Quantum Chemical Mass Spectrometry: Verification and Extension of the Mobile Proton Model for Histidine

Research Article

Abstract

The quantum chemical mass spectrometry for materials science (QCMS2) method is used to verify the proposed mechanism for proton transfer – the Mobile Proton Model (MPM) – by histidine for ten XHS tripeptides, based on quantum chemical calculations at the DFT/B3LYP/6-311+G* level of theory. The fragmentations of the different intermediate structures in the MPM mechanism are studied within the QCMS2 framework, and the energetics of the proposed mechanism itself and those of the fragmentations of the intermediate structures are compared, leading to the computational confirmation of the MPM. In addition, the calculations suggest that the mechanism should be extended from considering only the formation of five-membered ring intermediates to include larger-ring intermediates.

Graphical Abstract

Keywords

Quantum chemical mass spectrometry (QCMS2Mobile proton model Density functional theory Tripeptides 

Introduction

The analysis of peptide fragmentation in tandem mass spectrometry (MS/MS) has become the method of choice for protein identification [1, 2]: the sequence of peptides can be determined based on their fragmentation patterns and sequence information from databases, and the final protein identification can be achieved using one of a variety of search algorithms like SEQUEST [3, 4] and Mascot [5]. However, studies have shown that 40–70% of high-signal/noise MS/MS spectra cannot be matched to predicted protein spectra or are even misidentified by these widely used search algorithms [6]. This is mainly due to the fact that the complete set of fragmentations of any given peptide is not a priori known, which leads to the conclusion that any method allowing to obtain more detailed knowledge of the fragmentation mechanisms of peptides will be key to considerable improvements to the search algorithms mentioned above and, from there, to highly reliable protein identification.

In this respect, the Mobile Proton Model (MPM) [7, 8, 9, 10, 11, 12, 13, 14] is an important concept since this is the most comprehensive model currently available to describe peptide fragmentation, and forms an important part of the fundamentals of the currently available tools. The MPM states that following ion activation, the proton(s) added to a peptide will migrate from the initial protonation site to various other sites within the peptide to induce charge-directed fragmentation at the backbone, resulting in the formation of the typical b- and y-ions. The details of the mechanism of the proton transfer and subsequent formation of b- and y-ions depend on the sequence of the peptide in question. The MPM has been supported experimentally [11] using deuterium labeling techniques [15, 16, 17, 18], (statistical) analysis of mass spectra [19, 20, 21, 22, 23, 24, 25], and fragmentation efficiency curves [8, 9, 10, 12, 13, 26].

Attempts have also been made to validate the MPM using quantum chemical calculations. The first were based on MNDO (Modified Neglect of Differential Overlap) calculations of the bond orders of the bonds in the various protonated forms (protomers) of small peptides [27, 28]. The predicted differences in bond strength for the different protomers indicated that also those whose formation is not preferred energetically should be considered in peptide fragmentation, which forms the basis of the MPM. More sophisticated calculations were performed by Paizs and Csonka et al. who studied the structures of and the energetics associated with the various protomers of model peptides [29, 30, 31, 32]; calculated transition-state energies were subsequently used in conjunction with the Rice-Ramsperger-Kassel-Marcus (RRKM) formalism to generate unimolecular reaction rates and understand the time scales of the underlying processes. The results were in agreement with the MPM. More recently, the peptides’ charge distributions were taken into account in the calculations [33, 34, 35, 36, 37]: the total charge of carbon (QC) and nitrogen (QN) atoms in the amide bonds was calculated and it was shown that an increase of the QC/QN ratio results in a high fragmentation efficiency of a peptide. These calculations made the relationship between charge distribution and peptide fragmentation more explicit and thus led to a considerably greater understanding of the MPM. Calculations on energy barriers for proton transfer [38] and chemical dynamics simulations [24] have shown that the MPM is necessary to understand peptide fragmentation.

