B American Society for Mass Spectrometry, 2014

J. Am. Soc. Mass Spectrom. (2014) 25:1029Y1039 DOI: 10.1007/s13361-014-0855-6

RESEARCH ARTICLE

Influence of Metal–Peptide Complexation on Fragmentation and Inter-Fragment Hydrogen Migration in Electron Transfer Dissociation Daiki Asakawa,1 Takae Takeuchi,2 Asuka Yamashita,2 Yoshinao Wada1 1 2

Department of Molecular Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health, Osaka, Japan Department of Chemistry, Faculty of Science, Nara Women’s University, Nara, Japan Abstract. The use of metal salts in electrospray ionization (ESI) of peptides increases the charge state of peptide ions, facilitating electron transfer dissociation (ETD) in tandem mass spectrometry. In the present study, K+ and Ca2+ were used as charge carriers to form multiply-charged metal–peptide complexes. ETD of the potassium- or calcium-peptide complex was initiated by transfer of an electron to a proton remote from the metal cation, and a c'-z• fragment complex, in which the c' and z• fragments were linked together via a metal cation coordinating with several amino acid residues, was formed. The presence of a metal cation in the precursor for ETD increased the lifetime of the c'-z• fragment complex, eventually generating c• and z' fragments through inter-fragment hydrogen migration. The degree of hydrogen migration was dependent on the location of the metal cation in the metal-peptide complex, but was not reconciled with conformation of the precursor ion obtained by molecular mechanics simulation. In contrast, the location of the metal cation in the intermediate suggested by the ETD spectrum was in agreement with the conformation of “proton-removed” precursors, indicating that the charge reduction of precursor ions by ETD induces conformational rearrangement during the fragmentation process. Key words: Metal coordination, Conformation rearrangement, Lifetime of intermediate, Metal cation location Received: 7 January 2014/Revised: 6 February 2014/Accepted: 9 February 2014/Published Online: 27 March 2014

Introduction

E

lectrospray ionization (ESI)-based electron-mediated dissociation methods in tandem mass spectrometry, such as electron capture dissociation (ECD) [1] and electron transfer dissociation (ETD) [2], are widely used for peptide sequencing [3]. ECD/ETD utilize electron attachment/transfer to multiply protonated molecules and produce mainly the c' and z• fragments through hydrogen-abundant peptide radicals [4]. In particular, the labile group or bonds in the post-translational modifications of peptides and proteins were left intact during ECD/ETD process [5, 6]. Since ECD/ETD provide information on structure complementary to that obtained by conventional collision induced dissociation (CID), the combined use of ECD/ETD and CID

Electronic supplementary material The online version of this article (doi:10.1007/s13361-014-0855-6) contains supplementary material, which is available to authorized users. Correspondence to: Daiki Asakawa; e-mail: [email protected], Yoshinao Wada; e-mail: [email protected]

improves the sequence coverage of protein digests [7, 8]. In general, the efficiency of the ion/electron reaction for ECD/ETD is enhanced by increasing the charge state of precursor ions. The charge state distribution of multiply-protonated peptides is dependent on the peptide sequence, especially the number of basic residues [9]. ESI mainly produces doubly protonated species of tryptic peptides, since protonation preferentially occurs at the Nterminal amino group and the C-terminal basic residue, either lysine or arginine. However, double protonation is often insufficient for the ion/electron reaction to provide better sequence coverage, and a method of increasing the charge state of precursor ions is, therefore, desired for ECD/ETD. For peptides barely yielding multiply-charged precursors, use of trivalent metal (MT) cations as a charge carrier to generate multiply charged metal–peptide complexes such as [M–H+MT]2+ or [M+MT]3+ has been reported [10]. Generation of doubly- or triply charged trivalent metal–peptide complexes by ESI depends on peptide size. Typically, a doubly charged species, [M–H + MT]2+, is formed for peptides less than 1000 Da, whereas large peptides generate

