling. The methodology developed in this paper could be used to determine the cysteine connectivity of a natural occurring peptide bearing two disulfide bonds. Most of these peptides having a 1,3/2,4 or a 1,4/2,3 connectivity.
clearly between the proton-transfer and the electron-transfer products. For the −33 Da neutral loss, it is detected more abundant in the peak at 3.8 ms than in the one at 3.4 ms (data not shown). Case of Peptide 3 (C1−C3/C2−C4). Concerning the peptide 3, Figure 7 shows that the peaks at 3.4 ms and 3.8 ms are mainly composed of a proton-transfer product (in blue) and an electron-transfer product (partially opened species, in green), respectively. CID experiments demonstrate that the peak at 3.8 ms is clearly associated with the opening of the C1−C3 disulfide bond (Supporting Information Figure SI 4). ATD contributions of some fragment ions, depicted in the Supporting Information (Figure SI 5), show that a wide range of ions are specific to the 3.8 ms peak (y15+, y14+, y13+, b13+). Contribution of the −33 Da ion indicates that the two partially opened structures can be identified (see Supporting Information Figure SI 5). Case of Peptide 4 (C1−C4/C2−C3). Figure 7 shows that proton-transfer and electron-transfer products are not wellresolved using ion mobility. The isotopic deconvolution confirms that the partial reduced species is shifted of only 0.1 ms from the proton-transfer product. Fragmentation experiments reveal some ions (b132+, y13+, y14+, y15+, ...) indicating the presence of a partially opened peptides (see Supporting Information Figure SI 6). Discussion. For all peptides, the electron transfer cleaves disulfide bonds. The reduction of such bonds is demonstrated with the peptide 1, particularly suited for the presented workflow because C2 and C3 are vicinal and C1−C2/C3−C4 connectivity establishes two loops of different lengths. The opening of one of the two disulfides creates differences in CCS that are enough to separate the species in our experimental conditions of IMS. Online CID is, however, required to characterize unambiguously each of the generated species. For the peptides 2 and 3, only the opening of the largest loop can be clearly observed, especially for the peptide 3. Nevertheless, for peptides bearing two disulfide bonds, the information about one bond is enough to perform the cysteine connectivity identification. Concerning the peptide 4, the drift time variation
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CONCLUSIONS
The aim of this study was to develop a new gas-phase method for disulfide bond assignment of peptides bearing two disulfide bonds. Electron-transfer dissociation being able to cleave the disulfide bonds in the gas phase, this method was based on a combined use of ETD, ion mobility, and CID. To develop this technique, four peptides displaying various cysteine connectivities where used in order to visualize the efficiency of the technique depending on the connectivity. Ion mobility clearly showed an opening of structure when ETD is performed on a disulfide bond peptide (thanks to a comparison of the ATDs obtained with and without ETD). Concerning the 1,3/2,4 pattern, opening of the larger disulfide bond is clearly detected, revealing the advantage of using ion mobility to separate proton-transfer and electron-transfer species. CID fragments, resolved in ion mobility, provide enough structural information to confirm the structures and the F
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cysteine pairing. Disulfide bond assignment of a 1,2/3,4 pattern peptide has been also successfully achieved. However, the opening of one disulfide bond in a hairpin-fold peptide (1,4/ 2,3) is not clearly visualized in our experimental conditions. This result, assimilated to a signature of such peptides, can nevertheless be exploited to identify hairpin-fold peptides. In all experiments, neutral losses of 33 (SH) Da indicate the opening of an S−S bond after the ETD reaction. There is a place for method improvement. For example, ion mobility suffers of limited resolution that could be increased to visualize smaller CCS variations. ETD reaction efficiency can also be improved, by adjusting the reaction time (ion traps) or the nature of the ETD reagent, in order to obtain a more favorable electron-transfer/proton-transfer ratio. The workflow developed will be applied soon on peptides bearing three disulfide bonds. The biggest challenge for this type of peptide is that, after the reduction of one disulfide, two disulfide bonds remain in the sequence and three different connectivities are still possible. Species with two disulfideopened species have to be isolated, and ion mobility spectrometry has to have a sufficient resolution to isolate these partially opened species. Another possibility could take into account the reduction, by ETD, of two disulfides among the three. This system, more or less similar to our previous work, could probably be easier to characterize, but it is highly dependent on the reduction efficiency. However, after ETD, a mixture of peptides displaying three, two, and one disulfide bonds would be probably produced in various amounts. This method could also be tested in the future on proteins. The biggest challenge would be to deal with the large amounts of disulfide bonds found in proteins, IMS resolution and ETD efficiency being currently the limiting parameters of our methodology.