Despite the efforts described above, the mechanism of the MPM has not yet been evaluated as a whole and in sufficient detail using quantum chemical calculations, although, considering its importance, it deserves such a treatment. Also, the precise sequence of amino acids in the peptide is important since the initial protonation site influences the subsequent steps in the mechanism [11]. When histidine in particular is part of a peptide, a variation of the MPM, presented in Figure 1, is proposed to account for the formation of histidine b-ion after the transfer of the mobile proton [13, 39]: after protonation of the histidine side chain, an intramolecular transfer of one proton from the imidazole moiety of histidine to its carbonyl oxygen atom occurs (step 1 in Figure 1), resulting in an electropositive histidine carbonyl carbon atom that can be attacked by the nucleophilic histidine imino nitrogen atom generating a “fused bicyclic five-membered ring structure” (step 2 in Figure 1), which is rearranged in an intramolecular atom shuttling giving rise to a unique, protonated histidine b-ion and a neutral C-terminal fragment (step 3 in Figure 1).
Figure 1

The proposed mechanism for the MPM by histidine for XHS

This mechanism has been supported experimentally by such observations as the fact that alkylation of the histidine side chain blocks the subsequent hydrogen transfer and changes the appearance of the mass spectrum [39], but computational support has been limited to confirming the structure of the b-ion, in comparison to other typical b-ions such as oxazolone or acylium ions [40]. Considering the importance of obtaining more detailed insight into the fragmentation mechanisms of peptides in order to improve the above mentioned computational tools for protein identification, we have performed a detailed analysis of the full mechanism of the MPM for histidine.

Recently, we developed a new and generally applicable ab initio method for the prediction of fragmentation routes named quantum chemical mass spectrometry for materials science (QCMS2) [41], based on straightforward calculations of bond orders and energies of fragments: QCMS2 has been used to successfully predict the main features in the mass spectra of organic compounds and, more importantly, has led to the discovery of new fragmentation mechanisms. In our analysis of the MPM, QCMS2 is used to corroborate the various steps of the mechanism proposed for histidine as the central amino acid in ten tripeptides X-His-Ser with X = Asn, Asp, Gln, Glu, His, Pro, Lys, Ser, Trp, and Tyr. The energetics of the three steps in the MPM are compared with those of competing non-MPM fragmentations. Furthermore, the results of the calculations suggest that when interactions occur between the mobile proton in the imidazole moiety of histidine and the oxygen atoms of the carbonyl groups of the other amino acids in the backbone, the mechanism should be extended to include larger-ring intermediates.

Experimental

Conformational analyses were performed using the simulated annealing procedure [42] implemented in AMPAC 10.1.9 [43] with the semi-empirical AM1 method [44]. Quantum chemical calculations, i.e., the calculation of the molecular electrostatic potential (MEP), the geometry optimizations of the protonated tripeptides and the cyclic intermediates, and the energetics of the proposed MPM with histidine, were performed using the Gaussian 09 suite of programs [45], at the level of Density Functional Theory (DFT), using the B3LYP functional [46, 47, 48, 49, 50], applying both the restricted and unrestricted formalisms, and the 6-311+G* basis set [51]; the functional and basis sets were used as they are implemented in the program. Harmonic frequency calculations were performed to verify that the resulting structures are minima on the Potential-Energy Surface (PES). The QCMS2 method is automated and has been implemented into the BRABO [52] and STOCK [53] software packages, and the calculations of the fragmentation pathways were also performed at the DFT/B3LYP/6-311+G* level of theory. Bond orders were calculated using the Fractional Occupation Hirsfeld Iterative (FOHI) formalism [54], which is based on the Hirshfeld partitioning of the electron density [55, 56, 57, 58, 59, 60, 61]. Transition-state energies were calculated using the Synchronous Transit-Guided Quasi-Newton (STQN) method (QST3) [62, 63] implemented in Gaussian 09. The AIM2000 program [64, 65, 66], based on Bader’s Quantum Theory of Atoms In Molecules (QTAIM) [67], was used to calculate the electron densities in the bond critical points (BCPs) associated with the intramolecular interactions.