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triply charged ions [M+MT]3+ [10]. Unfortunately, ECD of the triply charged ion [M+MT]3+ does not improve the sequence coverage as compared with the corresponding doubly protonated molecule [M + 2H] 2+ [10], but the trivalent metal-aided method is still expected to be useful for ECD/ETD of small peptides. A line of studies demonstrated that ECD/ETD of metal–peptide complexes produce both metallated and protonated fragments [10-15]. However, since excess protons do not exist in peptide precursors, this phenomenon was attributed to the zwitterion structure formed by the negatively charged group and remote protons [10, 12]. ECD/ETD of metal–peptide complexes lead to the c'/z• ion formation for most metal cations other than Cu2+ and Eu3+ because these cations are directly reduced by ECD/ETD because of their electrochemical property [10, 13, 15], and the metal cation/ electron recombination energy is redistributed throughout the peptide to give b/y ions or neutral loss instead of c'/z• ion formation. Afonso et al. investigated the ECD processes of Cu2+-cyclic peptides in detail [16]. The fragment formation in ECD is mainly driven by the substantial excitation attributable to recombination energies of the complex and electron, which is in the 8– 10 eV range. Electron capture of Cu2+-cyclic peptide provides internal excitation in the charge-reduced complexes to break two bonds in the cyclic peptide to produce fragment ions. By contrast, metal–peptide complexes give c'/z• fragment pair by ECD/ETD for most metal cations other than Cu2+ and Eu3+ , as well as protonated precursor. Therefore, the proposed mechanisms to explain the N − Cα bond cleavage in ECD/ETD of protonated precursor can be envisaged for ETD of metal–peptide complex. According to the classical ECD mechanism [1], the ECD/ ETD processes of metal–peptide complexes would be initiated by electron attachment of a proton remote from the metal cation. Alternatively, Syrstad et al. propose that ECD/ETD processes produce amide anion-radical intermediate [17]. The mechanism proposed by Zubarev and coworkers involves the formation of a negative charge on the amide nitrogen and aminoketyl radical by electron capture at a hydrogen bond between the backbone nitrogen and carbonyl groups [18]. Despite their differences, those mechanisms involve the formation of aminoketyl radical intermediate, which eventually leads the N − C α bond cleavage. Intriguingly, while the N–C α bond cleavage generates the c' and z• fragment pair [19], ECD of alkaline-earth metal-peptide complexes generates mainly the c• and z' fragments [12], which are the products of inter-fragment hydrogen migration between c' and z• fragments [20]. In an ECD study by O’Connor et al. [21], the source of hydrogen in this process was an α-carbon of the c' fragment. The hydrogen migration is exothermic reaction and its transition energy is usually higher than that of c'/z• ion

formation [22, 23], However, increasing the internal energy of the precursor by infrared laser irradiation can suppress the formation of c• and z' fragments [24]. The high abundance of c• and z' fragments is generally rationalized on the basis of the long c'-z• complex lifetime to allow inter-fragment hydrogen migration. The inter-fragment hydrogen migration in ECD could be suppressed by increasing the charge-state of precursor ions to shorten the lifetime of the c'-z• complex [25]. These studies suggested that inter-fragment hydrogen migration during ECD/ETD would provide insights into the conformation of peptides or metal–peptide complexes in the gas phase, although multiple isotopes for metallic elements make it difficult to distinguish between two fragment pair types, c'/z• or c•/z'. To understand the behavior of metal cations in the precursor ions in ECD/ETD in detail, the present study focused on the inter-fragment hydrogen migration in the ETD processes of metal-peptide complexes. ETD of metal–peptide complexes produced abundant c• and z' ion species, a useful probe for delineating gas-phase conformations of intermediates in ETD. The ETD mass spectrum of metal–peptide complexes represented the lowest energy conformation of their proton-removed forms. In addition, complexation with metal ions increased the charge state of tryptic peptides containing acidic residues, and rendered ETD spectra more informative than those obtained from the protonated molecules with lower charge states.

