Article pubs.acs.org/JPCB
Kinetic Ion Thermometers for Electron Transfer Dissociation Robert Pepin and František Tureček* Department of Chemistry, Bagley Hall, Box 351700, University of Washington, Seattle, Washington 98195-1700, United States S Supporting Information *
ABSTRACT: Peptide fragment ions of the z-type were used as kinetic ion thermometers to gauge the internal energy of peptide cation-radicals produced by electron transfer in the gas-phase. Electron transfer dissociation (ETD)-produced z2 ions containing the leucine residue, z2(Leu-Lys) and z2(Leu-Arg), were found to undergo spontaneous dissociation by loss of C3H7 that was monitored by timeresolved kinetic measurements on the time scale of the linear ion trap mass spectrometer. Kinetic modeling of the dissociations, including collisional cooling and product loss by neutralization, provided unimolecular rate constants for dissociation that were converted to the z ion internal energies using RRKM theory. The internal energy of z2(Leu-Lys) and z2(Leu-Arg) fragment ions was found to decrease with the increasing size of the precursor peptide ion, indicating vibrational energy partitioning between the ion and neutral fragments and ergodic behavior. The experimentally determined excitation in the peptide cation-radicals upon electron transfer (285−327 kJ mol−1) was found to be lower than that theoretically calculated from the reaction exothermicity. The reasons for this missing energy are discussed.
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INTRODUCTION Electron transfer in a gas-phase cation−anion reaction is a highly exothermic process that results in excitation of the products and often induces their dissociation. A particularly important example of an ion−ion reaction concerns electron transfer from an anionic molecular electron donor to a multiply charged peptide ion which is implemented in electron transfer dissociation (ETD) mass spectrometry (Scheme 1).1 ETD has been studied extensively from both analytical and fundamental points of view2 and shown to trigger multiple dissociations of peptide ions thus providing information on the peptide sequence.3 A fundamental aspect of ETD that has not been addressed in detail concerns the internal energy of the charge-reduced peptide cation-radical formed by electron transfer. According to a simplified analysis, the energetics of electron transfer can be expressed by an energy balance (ΔEpf) eq 1 where REad,p is the adiabatic recombination energy of the multiply charged peptide ion and EAad,f is the electron affinity of the neutral counterpart of the electron donor, both taken as positive values: ΔEpf = REad,p − EA ad,f > 0
the ground electronic states of the ions and their neutral counterparts. However, an avoided crossing from the reactant potential energy surface can presumably proceed to an excited electronic state of one or both products, leading to eq 2 (Figure 1): ΔEp = REad,p − (EA ad,f + Eexc,f )
where ΔEp is the vibronic energy available to be deposited in peptide product p, and Eexc,f is the electronic excitation energy of product f. For example, fluoranthene, which is the neutral counterpart of the anion-radical frequently used in ETD, has several singlet excited electronic states of excitation energies Eexc,f = 333, 417, and 520 kJ mol−1 (3.45, 4.32, and 5.39 eV).5 If an excited state of fluoranthene was formed, the ΔEp available to the charge reduced peptide ion would be substantially reduced. To evaluate Eexc, one would have to determine ΔEp by measuring the internal energy of the charge reduced peptide ion and combine it with the known EAad of fluoranthene (58 kJ mol−1, 0.6 eV)6 and the REad of the peptide ion. The latter value can be obtained by electron structure calculations as a difference in the potential and zero-point vibrational energies of the multiply charged peptide ion and the associated chargereduced cation-radical.2,7 Internal energies of gas-phase ions are not directly measurable and their determination must be approached by indirect means. The energetics of electron transfer in fast collisions has previously been addressed by two methods. One relied on a “thermometer” system consisting of W(CO)6 where
(1)
This energy is available to be deposited in the products (denoted by p and f) in the form of electronic or vibrational excitation. The adiabatic nature of the electron transfer process follows from a collision model that presumes an orbiting complex in which the reactants approach each other along a long-range attractive potential energy surface (Figure 1).4 Upon approach, electron transfer occurs at some distance whereby the reactants undergo avoided crossing transition to the potential energy surface of the products. Equation 1 is typically enumerated using standard values of EA and RE that refer to © XXXX American Chemical Society