Results and Discussion

The two steps in the procedure involving the QCMS2 method when applied to the fragmentations of tripeptides are presented in Figure 2: in the following sections the ionization step and the fragmentation step will be discussed separately.
Figure 2

The two steps in the procedure involving the QCMS2 method applied to tripeptides: ionization and fragmentation

Ionization Step

The ionization step naturally depends on the precise ionization technique, which is used in practice. For electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI), the execution of this step is more time-consuming than for instance for electron ionization (EI), since the most favorable protonation sites must be determined. For ESI, the ionization technique used for peptides here, the ionization step has been further subdivided into three substeps, i.e., two conformational analyses separated by the determination of the protonation site(s) (Figure 2).
  1. (1)

    The first conformational analysis is performed on the neutral tripeptide and only takes one single conformer – the most stable – into account for determining the protonation site(s): other (stable) conformers will be considered in the second conformational analysis.

     
  2. (2)
    The following methodology, previously described [68], was used to determine the different protomer(s) of the tripeptide. The molecular electrostatic potential (MEP) was calculated for the most stable conformer obtained in step (1), which shows the preferred sites for protonation. This logically leads to a number of possible protomers, the total energies of which are obtained from a geometry optimization. More protomers than only the most energy-favorable one have to be taken into account if the Boltzmann distribution (at the temperature of the ionization source, i.e., 473–523 K) of the possible protomers reveals that the ratio of two protomers is higher than the detection limit of the mass spectrometer (3%) [69, 70]: this means that the mass spectrometer is sensitive enough to differentiate between two protomers. This leads to an energy cutoff of 15.25 kJ.mol−1. The data in Table 1 show that for the 10 studied tripeptides, the imidazole ring of the central histidine is always the most stable protonation site, except for HHS, for which it is the peripheral His; taking the energy cutoff into account, only this protomer needs to be considered. In the case of PHS, both the His-protonated and the protomer with protonation on the N-terminus have to be taken into account.
    Table 1

    Calculated Energies (ΔE in kJ.mol–1) of the Second-Most Stable Protomers of the ten Studied Tripeptides, Relative to the Energy of the Protomer Obtained after Protonation of the Imidazole Moiety of the Central Histidine; for HHS Protonation on the Peripheral Histidine is Favored

    Tripeptides

    Protonation site

    ΔE

    NHS

    N-terminus

    53.67

    DHS

    N-terminus

    40.66

    QHS

    N-terminus

    68.98

    EHS

    N-terminus

    87.56

    HHS

    Histidine imidazole (central)

    24.54

    PHS

    N-terminus

    15.19

    KHS

    Lysine amino group

    32.50

    SHS

    N-terminus

    60.76

    WHS

    N-terminus

    30.77

    YHS

    N-terminus

    43.77

     
  3. (3)

    In the second conformational analysis performed on the protonated tripeptide(s), only the lowest-energy conformers are taken into account: this means only those conformers that have a lower energy than the largest increase in relative energy. Conformers that only differ in the rotation of an X–H bond are also omitted from this selection.

     

The above leads to one conformer being considered for YHS, two for QHS, HHS, SHS, and WHS, four for NHS and EHS, five for DHS, seven for KHS, and two times two for PHS.

Fragmentation Steps

QCMS2 Method

The fragmentation of the tripeptides is simulated using the QCMS2 method, the details of which are described in detail in Reference 41. Using a set of rules the most likely fragmentation pathways are identified based on calculated reaction energies ΔE when bond cleavage is considered and on activation energies ΔE when rearrangements are taken into account. When QCMS2 was initially applied to the ten tripeptides, the traditional a- and x-ions were predicted and fragmentation of the side chain was observed [71], but the traditional b- and y-ions were not observed since these are the result of breaking strong bonds [41]. To compensate for this, the MPM has been explicitly incorporated into QCMS2 in the form of an additional subroutine, so that all relevant fragmentations are taken into account: in the following the energetics of the proposed mechanism for the formation of the histidine b-ion will be compared with the energetics of the other, non-MPM fragmentation routes in order to corroborate the mechanism. Considering that the three steps in the MPM are kinetically controlled we focus on activation energies.