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b00245.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank the FRS-FNRS for the financial support (FRIA and instrumentation), the Fonds Européen de développement regional (FEDER), the Walloon region, and the European commission (F.P. 7 VENOMICS project) for financial support.
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REFERENCES
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Combined Use of Ion Mobility and Collision-Induced Dissociation To Investigate the Opening of Disulfide Bridges by Electron-Transfer Dissociation in Peptides Bearing Two Disulfide Bonds Philippe Massonnet,† Gregory Upert,‡ Nicolas Smargiasso,† Nicolas Gilles,‡ Loïc Quinton,† and Edwin De Pauw*,† †
Laboratory of Mass Spectrometry, Department of Chemistry, GIGA-R, University of Liege, Allée de la Chimie 3, B-4000 Liege, Belgium ‡ Commissariat à l’Energie Atomique, DSV/iBiTec-S/SIMOPRO, F91191 Gif-sur-Yvette, France S Supporting Information *
ABSTRACT: Disulfide bonds are post-translational modifications (PTMs) often found in peptides and proteins. They increase their stability toward enzymatic degradations and provide the structure and (consequently) the activity of such folded proteins. The characterization of disulfide patterns, i.e., the cysteine connectivity, is crucial to achieve a global picture of the active conformation of the protein of interest. Electrontransfer dissociation (ETD) constitutes a valuable tool to cleave the disulfide bonds in the gas phase, avoiding chemical reduction/alkylation in solution. To characterize the cysteine pairing, the present work proposes (i) to reduce by ETD one of the two disulfide bridges of model peptides, resulting in the opening of the cyclic structures, (ii) to separate the generated species by ion mobility, and (iii) to characterize the species using collision-induced dissociation (CID). Results of this strategy applied to several peptides show different behaviors depending on the connectivity. The loss of SH· radical species, observed for all the peptides, confirms the cleavage of the disulfides during the ETD process.
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different excitation methods such as UV photons,17,18 radical oxygen species (ROS), 19 electron-capture-dissociation (ECD),20 or electron-transfer dissociation (ETD).21 Electron-based techniques (ETD22 and ECD23) have been widely exploited due to the high electron affinity of the S−S bond24,25 and to their ability to maintain labile posttranslational modifications.26,27 The efficiency of the ETD is, however, limited by a side reaction that transfers one proton from the peptide to the ETD reagent. This reaction, that competes with the electron transfer,28−30 leads to a decrease of the ETD efficacy (Figure 1). In addition to what can be found in Figure 1, Cole et al.31 showed that backbone cleavages occur on peptides bearing a single disulfide bridge by a two-step process. ETD reagent transfers first an electron to the peptide, creating a backbone radical that reacts in a second step with the disulfide bond. The result of this reaction is the generation of specific losses from cand z-ion types (c − 33, c + 32, z, z − 32, z + 33).31 Tan et al. applied ETD to assign cysteine pairing in peptides bearing two disulfide bonds.32 They confirmed the occurrence of the mechanism described by Cole et al.