(2)
Received: October 10, 2014 Revised: January 5, 2015
A
DOI: 10.1021/jp510244d J. Phys. Chem. B XXXX, XXX, XXX−XXX
The Journal of Physical Chemistry B
Article
Scheme 1
given internal energy. The other method, also applied to fast collisional electron transfer, relied on the analysis of dissociation kinetics.12−14 In this kinetic method, experimental branching ratios for the competitive formation of dissociation products were fitted with those calculated by convoluting theoretical (RRKM) rate constants with an energy distribution function. The parameters defining the energy distribution function then produced the most probable and mean internal energy of the dissociating species.14 Both methods implied that the time scale for the dissociations was commensurate with the time scale of the experiment. Unfortunately, direct kinetic measurements are not feasible under ETD conditions in ion traps. This is because the lifetimes of charge-reduced peptide ions are much shorter than the ETD experimental time scale, which includes the time for the ion− ion reaction, and product analysis that cannot be shortened below approximately 1 ms. With these restrictions in mind, we now propose and report a new indirect method that utilizes the dissociation kinetics of primary ETD products to determine their internal energy and relate it to the internal energy of the dissociating charge-reduced peptide ions. Consider an electron transfer dissociation of a doubly charged peptide ion forming a C-terminal fragment ion of the z-type and its N-terminal neutral counterpart of the c type (Scheme 1). The dissociation is driven by the internal energy of
Figure 1. Potential energy diagram for electron transfer from a molecular anion-donor to a doubly charge peptide cation. The color coding is as follows: Blue curve represents the interaction potential for the reactants, the green curve depicts the potential for the ground states of the products, and the red curve depicts the potential for the product excited electronic states.
the stepwise dissociation by loss of CO can be associated with an estimated bonding energy for each step. 8−11 The distribution of fragments formed upon electron transfer then can be used to estimate the fraction of reactive molecules of B
DOI: 10.1021/jp510244d J. Phys. Chem. B XXXX, XXX, XXX−XXX
The Journal of Physical Chemistry B
Article
the charge-reduced peptide ion that was acquired by electron transfer in combination with internal changes on the potential energy surface such as those from exothermic isomerizations.2 Backbone dissociations typically have very small threshold potential energies,2 implying that most of the nonfixed energy in the dissociating ion flows into the internal energy of the products. An additional aspect of these dissociations is that backbone fragments of the c and z type are engaged in relatively long-lived ion−molecule complexes15−17 that allow the vibrational energy to be redistributed between the fragments, which presumably occurs proportionally to their heat capacities. Hence, the internal energy of the z fragment ion is related to the internal energy of the dissociating charge-reduced peptide cation-radical. The z ion internal energy can be gauged by measuring the kinetics of its spontaneous dissociations if those occur on the time scale of the instrument. To this end, we designed peptide kinetic ion thermometers (KIT) which are z2 fragment ions from electron transfer dissociation of (Ala)nLeuArg and (Ala)nLeuLys dications where n = 2−5. The Leu N-terminated z2 ions were selected on the basis of their dissociation energetics and kinetics (Scheme 2).
The method we report consists of experimental measurements of time-dependent relative intensities of (A+ + A′+) and B+ ions and extracting the dissociation rate constants k1 for several peptide ions differing in the cn and z2 parts. The experimental values are compared to unimolecular dissociation rate constants, k1(E), calculated by RRKM on high-quality ab initio or DFT potential energy surfaces. Matching of the experimental and RRKM k1 values is then used to assign internal energy (E) to dissociating z2 ions and hence to gauge the internal energy of the peptide cation-radical precursors.