MPM Mechanism for Histidine

The verification of the proposed mechanism (Figure 1) will be discussed in detail for EHS; for the other tripeptides the data is presented in the Tables. For EHS, four conformers were taken into account and these are presented in Figure 3. In order for an intramolecular transfer of a mobile proton from the imidazole moiety of the central histidine to the carbonyl oxygen atom of that same histidine, i.e., the first step in the proposed mechanism (step 1 in Figure 1), to occur, there must be an interaction between them. For two of the four conformers of EHS (conformer 3 in Figure 3c and conformer 4 in Figure 3d), there is indeed such an interaction, considering the H…O distances of 2.271 Å (conformer 3) and 2.266 Å (conformer 4), which are lower than the sum of the Van der Waals radii (2.72 Å). The nature of these interactions was further corroborated based on a topological analysis of the total molecular electron density using the atoms-in-molecules (AIM) theory [67], which provides a definition of the chemical bond based on physical observables: two atoms are bonded when there is a BCP that gives rise to a bond path connecting these atoms. The values of the electron density in these BCPs are listed in Table 2 for the total set of conformers of each of the ten tripeptides. They are typical of strong hydrogen bonds (the value of the electron density in the BCP of the NH…O interaction between methylamine and water is 0.0183 a.u.) and it is clear that all tripeptides display an interaction between the mobile proton and the carbonyl group of the central histidine itself.
Figure 3

The four conformers of EHS in increasing order of energy from (a) to (d); the red dashed lines indicate the intramolecular H…O interactions with the carbonyl groups in the backbone (see text for details)

Table 2

Electron densities (in a.u.) in the BCPs of the Intramolecular Interactions Between the Imidazole Moiety of the Central Histidine and Each of the Three Carbonyl Oxygen Atoms of the Backbone in the Conformers Considered for Each of the ten Tripeptides; an Empty Field Means No Interaction

Tripeptides

N-terminus

Central

C-terminus

NHS

0.0228

0.0143

 

DHS

0.0171

0.0144

 

QHS

 

0.0166

0.0163

EHS

0.0132

0.0134

0.0179

HHS

0.0160

0.0164

 

PHS

 

0.0225

 

KHS

 

0.0164

0.0166

SHS

 

0.0170

0.0165

WHS

 

0.0167

0.0162

YHS

 

0.0224

 
The energetics associated with the proton transfer (step 1 in Figure 1) have been summarized in Table 3. These activation energies can be compared with the reaction energies of the lowest-energy non-MPM fragmentation routes given in Table 4 as ΔEfrag1 and graphically presented in Figure 4. It is clear that for EHS, the proton transfer (ΔE 1 = 143.46 kJ.mol−1, Table 3) is a more favored process than the lowest-energy fragmentation, in this case a heterolytic cleavage with ΔEfrag1 = 207.46 kJ.mol−1 (Table 4). The data in Tables 3 and 4 indicate that this is true for all 10 tripeptides, which means that the calculations corroborate the first step of the MPM.
Table 3

Activation (ΔE) Energies (in kJ.mol–1) of the Three Consecutive Steps in the MPM Mechanism for Histidine

Tripeptides

Step 1

Step 2

Step 3

ΔE 1

ΔE 2

ΔE 3

NHS

141.34

15.93

194.19

DHS

99.62

64.47

160.12

QHS

178.74

42.02

186.45

EHS

143.46

52.70

158.40

HHS

177.52

12.67

211.80

PHS

118.11

41.54

168.14

KHS

150.19

46.22

194.30

SHS

149.75

85.71

172.64

WHS

117.95

67.69

180.45

YHS

153.09

69.69

183.79

Table 4

Reaction Energies of the Lowest-Energy non-MPM Fragmentation Routes of the Initial N-Protonated Tripeptides (ΔEfrag1 in kJ.mol–1), of the O-Protonated Tripeptides (ΔEfrag2 in kJ.mol–1) and of the Cyclic Intermediates (ΔEfrag3 in kJ.mol–1)