isulfide bonds are post-translational modifications (PTMs) ensuring the stability of the structure and the activity of peptides or proteins.1,2 Intrachain disulfide bonds are encountered in many natural peptides including defensines,3 cyclotides,4,5 or many animal toxins.6,7 The identification of cysteine pairing is still very challenging in the presence of multiple disulfide bridges. Therefore, different protocols have been developed based on a combination of enzymatic digestion, chromatographic separation, and mass spectrometry analysis.7−9 They suffer several drawbacks such as the amount of sample needed to perform the characterization or the lack of enzymatic cleavage sites between the cysteines. The cleavage sites are mandatory to identify the connectivity. Another problem often encountered in the study of the cysteine pairing is the occurrence of disulfide bonds scrambling. Scrambling has already been observed in solution,10,11 in MALDI12 (matrixassisted laser desorption ionization), as well as in ESI analysis (electrospray ionization).13 An elegant way to overcome part of these problems consists in performing the analysis of disulfide-bridged peptides directly in the gas phase. Previous studies focused on the reactivity of peptides containing one disulfide bond regarding to collisioninduced dissociation (CID).14−16 These studies pointed out the fact that symmetric16 cleavages (i.e., at the S−S bond) as well as asymmetric15,16 cleavages (at the C−S bond) can occurs. Disulfide bond gas-phase reactivity was also tested using © XXXX American Chemical Society
Received: January 19, 2015 Accepted: April 27, 2015
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Figure 1. Reaction pathways when ETD is performed on a peptide containing one S−S bond (ref 21).
protection except for cysteine residues, on a Prelude synthesizer (Protein Technologies) using ChemMatrix Rink amide resin (Sigma-Aldrich, St. Quentin en Fallavier). HCTU (Activotec, Cambridge, U.K.) was used as a coupling reagent in the presence of N-methylmorpholine (NMM, Sigma-Aldrich, St. Quentin en Fallavier). Each coupling step was carried out twice for 5 min using a mixture 10 equiv/10 equiv/20 equiv FmocAA/HCTU/NMM in N-methyl-2-pyrrolidinone (NMP, 0.1 M final) followed by a capping step of 5 min with a solution of Ac2O/NMM 1/1 v/v in NMP (2.5 mL). Fmoc deprotection was performed twice for 2 min using 2 mL of a 20% piperidine solution in NMP. Cysteine residues were protected in pairs with either S-trityl (Trt) or S-acetamidomethyl (Acm) groups, see Table 1. The peptides were cleaved from the resin and unprotected using the cocktail TFA/H2O/anisole/TIS 87.5/5/ 5/2.5 (8 mL per 50 mg of resin). The peptides were then precipitated in cold Et2O and centrifuged (three cycles). The linear peptide bearing free cysteine residue (Trt-protected during the synthesis) and Acm-protected was then purified by high-performance liquid chromatography (HPLC). A two-step oxidation protocol was used to selectively fold the peptides. The first disulfide bridge was formed by dilution of the peptide (final concentration of 20 μM) into 0.1 M ammonium carbonate buffer, pH 7.5, containing 10% DMSO for one night, and the cyclic peptide was purified by reversed-phase HPLC and lyophilized. Simultaneous deprotection and cyclization of the second disulfide bridge was carried out for 1 h using 10 equiv of I2 in acetic acid/water 4/1 v/v mixture (1 mg of peptide/mL). The solution was 3-fold diluted using water and washed three times with equal volume of CH2Cl2, and the final peptide was obtained after HPLC purification. Reagents. Formic Acid (FA) was purchased from SigmaAldrich (Saint Louis, MO, United States), and acetonitrile (ACN) was purchased from Biosolve (Dieuze, France). Peptide 1 was analyzed at a concentration of 1 μM in a mixture of FA 0.1%/ACN 50/50 (v/v), while peptides 2, 3, and 4 were analyzed at a concentration of 5 μM in the same mixture of solvent. Mass Spectrometry. Instrument and Ionization Settings. All the experiments were performed on a SYNAPT-G2 mass spectrometer (Waters, Manchester, United Kingdom) in the “resolution mode” equipped with an electrospray ion source. Capillary and sampling cones were set at 3 kV and 10 V, respectively.
To investigate more deeply the ETD-generated species, ion mobility seems useful. Ion mobility mass spectrometry has already been described as an efficient tool to achieve the separation of partially opened species.13 In this previous study, oxidation and partial reduction of the disulfides bonds were performed in solution. “In solution” incomplete reductions highlighted unfortunately disulfide scrambling, making the attribution of the cysteine pairings tricky. On the basis of these results, a workflow was designed to avoid the limitations stressed above. Our study aims at combining the opening one of the two disulfide bond using ETD, then isolating the species using ion mobility and characterizing finally each of them by CID. This workflow obviously assumes that disulfide opening in the gas phase limits/avoids disulfide scrambling, and this aspect was evaluated. The proof of concept of this strategy is first demonstrated using a synthetic peptide (named as P1) that was designed for this purpose. P1 bears two independent disulfide bonds, forming one small loop (8 amino acids) and a larger one (15 amino acids). With this design, the opening of the bigger loop should cause a larger collision cross section (CCS) variation than the one of the smaller loop. The cross section of the two partially opened structures should be different enough to allow their separation by ion mobility spectrometry (IMS) and their individual MS/MS using CID. On the basis of these expected results, other peptides displaying various cysteine connectivities have been analyzed as well.