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EXPERIMENTAL SECTION Materials and Methods. All measurements were performed on an LTQ-XL ion trap mass spectrometer (Thermo Electron Fisher, San Jose, CA) equipped with an auxiliary chemical ionization source for the production of fluoranthene anions for electron transfer dissociation (ETD). (A)nLK and (A)nLR peptides were synthesized on a solid support (Wang resin, Bachem Americas, Torrance, CA, USA) using the standard fmoc technology.18 The fmoc protected amino acids were purchased from Sigma-Aldrich. Dications were produced by electrospray ionization from methanol−water−acetic acid (50/50/1) solutions, selected by mass and subjected to ETD at ion−ion reaction times that were varied from 1 to 500 ms. The relative intensities of the z2 ions, m/z 272 and 244 for LR and LK, respectively, and their respective w2 fragments at m/z 229 and 201 ions were monitored as a function of the ion−ion interaction time. The neutralization depletion rates of z2 and w2 ions were measured by generating the pertinent ions by ETD or ETD/CID, selecting them by mass and titrating them with fluoranthene anion-radicals as a function of the ion−ion reaction time. Calculations. All electron structure calculations were carried out with the Gaussian 09 suite of programs.19 Exhaustive conformational analysis of the (AALR + 2H)2+ precursor ions was performed using the ConformSearch engine described previously.20 Conformational analysis of the (AAALR + 2H)2+ precursor ions started from the lowest free-energy conformers of the related (AAVAR + 2H)2+ ions21 in which the side-chains of the Ala and Val residues were appropriately modified and the structures were fully optimized. The optimized ion structures in the Cartesian coordinate format are included in the Supporting Information (Table S1−S12) and supplied with the calculated total and zero-point vibrational energies. Conformational analysis of the z2(LR) and z2(LK) ions and transition states (TS) was performed by full geometry optimizations of starting structures in which multiple torsional angles in the Leu residue were rotated by 60 degrees. All geometry optimizations were carried out with the B3LYP22,23 and M06-2X24 density functional theory methods using the 631+G(d,p) basis set. The stationary points were characterized by harmonic frequency analysis to have the appropriate number of imaginary frequencies (0 for local energy minima, 1 for the TS). Further sets of energies were obtained by single-point B3LYP, M06-2X, and perturbational Møller−Plesset (MP2, frozen core)25 calculations using the larger 6-311++G(2d,p) and aug-cc-pVTZ basis sets.26 Additional energies were obtained with coupled-cluster single-point calculations27 with single, double, and perturbational triple excitations (CCSD(T),28 using the 6-31G(d) basis set, and these were expanded to effective CCSD(T)/aug-cc-pVTZ energies using the standard linear formula, E[CCSD(T)/aug-cc-pVTZ] ≈ E[CCSD(T)/6-31G(d)] + E[MP2/aug-cc-pVTZ] − E[MP2/6-31G-
Scheme 2
The z2(LR) and z2(LK) ions, denoted by A+, undergo uniform dissociations by loss of C3H7 radicals from the Leu side chain forming w2 ions (B+), see also Figure S1, Supporting Information. The dissociation is characterized by rate constant k1. Competing with dissociation, ions A+ undergo cooling collisions with the He gas in the ion trap, forming a fraction of nonreactive ions (A′+). The cooling is characterized by rate constant kcool. In addition, A+, A′+, and B+ are depleted by neutralizing ion−ion reactions with fluoranthene anion radicals that are characterized by the respective rate constants k2N and k3N for pseudo-first order reactions. The reaction kinetics in Scheme 2 is described by eq 3−5 that have closed-form solutions (eq 6−8), where K = k1 + k2N + kcool: dA+ = −(k1 + k 2N + kcool)A+ dt