Experimental Materials and Methods Synthetic nonapeptides containing an Asn bearing a single N-acetylglucosamine (GlcNAc) were purchased from the Peptide Institute Inc. (Osaka, Japan). The sequences were derived from residues 293-301 of the Fc regions of four different subclasses of human IgG. Lactalbumin was purchased from Sigma-Aldrich (St. Louis, MO, USA). All reagents were used without further purification. All of the solvents used were HPLC grade in quality except for water from the Milli-Q purification system (Millipore, Billerica, MA, USA). Lactalbumin was reduced by 0.13 M dithiothreitol in a 0.5 mL solution of 6 M guanidine and 0.25 M Tris-HCl at pH 8.0 at 56°C for 1 h and then Scarbamidomethylated by 0.22 M iodoacetamide for 30 min at room temperature. These chemicals were removed by gel filtration using a NAP5 column (GE Healthcare) equilibrated with 0.05 N HCl, and then the eluted solution was adjusted to pH 8.2 by the addition of 1.5 M Tris. A total of 0.5 μg each of lysylendopeptidase (Achromobacter protease I; Wako Pure Chemical) and trypsin (sequence grade modified

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Figure 1. (a) ESI mass spectrum of synthetic IgG1 glycopeptide EEQYN(GlcNAc)STYR. ETD mass spectra of the (b) [M+2H]2+, (c) [M+ H+K]2+, (d) [M+2H+K]3+, and (e) [M+H+2 K]3+. Asterisk (*) and dagger (†) indicate precursor and charge-reduced ions, respectively

trypsin, from porcine pancreas, Promega) was added to the solution, followed by incubation at 37°C for 15 h. The digested peptides were purified employing C18 ZipTip (Millipore).

reaction and the ion/ion reaction time was optimized in order to achieve a high signal-to-noise ratio for the ETD fragment ion peaks. The ion/ion reaction time exerted its effect on the intensity ratio of the precursor/product but did not influence the intensity ratio of c•/c' and z•/z'.

Mass Spectrometry The peptides were dissolved in 0.1% m-nitrobenzyl alcohol and 50% methanol solution at a concentration of 10 μM, and directly infused into an LTQ XL ion trap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) for nanoESI MS. For the ETD experiments, 10 6 anions of fluoranthene were used for

Molecular Mechanics Simulation To obtain a better initial idea of the most probable 3D conformations of the glycopeptide, EEQYN(GlcNAc) STYR, we calculated the optimal structure, the dominant conformers, and their steric energies for the following ions: [M + H + K]2+, [M + 2H + K]3+,

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Figure 2. ETD mass spectra of charge-reduced ions from (a) [M+H+K]2+, (b) [M+2H+K]3+, and (c) [M+H+2 K]3+ of synthetic IgG1 glycopeptide EEQYN(GlcNAc)STYR

[M + H + 2 K] 3+ and their corresponding "proton-removed" precursors: [M+K]+, [M+H+K]2+, [M+2 K]2+. This was performed using force fields at the MMFF94S level with CONFLEX 7 software (Conflex Co., Tokyo, Japan). The optimal geometries and their

energies for the first 10 conformations of [M+H+K]2+, [M+2H+K]3+, [M+H+2 K]3+, [M+K]+, and [M+2 K]2+ were calculated using the B3LYP/6-311 + G(2d,p)// B3LYP/6-31G(d) level with the Gaussian 09 program [26].

Table 1. Observed Fragment Ions in ETD Mass Spectra of Glycopeptides with Different Precursors and Their Ht Values. The Abundances of c' and z' Ions were Calculated Using Theoretical Isotopic Contributions of c• and z• Ions, Respectively. The Ht Values were Determined by Averaging Three ETD Mass Spectra

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Figure 3. Lowest energy conformations found in the molecular mechanics simulation of analyte peptides containing two potassium cations, (a) [M+H+2 K]3+ and (b) [M+2 K]2+

Notation In the present study, Zubarev’s unambiguous notation was adopted for peptide fragment ions [4]. According to this notation, homolytic N–Cα bond cleavage yields the radical fragments c• and z•, and addition of a hydrogen atom to c• or z• fragments produces a c' or z' fragment, respectively.