Tripeptides

ΔEfrag1

ΔEfrag2

ΔEfrag3

NHS

263.73

266.06

384.44

DHS

261.81

294.38

345.22

QHS

278.78

110.43

323.80

EHS

207.46

279.40

300.56

HHS

198.87

231.73

350.02

PHS

267.56

286.50

316.42

KHS

281.22

112.06

303.99

SHS

246.73

291.57

249.86

WHS

284.87

197.01

261.22

YHS

274.23

238.98

257.22

Figure 4

Reaction energies (ΔE in kJ.mol−1) of the lowest-energy non-MPM fragmentation routes for EHS

The second step of the mechanism comprises the attack of the nucleophilic imino nitrogen atom of histidine on the electropositive histidine carbonyl carbon atom (step 2 in Figure 1) resulting in what is commonly called a “fused bicyclic five-membered ring structure”, but which is formally a bicyclo[3.3.0]diazaoctane (BCO) derivative. The activation energies associated with this second step are also listed in Table 3 and are quite low: for EHS it is ΔE 2 = 52.70 kJ.mol−1, which means that the formation of the cyclic structure proceeds very easily. Since the lowest-energy fragmentation of the O-protonated EHS (a heterolytic cleavage) has a reaction energy of ΔEfrag2 = 279.40 kJ.mol−1 (Table 4), it is clear that the more favored process is the formation of the cyclic structure. The data in Tables 3 and 4 indicate that this is true for all ten tripeptides, which means that the calculations also corroborate the second step of the MPM. Indeed, for all ten tripeptides the activation energy of the second step is low compared with the other two steps indicating a particularly efficient process.

The third and final step of the MPM mechanism is a rearrangement that induces cleavage forming a unique, protonated histidine b-ion and a neutral C-terminal fragment (step 3 in Figure 1). For EHS, this step has ΔE 3 = 158.40 kJ.mol−1 (Table 3). Since the lowest-energy non-MPM fragmentation of the cyclic intermediate of EHS (a heterolytic cleavage) has a reaction energy of ΔEfrag3 = 300.56 kJ.mol−1 (Table 4), it is clear that the more favored process is the break-up of the cyclic structure as defined in the MPM. The data in Tables 3 and 4 indicate that this is true for all ten tripeptides, which means that the calculations also corroborate the final step of the MPM.

Extension of the Proposed Mechanism

Now that the MPM for histidine as presented in the literature has been confirmed computationally, it seems that there is room for extension of the model. Indeed, the data in Table 2 suggest that depending on the precise sequence of amino acids one or two additional hydrogen bonds can be created in the conformers considered for each of the ten tripeptides, next to the one between the mobile proton and the carbonyl group of the central histidine itself: four tripeptides allow a second interaction with the carbonyl group at the N-terminus and five a second interaction with the carbonyl group at the C-terminus. Just one tripeptide (EHS) allows all three possible interactions. Figure 3b, c, and d illustrate the presence of H…O interactions between the imidazole moiety of the central histidine and the other carbonyl oxygen atoms of the backbone in three of the four conformers of EHS. Conformer 2 (Figure 3b) displays an interaction between the mobile proton and the carbonyl oxygen atom of the N-terminus with an H…O distance of 2.265 Å, while conformers 3 (Figure 3c) and 4 (Figure 3d) display interactions between the mobile proton and the carbonyl oxygen atom of the C-terminus with H…O distances 2.067 Å and 2.043 Å, respectively; all distances are below the sum of the Van der Waals radii. Despite the presence of these intramolecular interactions, however, the energetics will ultimately determine whether the associated processes are favorable in comparison with the alternatives.