(3)
dA′+ = kcool A+ − k 2N A′+ dt
(4)
dB+ = k1 A+ − k 3N B+ dt
(5)
A+ = A +0 e−(k1+ k 2N + kcool)t = A +0 e−Kt
(6)
A′+ =
B+ =
A +0 kcool −k 2Nt [e − e−Kt ] K − k 2N
A +0 k1 [e−k3Nt − e−Kt ] K − k 3N
(7)
(8) C
DOI: 10.1021/jp510244d J. Phys. Chem. B XXXX, XXX, XXX−XXX
The Journal of Physical Chemistry B
Article
(d)].29 The MP2 energies were corrected for contamination by higher spin states using the standard spin projection procedure30,31 and are labeled as PMP2. All reported energies include zero-point vibrational energy (ZPVE) corrections. The structure and energy data are compiled in Tables S13−S29 of the Supporting Information. The TS energies, scaled harmonic frequencies and principal moments of inertia were used for Rice-Ramsperger-Kassel-Marcus theory (RRKM) calculations.32 These were performed with the QCPE program33 that was recompiled for Windows XP and Windows 7, as described previously.34
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RESULTS Kinetic Measurements. The z2 ions generated by ETD from AnLK and AnLR peptide ions undergo a single channel dissociation by loss of C3H7 radicals from the leucine side chain, forming the pertinent w2 fragment ions. This was established by generating the z2 ions by ETD, selecting the stable fraction by mass, and collisionally activating them at different levels of excitation (Figure S1, Supporting Information). These experiments established that the z 2 ion dissociation was dominated by a single reaction channel, and its kinetics depended on a single rate constant (k1). Timedependent ETD measurements were carried out for z2(LR) ions generated from doubly charged AALR, AAALR, and AAAALR ions and for analogous z2(LK) ions generated from doubly charged AAALK and AAAALK ions to produce timeresolved ion intensity profiles analogous to breakdown diagrams. These are illustrated with the breakdown diagram for the z2 and w2 ions from AAALK (Figure 2); the others are given in Figures S2−S4 in the Supporting Information. Figure 2 graphs show that the time−resolved breakdown diagram for the z2 and w2 ions consists of two components. The first component (bottom panel in Figure 2) represents a fast depletion of the z2 ions and formation of the w2 ions which occurs on the time scale of
Kinetic Ion Thermometers for Electron Transfer Dissociation Robert Pepin and František Tureček* Department of Chemistry, Bagley Hall, Box 351700, University of Washington, Seattle, Washington 98195-1700, United States S Supporting Information *
ABSTRACT: Peptide fragment ions of the z-type were used as kinetic ion thermometers to gauge the internal energy of peptide cation-radicals produced by electron transfer in the gas-phase. Electron transfer dissociation (ETD)-produced z2 ions containing the leucine residue, z2(Leu-Lys) and z2(Leu-Arg), were found to undergo spontaneous dissociation by loss of C3H7 that was monitored by timeresolved kinetic measurements on the time scale of the linear ion trap mass spectrometer. Kinetic modeling of the dissociations, including collisional cooling and product loss by neutralization, provided unimolecular rate constants for dissociation that were converted to the z ion internal energies using RRKM theory. The internal energy of z2(Leu-Lys) and z2(Leu-Arg) fragment ions was found to decrease with the increasing size of the precursor peptide ion, indicating vibrational energy partitioning between the ion and neutral fragments and ergodic behavior. The experimentally determined excitation in the peptide cation-radicals upon electron transfer (285−327 kJ mol−1) was found to be lower than that theoretically calculated from the reaction exothermicity. The reasons for this missing energy are discussed.