Results and Discussion Initial Step in ETD of Potassium–Peptide Complexes A glycopeptide, EEQYN(GlcNAc)STYR, of human IgG1 was analyzed by ESI MS. As shown in Fig. 1a, ESI generated a doubly protonated molecule [M+2H]2+ with good intensity, and the protonated sites of this glycopeptide were the guanidinium group of the C-terminal Arg and N-terminal amino group. In addition, two triply charged ions, [M+2H+K]3+ and [M+H+2 K]3+, were generated probably because of high affinity for potassium cations, whereas the triply protonated molecule [M+3H]3+ was not observed. ETD of the precursor ions, [M+2H]2+, [M+H+K]2+, [M+2H+K]3+, and [M+H+2 K]3+, yielded c'/c• and z•/z'

Figure 4. Lowest energy conformations in different charge states from the molecular mechanics simulation for peptides containing a potassium cation, (a) [M+2H+K]3+, (b) [M+H+ K]2+ and (c) [M+K]+

fragments as shown in Fig. 1b–e. ETD of potassium–peptide complexes would be initiated by electron transfer to a proton in the complex, giving radical intermediates. Subsequently, the intermediates underwent radical-induced dissociation of N–Cα bonds. On the other hand, reduction of metal cations occurs on K+ in the potassium–peptide complex, but this process does not mediate pairing of the c' and z• fragments [10, 13, 15]. Flick et al. reported the ECD phenomena of lanthanide–peptide complexes to be based on the electrochemical properties of lanthanide cations in

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Figure 5. ESI mass spectra of tryptic digests of lactalbumin in the absence (a) and presence (b) of 1 mM CaCl2. Asterisks (*) in (b) indicate protonated molecules observed in (a)

aqueous solution [10]. According to their report, the alkali metal cations bound to peptide would barely be reduced compared with lanthanide metal cations in ETD, since the electron reduction potentials of alkali metal cations in aqueous solution are lower than that of lanthanide cations. Therefore, the potassium–peptide complex was probably initiated by reduction of protons to form hydrogen-abundant intermediate radicals. To address the initial step in ETD of potassium–peptide complexes, we first looked at the charge-reduced precursors observed in the mass spectrum (Fig. 2). Although the ETD of [M+H+K]2+ resulted in the formation of [M+K]+ by electron transfer with a subsequent hydrogen radical loss or proton transfer, potassium loss was not observed (Fig. 2a). Similarly, ETD of the triply charged complex, [M+2H+K]3+, yielded singly charged [M+K]+ by loss of two protons (Fig. 2b). These findings indicated remote protons in potassium–peptide complexes to be reduced by ion/ion reaction, leading to the formation of intermediate radicals carrying a charge on potassium. Notably, ETD of [M+H+2 K]3+ yielded charge-reduced species lacking hydrogen, [M+2 K]+• and [M– H+2 K]+, as well as [M+H+2 K]+••, indicating that two protons in the precursor were reduced by ETD although [M+ H+2 K]3+ had one excess proton (Fig. 2c). The result suggested that [M+H+2 K]3+ took a zwitterion form in which the acidic group was deprotonated and that a proton was present elsewhere in the peptides.