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MATERIALS AND METHODS Materials. Peptide Synthesis. The peptides used in this study are listed in Table 1. Peptides were synthesized on a 25 μmol scale using Fmoc chemistry standard side chain Table 1. List of the Peptides Used in This Studya
A * indicates a C‑ter amidation. Cysteines are presented in bold and Acm-protected cysteines (during synthesis) are depicted in red.
a
B
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Figure 2. Workflow used in this study. This workflow has been performed on a SYNAPT-G2 mass spectrometer.
Figure 3. (I) ATD of the 3+ species of the peptide 1 before and after ETD; corresponding possible structures are depicted above the peaks. (II) Corresponding isotopic profiles and theoretical isotopic profiles of the “ETD opened” and “ETD not opened” species.
Electron-Transfer Dissociation, Ion Mobility, and CID Fragmentation. ETD reaction was performed before the ion mobility cell, in the so-called “trap cell”. The signal intensity of bicyanobenzene, used as the ETD reagent, was optimized according to the manufacturer’s protocol (to reach roughly 106 in the glow discharge mode) before each reaction. In each case, the ETD reaction considered was the one that reduces the peptide from the 4+ to the 3+ charge state. The ion mobility contribution of the 3+ species is always analyzed in the following work. For all peptides, the wave velocity and the wave height in the ion mobility cell were, respectively, set at 1250 m/ s and 40 V, while the wave height and the wave velocity in the transfer cell (just after the ion mobility cell) were, respectively, settled at 508 m/s and 0.1 V. The wave velocity in the trap was fixed at 300 m/s while, for each peptide, the trap wave height had to be optimized. For the peptide 1, the IMS cell pressure (N2) was 1.69 mbar while the trap and the transfer pressures were 0.756 mbar. For peptides 2, 3, and 4, IMS pressure (N2) was 1.52 mbar while the trap and transfer pressure were 0.756 mbar. For all experiments, the He cell pressure was of 0.16 mbar. CID fragmentation was performed in the transfer cell. The accelerating voltage in this cell was set between 35 and 45 V depending of the peptide. For all the experiments involving ETD and ion mobility, the trapping release time, the mobility trap height, and mobility extract height parameters were, respectively, of 100 μs and 0.5 and 40 V. A summary of what has been done in the SYNAPT-G2 is depicted in Figure 2.
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Figure 4. Arrival time distribution (ATD) of the 3+ species of the peptide 1. An isotopic deconvolution of the 3+ charge state has been performed at each drift time in order to have access, at each drift time, to the ratio proton transfer/electron transfer.
P3, and P4), aiming at studying the differences in drift times induced by different cysteine connectivities in a same peptide sequence. Part 1: Proof of Concept on a Designed Peptide. The ETD reaction was performed on the 4+ charge state, and the total arrival time distribution (ATD) of the resulting 3+ ions was recorded. To visualize the effect of ETD on disulfide-bonded peptides, the ATD of the 3+ species (after ETD reaction on the 4+) was compared to the 3+ of the native peptide (without ETD). This comparison, depicted in Figure 3, shows the appearance of species with higher drift times (i.e., larger collisional cross sections) when ETD is performed (peaks B
RESULTS AND DISCUSSION
Results. The first part of the results is devoted to the peptide 1 (P1, Table 1), this peptide being a model peptide used for the demonstration of the proof of concept. The second part of the discussion is focused on the peptides 2, 3, and 4 (P2, C
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Figure 5. CID spectra of the ion mobility-separated species (35 V CID) for the peptide 1. The ATD of the reduced species (3+ charge state) is presented in the upper part. The corresponding CID spectra are presented below. (Remark: the CID experiment has also been performed on the 3+ obtained without ETD. The corresponding spectrum is similar to the spectrum A). Structures that are presented are for the purpose of illustration.