For conformer 2 of EHS, the transfer of the mobile proton to the carbonyl oxygen atom of the N-terminus has ΔE = 187.24 kJ.mol−1 (Table 5), and this value is not much higher than, but comparable to, that found for the original MPM (ΔE 1 = 143.46 kJ.mol−1, Table 3). When the nucleophilic imino nitrogen atom of histidine attacks the electropositive carbonyl carbon atom, the result is a bicyclic intermediate containing a six-membered ring, which is formally a bicyclo[4.3.0]diazanonane (BCN) derivative. The activation energy is low: for EHS it is 46.07 kJ.mol−1 (Table 5), and this value is again comparable to that of the original MPM (ΔE 2 = 52.70 kJ.mol−1, Table 3).
Table 5

Activation Energies (ΔE in kJ.mol–1) of the First Two Steps in the “Six-Membered-Ring MPM” Mechanism for Histidine, and Reaction Energies of the Lowest-Energy Non-MPM Fragmentation Routes of the O-Protonated Tripeptides (ΔEfrag4 in kJ.mol–1)

Tripeptides

Step 1

Step 2

ΔEfrag4

ΔE

ΔE

NHS

98.71

78.98

279.57

DHS

134.96

37.04

163.65

EHS

187.84

46.07

242.44

For conformers 3 and 4 of EHS a transfer of the mobile proton to the carbonyl oxygen atom of the C-terminus can be considered. In the following discussion we limit ourselves to conformer 3. For this conformer, the transfer of the mobile proton to the carbonyl oxygen atom of the C-terminus has ΔE = 275.82 kJ.mol−1 (Table 6), and this value is quite a bit higher than that found for the original MPM (ΔE 1 = 143.46 kJ.mol−1, Table 3). When the nucleophilic imino nitrogen atom of histidine attacks the electropositive carbonyl carbon atom, the result is a bicyclic intermediate containing an eight-membered ring, which is formally a bicyclo[6.3.0]diazaundecane (BCU) derivative. Again, the activation energy is low: for EHS it is 66.85 kJ.mol−1 (Table 6), and this value compares well with that of the original MPM (ΔE 2 = 52.70 kJ.mol−1, Table 3).
Table 6

Activation Energies (ΔE in kJ.mol–1) of the First Two Steps in the “Eight-Membered-Ring MPM” Mechanism for Histidine, and Reaction Energies of the Lowest-Energy Non-MPM Fragmentation Routes of the O-Protonated Tripeptides (ΔEfrag5 in kJ.mol–1)

Tripeptides

Step 1

Step 2

ΔEfrag5

ΔE

ΔE

QHS

243.00

40.59

254.74

EHS

275.82

66.85

321.44

KHS

219.20

2.35

203.68

SHS

206.83

52.38

283.98

WHS

196.73

51.89

263.54

For both the “six-membered ring MPM” (formation of a BCN) and the “eight-membered ring MPM” (formation of a BCU), the energy of the proton transfer step is smaller than or comparable to the reaction energy of the lowest-energy fragmentation route for EHS (ΔE = 207.46 kJ.mol−1, Table 4). For the six-membered ring MPM, the lowest-energy fragmentation route of the N-terminal O-protonated EHS is a heterolytic cleavage with ΔEfrag4 = 242.44 kJ.mol−1 (Table 5). For the eight-membered ring MPM, the lowest-energy fragmentation route of the C-terminal O-protonated EHS is a heterolytic cleavage with ΔEfrag5 = 321.44 kJ.mol−1 (Table 6). In both cases, the formation of the ring structure is clearly preferred.

When the energetics of the original (five-membered ring) MPM, the six-membered ring MPM, and the eight-membered ring MPM are compared graphically in Figure 5, their differences and similarities become clear. The first step, the proton transfer, has the lowest ΔE in the five-membered ring mechanism (ΔE = 143.46 kJ.mol−1, Table 3), but the value for the six-membered ring mechanism is not much higher (ΔE = 187.84 kJ.mol−1, Table 5); for the eight-membered ring mechanism, the value has increased considerably (ΔE = 275.82 kJ.mol−1, Table 6), but is still comparable (within the QCMS2 methodology [41]) with the energy of the lowest-energy non-MPM fragmentation (ΔE = 207.46 kJ.mol−1), which means that also the latter proton transfer can occur. The energetics of the second step, the formation of the BCO (ΔE = 52.70 kJ.mol−1, Table 3), the BCN (ΔE = 46.07 kJ.mol−1, Table 5), or the BCU (ΔE = 66.85 kJ.mol−1, Table 6), are even more similar, the largest difference being about 20 kJ.mol−1. The absence of any data on the third step reflects the current uncertainty on how these six- and eight-membered ring intermediates might fragment, and this remains the subject of ongoing work. Overall, the reported energy differences clearly indicate the similarity between the three mechanisms and demonstrate that all three are more or less equally feasible energetically for EHS. The data in Tables 5 and 6 indicate that this is true for all relevant tripeptides.
Figure 5