charged precursor was reduced by ion/ion reaction, and the resulting singly charged intermediate produced the z• ion and neutral c' fragments. The preferential formation of z•/z' ions but not c•/c' ions was attributed to the basic residue Arg at the C-terminus. On the other hand, the triply charged ion [M + 2H + K] 3+ produced doubly charged intermediates and, finally, generated both c' and z• fragments with a single charge (Fig. 1d), indicating that use of the [M + 2H + K]3+ precursor would improve the sequence coverage of peptides compared with the doubly charged precursors in Fig. 1b and c. By contrast, ETD of [M + H + 2 K]3+ produced fragments less abundantly than [M + 2H + K]3+, demonstrating the contribution of remote protons to radical intermediate formation. Increasing the number of K+ instead of H+ in precursors would, thus, impair the efficiency of ion/ ion reaction and reduce the fragment ion yield. As depicted in the insets of Fig. 1, c• and z' ions derived from hydrogen migration between c' and z• were abundant in ETD mass spectra of potassium–peptide complexes. The interfragment hydrogen migration in ETD is dependent on the amino acid sequence and precursor ion species, and is estimated as the “Ht, value” defined by Nishikaze, et al. [25]:  0   I zn I c•n    100;      100 Ht ¼  •  I zn þ I z0n I c•n þ I c0n

Inter-Fragment Hydrogen Migration During ETD Process ETD of doubly charged ions, [M + 2H]2+ and [M + H + K]2+, in Fig. 1b and c gave three and four different types of z•/z' ions, respectively. The proton of these doubly

where I is the intensity of fragment ions. The fragment ions observed in ETD mass spectra and their Ht values are summarized in Table 1.

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Figure 6. ETD mass spectra of (a) [M+2H]2+ and (b) [M+H+Ca]3+ of FLDDDLTDDIMC*VK (residues 80-93); and (c) [M+3H]3+ and (d) [M+2H+Ca]4+ of DDQNPHSSNIC*NISC*DK (residues 63-79). Daggers (†) and double daggers (‡) indicate precursor and charge-reduced ions, respectively. Asterisks on c' and z' fragments indicate Ca2+ adducts, [c'–H+Ca]+ and [z'–H+Ca]+, respectively, and c'2+ and z•2+ indicate [c'+Ca]2+ and [z•+Ca]2+, respectively

ETD of triply charged potassium–peptide complexes efficiently produced c• and z' ions (Fig. 1d and e), although hydrogen migration between complementary c' and z• fragments in ECD/ETD is mostly suppressed because of Coulomb repulsion in the peptide ions with a higher charge state [20, 25]. In contrast, ETD of the doubly charged peptides, [M+2H]2+ and [M+H+K]2+, involved less interfragment hydrogen migration (Fig. 1b and c). These unexpected findings are attributed to interactions between

peptide and potassium cation. Alkali metal cations coordinate with carbonyl oxygen and the carboxyl group [27]. Therefore, the c' and z• fragments generated from [M+2H+ K]3+ and [M+H+2 K]3+ were held together by ionic K+ bonding, which produces a long lifetime of the intermediate complex allowing inter-fragment hydrogen migration. The same results were obtained with other glycopeptides, EEQFN(GlcNAc)STFR, EEQYN(GlcNAc)STFR, and EEQFN(GlcNAc)STYR (Supporting Information,

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Table 2. Observed Fragment Ions in ETD Mass Spectra of Peptide Residues 80-93 with Different Precursors and Their Ht Values. The Ht Values Presented are the Average of Three ETD Mass Spectra. The Ht Values of c9'/c9• and c11'/c11• Could not be Determined Because of Overlapping with Other Fragment Ions

Figures S1–S3), whose sequences differed from each other at position 4 and/or 8, and these differences were unrelated to the inter-fragment hydrogen migration.

Conformation of Intermediate in ETD of Potassium–Peptide Complex To understand the process of inter-fragment hydrogen migration mediated by potassium cations in more detail, a molecular mechanics simulation was performed. First, we analyzed the [M+H+2 K]3+ ions, which preferentially produced c• and z' fragments as shown in Fig. 1e. Figure 3a shows the lowest energy conformations of the precursor ion, [M+H+2 K]3+.