■
INTRODUCTION Electron transfer in a gas-phase cation−anion reaction is a highly exothermic process that results in excitation of the products and often induces their dissociation. A particularly important example of an ion−ion reaction concerns electron transfer from an anionic molecular electron donor to a multiply charged peptide ion which is implemented in electron transfer dissociation (ETD) mass spectrometry (Scheme 1).1 ETD has been studied extensively from both analytical and fundamental points of view2 and shown to trigger multiple dissociations of peptide ions thus providing information on the peptide sequence.3 A fundamental aspect of ETD that has not been addressed in detail concerns the internal energy of the charge-reduced peptide cation-radical formed by electron transfer. According to a simplified analysis, the energetics of electron transfer can be expressed by an energy balance (ΔEpf) eq 1 where REad,p is the adiabatic recombination energy of the multiply charged peptide ion and EAad,f is the electron affinity of the neutral counterpart of the electron donor, both taken as positive values: ΔEpf = REad,p − EA ad,f > 0
the ground electronic states of the ions and their neutral counterparts. However, an avoided crossing from the reactant potential energy surface can presumably proceed to an excited electronic state of one or both products, leading to eq 2 (Figure 1): ΔEp = REad,p − (EA ad,f + Eexc,f )
where ΔEp is the vibronic energy available to be deposited in peptide product p, and Eexc,f is the electronic excitation energy of product f. For example, fluoranthene, which is the neutral counterpart of the anion-radical frequently used in ETD, has several singlet excited electronic states of excitation energies Eexc,f = 333, 417, and 520 kJ mol−1 (3.45, 4.32, and 5.39 eV).5 If an excited state of fluoranthene was formed, the ΔEp available to the charge reduced peptide ion would be substantially reduced. To evaluate Eexc, one would have to determine ΔEp by measuring the internal energy of the charge reduced peptide ion and combine it with the known EAad of fluoranthene (58 kJ mol−1, 0.6 eV)6 and the REad of the peptide ion. The latter value can be obtained by electron structure calculations as a difference in the potential and zero-point vibrational energies of the multiply charged peptide ion and the associated chargereduced cation-radical.2,7 Internal energies of gas-phase ions are not directly measurable and their determination must be approached by indirect means. The energetics of electron transfer in fast collisions has previously been addressed by two methods. One relied on a “thermometer” system consisting of W(CO)6 where
(1)
This energy is available to be deposited in the products (denoted by p and f) in the form of electronic or vibrational excitation. The adiabatic nature of the electron transfer process follows from a collision model that presumes an orbiting complex in which the reactants approach each other along a long-range attractive potential energy surface (Figure 1).4 Upon approach, electron transfer occurs at some distance whereby the reactants undergo avoided crossing transition to the potential energy surface of the products. Equation 1 is typically enumerated using standard values of EA and RE that refer to © XXXX American Chemical Society
(2)
Received: October 10, 2014 Revised: January 5, 2015
A
DOI: 10.1021/jp510244d J. Phys. Chem. B XXXX, XXX, XXX−XXX
The Journal of Physical Chemistry B
Article
Scheme 1
given internal energy. The other method, also applied to fast collisional electron transfer, relied on the analysis of dissociation kinetics.12−14 In this kinetic method, experimental branching ratios for the competitive formation of dissociation products were fitted with those calculated by convoluting theoretical (RRKM) rate constants with an energy distribution function. The parameters defining the energy distribution function then produced the most probable and mean internal energy of the dissociating species.14 Both methods implied that the time scale for the dissociations was commensurate with the time scale of the experiment. Unfortunately, direct kinetic measurements are not feasible under ETD conditions in ion traps. This is because the lifetimes of charge-reduced peptide ions are much shorter than the ETD experimental time scale, which includes the time for the ion− ion reaction, and product analysis that cannot be shortened below approximately 1 ms. With these restrictions in mind, we now propose and report a new indirect method that utilizes the dissociation kinetics of primary ETD products to determine their internal energy and relate it to the internal energy of the dissociating charge-reduced peptide ions. Consider an electron transfer dissociation of a doubly charged peptide ion forming a C-terminal fragment ion of the z-type and its N-terminal neutral counterpart of the c type (Scheme 1). The dissociation is driven by the internal energy of
Figure 1. Potential energy diagram for electron transfer from a molecular anion-donor to a doubly charge peptide cation. The color coding is as follows: Blue curve represents the interaction potential for the reactants, the green curve depicts the potential for the ground states of the products, and the red curve depicts the potential for the product excited electronic states.