and C on Figure 3). The analysis of isotopic profiles reveals a shift of one mass unit compared to the nonreduced peptide. These observations, regarding Figure 1, may be linked to a partial opening of the peptide structure (opening of one of the two disulfide bridges). The B peak could be associated with the opening of the small loop, while the C peak could be associated with the opening of the large loop (Figure 3). To study the nature of the species building the ATD distribution, each isotopic profile has been deconvoluted in order to determine the relative contribution of the protontransfer reaction (close species) compared to disulfide bond opening (partially open species) (Figure 4). This figure shows that the peak A (around 4.2 ms) is mainly composed of a proton-transfer species (about 90%) while B and C peaks are mainly composed of disulfide-bonded partially opened species. Isotopic analysis at ATD contribution shows that the first peak around 4.2 ms (Figure 3, peak A) is mainly constituted of a proton-transfer product (in blue). This species is identified, with a high confidence, to the fully folded peptide, displaying a reduced charge state. On the other hand, the peaks at 4.4 ms
(Figure 3, peak B) and 4.8 ms (Figure 3, peak C) are mainly due to partially disulfide-opened species (in green). According to this observation and to the structure of the peptide 1, several hypotheses are proposed: (i) the species A should correspond to the folded species, the most compact structure; (ii) the species B should be linked to the peptide with the smaller loop opened (C3−C4), creating a small increase of CCS and the corresponding arrival time; (iii) the species C should represent the peptide with the larger loop opened (C1−C2), displaying a larger variation of CCS, visualized by an important increase in drift time (∼0.6 ms). To verify these hypotheses, CID experiments were performed after the ion mobility separation on each of the three contributions A, B, and C setting the collision voltage at 35 V (Figure 5). According to the resulting tandem mass spectrum (MS/MS spectrum), the fragmentation pattern of the peak A is in agreement with a species that still has the two disulfide bonds intact (Figure 5A). Indeed, the two disulfides strongly stabilize the peptide resulting in the occurrence of only few fragments as D
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formation. The MS/MS spectrum extracted from the peak C (Figure 5C) is in agreement with the opening of the disulfide bond between C1 and C2. Indeed, the masses of the different fragments perfectly fit with the supposed sequence, i.e., classical y- and b-ions (and y-1 and b-1) and atypical a-2, b-2, and y-2 fragments for the sequence still embedded in the remaining disulfide. These observations clearly reinforce the initial assumptions. The variation of CCS observed by the opening of only one disulfide bridge is sufficient enough to isolate the partially opened and the proton-transfer species. The online MS/MS experiments provide enough information to confirm the identity of the different ETD-generated species. It is important to note that a loss of 33 mass units is observed from the [M + 4H]3+• species in the fragmentation spectra B and C. The mobility profiles of this species, as well as other selected fragment ions, are presented in Figure 6. This loss is associated with the generation of a dehydroalanine residue after the electron-transfer pathway presented in Figure 1.31 In this case, the loss of 33 Da confirms the presence of sulfur radicals in the peaks B and C and then the opening of the S−S bond by ETD. Figure 6 also presents the contributions of other fragment ions to the ATD. The results show that the b13 and y11 ions are mainly present in the peak C, confirming the opening of the disulfide bond between C1 and C2. This figure also shows that the b20 ion is mainly identified in the peak B, confirming the opening of the disulfide bond between C3 and C4 for this species. The presence of the specific [a6 − 2]+ species in the B peak additionally indicates that the remaining disulfide bond after ETD reaction (C3−C4) has been cleaved by CID (generation of thioaldehyde).33 The pathway that leads to the generation of this species is explained in the Supporting Information Figure SI 1. Figure 6 also reveals that the [M + 4H + 33]3+ species can be found in the peaks B and C, which confirms that they are both partially opened species. Influence of Disulfide Bond Connectivity on ETD-IMS Experiments. To investigate the influence of disulfide bond connectivity on ETD-IMS experiments, three peptides displaying the same amino acid sequence but three different cysteine connectivities were synthesized (Table 1). Each isoform was analyzed according to the same ETD-IMS-CID workflow previously described (Figure 7). From a general point of view, the three ATDs present roughly the same profile. They show a large and intense peak (Figure 7). However, the isotopic distribution analysis of the ATD clearly highlights different behaviors of the three species (Figure 7b). Case of Peptide 2 (C1−C2/C3−C4). For the peptide 2, isotopic deconvolution at each drift time shows that the first peak around 3.4 ms (in blue) is composed of a mixture of proton-transfer (in red) and electron-transfer products (see Figure 7). The CID spectra of each ATDs (Supporting Information Figure SI 2) confirms and demonstrates also that the peak around 3.8 ms is mainly composed of an opened species (in green). This open species is associated with the opening of the C1−C2 disulfide bridge, because (i) this bridge is the biggest of the two and is supposed to cause a significant drift time (DT) variation after reduction (ΔDT ≈ 0.4 ms) and (ii) several characteristic fragment ions such as y14+ are mainly found in the peak at 3.8 ms (see Supporting Information Figure SI 3). However, for the y15+ ion, the situation is less clear and seems to show a more complex situation in which the resolution of the separation is not sufficient to distinguish
Figure 6. ATD contributions of selected peaks depicted in Figure 5. All the contributions are compared to the total ATD contribution of the ETD charge-reduced species.
expected for such peptides fragmented under low-energy CID. The spectrum of the peak B and the peak C display many more fragment ions (b20, b21, y5 for peak B and y8, y10, y11, y13, y14, y17, y21, y22, b4, b10, b11, b13 for the C peak), indicating the probable opening of one disulfide bridge, as deduced from the ATD changes (Figure 4). The MS/MS spectrum extracted from the peak B contains less fragment ions than the one from the peak C. In this peptide, the opening of the larger loop will create a free chain of 15 amino acids that would probably lead to a longer sequence tag by CID. Moreover, for the peak B, few fragmentations are detected originating from the remaining disulfide bond (y6-2, b6-2). The pathways that leads to these ions are depicted in the Supporting Information (Figure SI 1).33 These ions are generated by the homolytic cleavage of the S−S bond followed by a backbone cleavage and a thioaldehyde E
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Figure 7. (a) ATD of the 3+ species of the peptides 2, 3, and 4 without ETD. (b) Deconvoluted ATD for the same peptide after charge reduction (4+ to 3+) using ETD. The black line represent the total ATD contribution, the blue line represent the proton-transfer contribution, and the green line represent the partially disulfide-opened species.
is quite weak; proton transfer and disulfide bond opened species are superimposed in the ATD. This result is supported by the CID fragmentation spectrum of this peptide (see Supporting Information Figure SI 6) in which the opening of the C1−C4 connectivity is detected. This property can also be seen as a signature of C1−C4/C2−C3 connectivity, also referred as to the “hairpin fold”. In all cases, presence of 33 Da neutral loss confirms the opening of the disulfides, and the intensity of this ion can be used to monitor this reaction.31 No scrambling phenomenon was detected during this study. This could suggest that the reaction time scale is not sufficient to visualize or to generate scrambling. Indeed, using this experimental setup, the reaction time is only around 100 μs while ETD reactions in ion trap can go to 100 ms long. One other explanation could come from the fact that the energy given to the system is not high enough to perform scrambling. The methodology developed in this paper could be used to determine the cysteine connectivity of a natural occurring peptide bearing two disulfide bonds. Most of these peptides having a 1,3/2,4 or a 1,4/2,3 connectivity.