Notably, all the low energy conformations were similar to each other structurally, with a proton at the C-terminal Arg that did not interact with potassium ions, and they were elongated because of the large Coulomb repulsion between potassium cations and the protonated Arg residue. However, in the ETD mass spectrum, an intense signal for z1' was observed as a result of hydrogen migration between c8' and z1• fragments, suggesting the c8'-z1• complex to be a long-lived radical intermediate held together through potassium–peptide binding. The abundant z1' ions in this ETD mass spectrum did not reconcile with the precursor ion conformation showing repulsion between K+ and protonated Arg. This finding suggested that the charge reduction of precursor ions caused substantial conformational rearrange-

Table 3. Observed Fragment Ions in ETD Mass Spectra of Peptide Residues 63-79 with Different Precursors and Their Ht Values. Ht Values of c11'/c11•, c15'/c15•, and z15•/z15' Could not be Determined Because of Overlapping with Other Fragment Ions. The Ht Values Presented are the Average of Three ETD Mass Spectra

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ment in order to stabilize the intermediate radical. O’Connor et al. have estimated that the lifetime of radical intermediates formed in ECD exceeds 1 ms [28], being long enough for conformational rearrangements. The proton bound to an analyte molecule was reduced by ETD, and the resulting intermediate, [M+H+2 K]2+•, was expected to have a conformation similar to that of the proton-removed precursor, [M+2 K]2+. In the conformation of [M+2 K]2+, most amino acids coordinated with potassium cations to form globular structures (Fig. 3b). If the conformation of [M+H+2 K]2+• was similar to that of [M+ 2 K]2+, all of the c'-z• complex intermediates would be longlived because of strong potassium–peptide interactions. Indeed, the Ht values were high for [M+H+2 K]3+ (Table 1), raising the possibility of the ETD spectra of this ion representing the lowest energy conformation of [M+K]2+. Next, we examined the conformations of intermediates from [M+2H+K]3+, which produced the four c'/c• and seven z•/z' fragments shown in Fig. 1d. Small z• type fragments, z1•–z4•, were observed as protonated forms, whereas their counterparts, c5'–c8', contained potassium cations. In addition, [z5•/z5'+H]+, [z7•/z7'+K]+ and [z8•/z8'+K]+ were observed. These findings indicated K+ to be localized at the residues between positions 1 and 4 in the intermediate radical, and that interaction between K+ and peptides increased the lifetime of the resulting c'-z• complex thereby allowing inter-fragment hydrogen migration. The formation of [z5•/z5'+H]+, [z7•/z7'+K]+, and [z8•/z8'+K]+ indicated that inter-fragment hydrogen migration occurred in the region from positions 1 to 4 and, thus, revealed the location of K+ in the intermediate. To understand the ETD process of [M+2H+K]3+, we searched for the lowest energy conformations for [M+2H+K]3+ and [M+H+K]2+. As shown in Fig. 4a, the N-terminal amino group and C-terminal Arg of [M+2H+ K]3+ were protonated and could not interact with K+ due to Coulomb repulsion. Therefore, coordination of K+ with Thr7 and Tyr8 was suggested, but this notion was inconsistent with the ETD spectrum. On the other hand, K+ was coordinated with Glu1, Glu2, and Tyr4 as well as Tyr8 in the [M+H+K]2+ ion containing protonated Arg (Fig. 4b), in agreement with the location of K+ described above for the intermediate [M+2H+ K]2+• generated by ETD of [M+2H+K]3+. ETD of [M+H+K]+ produced four z•/z' fragments, [z2•/ z2'+H]+, [z3•/z3'+H]+, [z4•/z4'+H]+, and [z8•+K]+, indicating K+ to be located in the N-terminal side of the intermediate radical (Fig. 1c). In the lowest energy conformations of the proton-removed precursor, [M+K]+, as shown in Fig. 4c, K+ was coordinated with the side chains of Thr7 and Arg9 and the carbonyl groups of Glu2, Ser6, Thr7, and Tyr8. Consistent with the results of triply charged potassium–peptide complexes presented in Fig. 1d and e, the Ht values for [M+H+ K]2+ could be explained by the lowest energy structure of the proton-removed precursor, [M+K]+ shown in Fig. 4c.