Comparison of the energetics of the first two steps [proton transfer (step 1) and formation of the cyclic intermediate (step 2)] in the (original) five- (red), and the new six- (blue) and eight-membered ring (green) MPMs for EHS (RP1 is the O-protonated intermediate, RP2 is the cyclic intermediate, and the TS are transitions states)

The Particular Case of HHS

For HHS, the mechanism and its extension are slightly different from those of the other tripeptides since it is the imidazole moiety of the peripheral histidine that is protonated rather than that of the central one. For the verification of the proposed mechanism, this means that the typical b-ion structure is smaller – it consists of only a single amino acid (the peripheral histidine) – than for the other tripeptides (for which it contains two amino acids). Its two conformers display two relevant H…O interactions: one between the mobile proton on the peripheral histidine with the oxygen atom of its own carbonyl group at the N-terminus and a second between the mobile proton and the oxygen atom of the central histidine (Table 2). The proton transfer associated with the latter has ΔE = 161.43 kJ.mol−1.

Then, there are two possibilities for the second step. The first involves the attack of the nucleophilic imino nitrogen atom of the peripheral histidine on the carbonyl group resulting in the formation of a BCU with ΔE = 113.39 kJ.mol−1 (eight-membered ring mechanism); note that the formation of this BCU differs from the ones formed for the other tripeptides since for these the interaction with the carbonyl oxygen atom of the C-terminus would lead to the formation of a BCU. The second involves the attack of the nucleophilic imino nitrogen atom of the central histidine on the carbonyl group resulting in the formation of a BCO with ΔE = 77.40 kJ.mol−1 (five-membered ring mechanism). The subsequent intramolecular atom shuttling (rearrangement) of the BCO (step 3) induces cleavage resulting in the unique b-ion structure and a neutral C-terminal fragment with ΔE = 165.55 kJ.mol−1. These fragments are naturally the same as the ones in the originally proposed mechanism. These energies are sufficiently low for both proton transfers to be favorable processes, which would result in two different b-ions.

Conclusions

The proposed mechanism for the Mobile Proton Model for histidine has been verified, as the results of the QCMS2 calculations show that the energetics of the three steps in the mechanism allow for a competition with non-MPM fragmentation routes, generally in favor of the MPM route: (1) strong H…O hydrogen bonds facilitate the transfer of the mobile proton from the initial protonation site on histidine to the oxygen atom of one of the carbonyl groups in the backbone, (2) the formation of the cyclic intermediates is an energy-favorable process, and (3) the fragmentations of the cyclic intermediates via rearrangements are also energy-favorable. The observation that the energetics of the formation and fragmentations of the BCO, BCN, and BCU intermediates are relatively similar suggests that next to the original five-membered ring mechanism, two additional mechanisms should be incorporated into MPM for histidine, i.e., the six- and the eight-membered ring mechanisms.

Notes

Acknowledgements

All calculations were performed using the Hopper HPC infrastructure at the CalcUA core facility of the University of Antwerp, a division of the Flemish Supercomputer Center VSC, funded by the Hercules Foundation, the Flemish Government (Department EWI), and the University of Antwerp. The authors also gratefully acknowledge Professor Dr. Christian Van Alsenoy for the many helpful discussions and his continued support. This research was funded by a Ph.D. grant (to J.C.) of the Agency for Innovation by Science and Technology (IWT).

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© American Society for Mass Spectrometry 2017

Authors and Affiliations

  1. 1.Department of ChemistryUniversity of AntwerpAntwerpBelgium

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