clearly between the proton-transfer and the electron-transfer products. For the −33 Da neutral loss, it is detected more abundant in the peak at 3.8 ms than in the one at 3.4 ms (data not shown). Case of Peptide 3 (C1−C3/C2−C4). Concerning the peptide 3, Figure 7 shows that the peaks at 3.4 ms and 3.8 ms are mainly composed of a proton-transfer product (in blue) and an electron-transfer product (partially opened species, in green), respectively. CID experiments demonstrate that the peak at 3.8 ms is clearly associated with the opening of the C1−C3 disulfide bond (Supporting Information Figure SI 4). ATD contributions of some fragment ions, depicted in the Supporting Information (Figure SI 5), show that a wide range of ions are specific to the 3.8 ms peak (y15+, y14+, y13+, b13+). Contribution of the −33 Da ion indicates that the two partially opened structures can be identified (see Supporting Information Figure SI 5). Case of Peptide 4 (C1−C4/C2−C3). Figure 7 shows that proton-transfer and electron-transfer products are not wellresolved using ion mobility. The isotopic deconvolution confirms that the partial reduced species is shifted of only 0.1 ms from the proton-transfer product. Fragmentation experiments reveal some ions (b132+, y13+, y14+, y15+, ...) indicating the presence of a partially opened peptides (see Supporting Information Figure SI 6). Discussion. For all peptides, the electron transfer cleaves disulfide bonds. The reduction of such bonds is demonstrated with the peptide 1, particularly suited for the presented workflow because C2 and C3 are vicinal and C1−C2/C3−C4 connectivity establishes two loops of different lengths. The opening of one of the two disulfides creates differences in CCS that are enough to separate the species in our experimental conditions of IMS. Online CID is, however, required to characterize unambiguously each of the generated species. For the peptides 2 and 3, only the opening of the largest loop can be clearly observed, especially for the peptide 3. Nevertheless, for peptides bearing two disulfide bonds, the information about one bond is enough to perform the cysteine connectivity identification. Concerning the peptide 4, the drift time variation
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CONCLUSIONS
The aim of this study was to develop a new gas-phase method for disulfide bond assignment of peptides bearing two disulfide bonds. Electron-transfer dissociation being able to cleave the disulfide bonds in the gas phase, this method was based on a combined use of ETD, ion mobility, and CID. To develop this technique, four peptides displaying various cysteine connectivities where used in order to visualize the efficiency of the technique depending on the connectivity. Ion mobility clearly showed an opening of structure when ETD is performed on a disulfide bond peptide (thanks to a comparison of the ATDs obtained with and without ETD). Concerning the 1,3/2,4 pattern, opening of the larger disulfide bond is clearly detected, revealing the advantage of using ion mobility to separate proton-transfer and electron-transfer species. CID fragments, resolved in ion mobility, provide enough structural information to confirm the structures and the F
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(6) Gehrmann, J.; Alewood, P. F.; Craik, D. J. J. Mol. Biol. 1998, 278, 401−415. (7) Calvete, J. J.; Schrader, M.; Raida, M.; McLane, M. A.; Romero, A.; Niewiarowski, S. FEBS Lett. 1997, 416, 197−202. (8) Bauer, M.; Sun, Y.; Degenhardt, C.; Kozikowski, B. J. Protein Chem. 1993, 12, 759−764. (9) Gorman, J. J.; Wallis, T. P.; Pitt, J. J. Mass Spectrom. Rev. 2002, 21, 183−216. (10) Wang, Y.; Lu, Q.; Wu, S.-L.; Karger, B. L.; Hancock, W. S. Anal. Chem. 2011, 83, 3133−3140. (11) Gilbert, H. F. In Methods in Enzymology; Lester, P., Ed.; Academic Press: San Diego, CA, 1995; pp 8−28. (12) Brgles, M.; Kurtović, T.; Halassy, B.; Allmaier, G.; MarchettiDeschmann, M. J. Mass Spectrom. 2011, 46, 153−162. (13) Echterbille, J.; Quinton, L.; Gilles, N.; De Pauw, E. Anal. Chem. 2013, 85, 4405−4413. (14) Papayannopoulos, I. A. Mass Spectrom. Rev. 1995, 14, 49−73. (15) Mormann, M.; Eble, J.; Schwöppe, C.; Mesters, R.; Berdel, W.; Peter-Katalinić, J.; Pohlentz, G. Anal. Bioanal. Chem. 2008, 392, 831− 838. (16) Lioe, H.; O’Hair, R. A. J. J. Am. Soc. Mass Spectrom. 