ETD of Peptides from Calcium-Binding Protein The calcium-binding protein α-lactalbumin was then analyzed as a metal-binding protein model. The calcium-

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binding site of α-lactalbumin is located in residues 78-89, among which the acidic residues Asp bind Ca2+. Tryptic digestion of α-lactalbumin produced two peptides, DDQNPHSSNIC*NISC*DK (residues 63-79) and FLDDDLTDDIMC*VK (residues 80-93) (C* for S-carbamidomethylated cysteine), sharing the calcium-binding site. The ESI mass spectra of the tryptic digest of α-lactalbumin with or without 1 mM CaCl2 were compared, as shown in Fig. 5. The doubly protonated species were mainly observed in Fig. 5a, since protonation preferentially occurs at the Nterminal amino group and the C-terminal basic residue. For peptides 78-89 and 99-108, triply protonated molecules were generated because the maximum charge of multiply-protonated peptides is dependent on the number of basic residues including His. As demonstrated in Fig. 5b, Ca2+ effectively bound to peptides 63-79 and 80-93 containing three and five Asp residues, respectively, and produced quadruply charged [M + 2H +Ca]4+ and triply charged [M +H + Ca]3+ ions, respectively, indicating that the addition of CaCl2 could increase the charge state of tryptic peptides containing a Ca2+ binding site. In the ETD mass spectra shown in Fig. 6a and b, a triply charged Ca2+-peptide (residues 80-93) complex, [M+H+ Ca]3+, produced 10 fragments each of c'/c• and z•/z' (Fig. 6b), whereas the doubly protonated peptide (residues 80-93) yielded only c13' and four z•/z' fragments (Fig. 6a), consistent with the general notion of better sequence coverage with higher charge state precursors. In the ETD mass spectrum of [M+H+Ca]3+, the cleavage at the Nterminal side of the Cys residue gave a w fragment but not z•/z', because of α-cleavage of z• fragments [29]. Next, the location of Ca2+ in the precursor ion could be determined based on the fragment ions and Ht values as follows. As shown in Table 2, five Ca2+-containing z•/z' type fragments, z9•/z9'–z13•/z13', were generated. More importantly, protonated c2• and c3• were observed and the Ht values were high for the N-terminal region from the first to the fifth residue, indicating that Ca2+ was localized in the N-terminal portion (residues 80-85) of the intermediate, [M+H+Ca]2+•. On the other hand, residues 86-89 did not coordinate with Ca2+ under these conditions, although the Ca2+ binding site was located in residues 78-89 of α-lactalbumin. In Fig. 5, ESI of peptide residues 78-89 generated a triply protonated molecule because of an additional basic residue (histidine) at position 6 (residue 68). ETD of this precursor produced 10 c'/c• and nine z•/z' fragments, but their yields were low (Fig. 6c). In contrast, the quadruply charged Ca2+peptide complex precursor, [M+2H+Ca]4+ displayed more efficient dissociation (Fig. 6d). The fragment ions and Ht values for [M+3H]3+, [M+H+Ca]3+, and [M+2H+Ca]4+ are summarized in Table 3. The Ht values for [M+2H+ Ca]4+ suggested Ca2+ to be localized in the N-terminal region (residues 63-70) of the radical intermediate, [M+ 2H+Ca]3+•, despite the actual Ca2+-binding site being at residues 78-89 of α-lactalbumin. These conflicting results were attributed to an excess proton being located on the C-

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terminal Lys residue in the intermediate, which is the most efficient protonation site in the peptide consisting of residues 63-79. In this peptide, Ca2+ would be coordinated with the N-terminal region of the peptide due to Coulomb repulsion. Ca2+ effectively bound to the digested peptides containing the Ca2+-binding site of proteins and formed multiply charged ions, which provided better sequence information than the multiply protonated peptide. This metal-aided ETD could be a useful method for sequencing of acidic peptides. However, the cleavage adjacent to the acidic residue in the metal–peptide complex produces long-lived c'-z• complexes, which are then transformed into the c• and z' fragment pair by inter-fragment hydrogen migration. These c' and z' fragments are heavier than the corresponding c• and z• fragments by 1.0078 Da, and the acidic residues (Asp and Glu) are heavier than their corresponding amide forms (Asn and Gln) by 0.9840 Da. The difference of 0.0238 (1.0078– 0.9840) Da is too small to discriminate between acid and amide residues when both c•/c' or z'/z• are present in the ETD mass spectrum. However, understanding of the interfragment hydrogen migration mechanism will predict this type of transformation and facilitate interpretation of the ETD spectra of metal–peptide complexes.

Conclusion

Mechanism of Doubly-Charged Fragment Formation

Acknowledgments

ETD of the triply charged glycopeptide [M + 2H + K]3+ produced doubly-charged z8• fragments derived from cleavage at Glu1–Glu2 (Fig. 1d). As explained above, cleavage of the Glu1–Glu2 bond yielded a long-lived intermediate and subsequent inter-fragment hydrogen migration to form the [z8'+ K]+ fragment. In contrast, mainly the [z8• + H + K]2+ fragment was observed instead of the [z8' + H + K]2+ fragment (inset of Fig. 1c). This result suggested that the formation of doubly charged z8• fragments occurred via a short-lived radical intermediate, which might have a conformation similar to that of the precursor ion. According to the molecular mechanics simulation illustrated in Fig. 4a, K+ in precursor ions does not interact with Glu residues near the N-terminus and, thus, the lifetime of the c1'-z8• complex is probably short. Similarly, as shown in Fig. 6d, [z15'–H + Ca]+ and [z14'–H + Ca]+ were formed from [M + 2H + Ca]4+ by inter-fragment hydrogen migration, whereas barely any [z15' + Ca]2+ and [z14' + Ca]2+ were produced. Furthermore, the degree of inter-fragment hydrogen migration differs between [z9•/z9' + H]+ and [z9•/z9'–H + Ca]+ (inset of Fig. 6d), indicating that the intermediates yielding these fragment ions differed from each other conformationally. This hypothesis requires further investigations such as a double resonance ECD experiment directly measuring the lifetimes of intermediates [28] to provide more information about the intermediates in ETD of metal–peptide complexes.

We investigated ETD of metal–peptide complexes. The use of a suitable metal salt in ESI would increase the charge state of tryptic peptide ions containing acidic residues and provide information useful for sequencing by ETD. Metal cations were coordinated with several amino acid residues to form metal–peptide complexes, and this metal–peptide binding increased the lifetimes of c'-z• fragment complexes, which were eventually converted to the c• and z' fragment pair by inter-fragment hydrogen migration. The degree of interfragment hydrogen migration observed in the ETD of metal– peptide complexes was supported by the conformation of “proton-removed” precursors but not by that of precursor ions, suggesting that the charge reduction of precursor ions by ETD induces conformational rearrangement to give a “proton-reduced” form. ETD provides snapshot information on the conformation of gas-phase metal–peptide complexes, whereas the ion size is obtained by a hyphenated instrument designed for ion mobility spectroscopy and MS [30, 31]. The combination of ETD, ion mobility spectroscopy, and molecular structure calculation would allow better understanding of the chemistry of gas-phase metal–peptide complexes.

D.A. gratefully acknowledges the research fellowship from the Japan Society for the Promotion of Science (JSPS) for Young Scientists (23-10272). T.T. acknowledges JSPS KAKENHI grant number 24619003 for supporting this work. The computations of molecular structures were partially performed using the Research Center for Computational Science, Okazaki, Japan.

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Influence of metal-peptide complexation on fragmentation and inter-fragment hydrogen migration in electron transfer dissociation.

The use of metal salts in electrospray ionization (ESI) of peptides increases the charge state of peptide ions, facilitating electron transfer dissoci...
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