2007, 18, 1109−1123. (17) Fung, Y. M. E.; Kjeldsen, F.; Silivra, O. A.; Chan, T. W. D.; Zubarev, R. A. Angew. Chem. 2005, 117, 6557−6561. (18) Agarwal, A.; Diedrich, J. K.; Julian, R. R. Anal. Chem. 2011, 83, 6455−6458. (19) Stinson, C. A.; Xia, Y. Analyst 2013, 138, 2840−2846. (20) Zubarev, R. A.; Kruger, N. A.; Fridriksson, E. K.; Lewis, M. A.; Horn, D. M.; Carpenter, B. K.; McLafferty, F. W. J. Am. Chem. Soc. 1999, 121, 2857−2862. (21) Chrisman, P. A.; Pitteri, S. J.; Hogan, J. M.; McLuckey, S. A. J. Am. Soc. Mass Spectrom. 2005, 16, 1020−1030. (22) Wiesner, J.; Premsler, T.; Sickmann, A. Proteomics 2008, 8, 4466−4483. (23) Ganisl, B.; Breuker, K. ChemistryOpen 2012, 1, 260−268. (24) Simons, J. Chem. Phys. Lett. 2010, 484, 81−95. (25) Tureček, F.; Julian, R. R. Chem. Rev. 2013, 113, 6691−6733. (26) Mikesh, L. M.; Ueberheide, B.; Chi, A.; Coon, J. J.; Syka, J. E. P.; Shabanowitz, J.; Hunt, D. F. Biochim. Biophys. Acta, Proteins Proteomics 2006, 1764, 1811−1822. (27) Syka, J. E. P.; Coon, J. J.; Schroeder, M. J.; Shabanowitz, J.; Hunt, D. F. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 9528−9533. (28) Gunawardena, H. P.; He, M.; Chrisman, P. A.; Pitteri, S. J.; Hogan, J. M.; Hodges, B. D. M.; McLuckey, S. A. J. Am. Chem. Soc. 2005, 127, 12627−12639. (29) Good, D. M.; Coon, J. J. BioTechniques 2006, 40, 783. (30) Coon, J. J.; Syka, J. E. P.; Schwartz, J. C.; Shabanowitz, J.; Hunt, D. F. Int. J. Mass Spectrom. 2004, 236, 33−42. (31) Cole, S.; Ma, X.; Zhang, X.; Xia, Y. J. Am. Soc. Mass Spectrom. 2012, 23, 310−320. (32) Tan, L.; Durand, K. L.; Ma, X.; Xia, Y. Analyst 2013, 138, 6759− 6765. (33) Chrisman, P. A.; McLuckey, S. A. J. Proteome Res. 2002, 1, 549− 557.
cysteine pairing. Disulfide bond assignment of a 1,2/3,4 pattern peptide has been also successfully achieved. However, the opening of one disulfide bond in a hairpin-fold peptide (1,4/ 2,3) is not clearly visualized in our experimental conditions. This result, assimilated to a signature of such peptides, can nevertheless be exploited to identify hairpin-fold peptides. In all experiments, neutral losses of 33 (SH) Da indicate the opening of an S−S bond after the ETD reaction. There is a place for method improvement. For example, ion mobility suffers of limited resolution that could be increased to visualize smaller CCS variations. ETD reaction efficiency can also be improved, by adjusting the reaction time (ion traps) or the nature of the ETD reagent, in order to obtain a more favorable electron-transfer/proton-transfer ratio. The workflow developed will be applied soon on peptides bearing three disulfide bonds. The biggest challenge for this type of peptide is that, after the reduction of one disulfide, two disulfide bonds remain in the sequence and three different connectivities are still possible. Species with two disulfideopened species have to be isolated, and ion mobility spectrometry has to have a sufficient resolution to isolate these partially opened species. Another possibility could take into account the reduction, by ETD, of two disulfides among the three. This system, more or less similar to our previous work, could probably be easier to characterize, but it is highly dependent on the reduction efficiency. However, after ETD, a mixture of peptides displaying three, two, and one disulfide bonds would be probably produced in various amounts. This method could also be tested in the future on proteins. The biggest challenge would be to deal with the large amounts of disulfide bonds found in proteins, IMS resolution and ETD efficiency being currently the limiting parameters of our methodology.
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in text. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b00245.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank the FRS-FNRS for the financial support (FRIA and instrumentation), the Fonds Européen de développement regional (FEDER), the Walloon region, and the European commission (F.P. 7 VENOMICS project) for financial support.
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REFERENCES
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