the stepwise dissociation by loss of CO can be associated with an estimated bonding energy for each step. 8−11 The distribution of fragments formed upon electron transfer then can be used to estimate the fraction of reactive molecules of B
DOI: 10.1021/jp510244d J. Phys. Chem. B XXXX, XXX, XXX−XXX
The Journal of Physical Chemistry B
Article
the charge-reduced peptide ion that was acquired by electron transfer in combination with internal changes on the potential energy surface such as those from exothermic isomerizations.2 Backbone dissociations typically have very small threshold potential energies,2 implying that most of the nonfixed energy in the dissociating ion flows into the internal energy of the products. An additional aspect of these dissociations is that backbone fragments of the c and z type are engaged in relatively long-lived ion−molecule complexes15−17 that allow the vibrational energy to be redistributed between the fragments, which presumably occurs proportionally to their heat capacities. Hence, the internal energy of the z fragment ion is related to the internal energy of the dissociating charge-reduced peptide cation-radical. The z ion internal energy can be gauged by measuring the kinetics of its spontaneous dissociations if those occur on the time scale of the instrument. To this end, we designed peptide kinetic ion thermometers (KIT) which are z2 fragment ions from electron transfer dissociation of (Ala)nLeuArg and (Ala)nLeuLys dications where n = 2−5. The Leu N-terminated z2 ions were selected on the basis of their dissociation energetics and kinetics (Scheme 2).
The method we report consists of experimental measurements of time-dependent relative intensities of (A+ + A′+) and B+ ions and extracting the dissociation rate constants k1 for several peptide ions differing in the cn and z2 parts. The experimental values are compared to unimolecular dissociation rate constants, k1(E), calculated by RRKM on high-quality ab initio or DFT potential energy surfaces. Matching of the experimental and RRKM k1 values is then used to assign internal energy (E) to dissociating z2 ions and hence to gauge the internal energy of the peptide cation-radical precursors.
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EXPERIMENTAL SECTION Materials and Methods. All measurements were performed on an LTQ-XL ion trap mass spectrometer (Thermo Electron Fisher, San Jose, CA) equipped with an auxiliary chemical ionization source for the production of fluoranthene anions for electron transfer dissociation (ETD). (A)nLK and (A)nLR peptides were synthesized on a solid support (Wang resin, Bachem Americas, Torrance, CA, USA) using the standard fmoc technology.18 The fmoc protected amino acids were purchased from Sigma-Aldrich. Dications were produced by electrospray ionization from methanol−water−acetic acid (50/50/1) solutions, selected by mass and subjected to ETD at ion−ion reaction times that were varied from 1 to 500 ms. The relative intensities of the z2 ions, m/z 272 and 244 for LR and LK, respectively, and their respective w2 fragments at m/z 229 and 201 ions were monitored as a function of the ion−ion interaction time. The neutralization depletion rates of z2 and w2 ions were measured by generating the pertinent ions by ETD or ETD/CID, selecting them by mass and titrating them with fluoranthene anion-radicals as a function of the ion−ion reaction time. Calculations. All electron structure calculations were carried out with the Gaussian 09 suite of programs.19 Exhaustive conformational analysis of the (AALR + 2H)2+ precursor ions was performed using the ConformSearch engine described previously.20 Conformational analysis of the (AAALR + 2H)2+ precursor ions started from the lowest free-energy conformers of the related (AAVAR + 2H)2+ ions21 in which the side-chains of the Ala and Val residues were appropriately modified and the structures were fully optimized. The optimized ion structures in the Cartesian coordinate format are included in the Supporting Information (Table S1−S12) and supplied with the calculated total and zero-point vibrational energies. Conformational analysis of the z2(LR) and z2(LK) ions and transition states (TS) was performed by full geometry optimizations of starting structures in which multiple torsional angles in the Leu residue were rotated by 60 degrees. All geometry optimizations were carried out with the B3LYP22,23 and M06-2X24 density functional theory methods using the 631+G(d,p) basis set. The stationary points were characterized by harmonic frequency analysis to have the appropriate number of imaginary frequencies (0 for local energy minima, 1 for the TS). Further sets of energies were obtained by single-point B3LYP, M06-2X, and perturbational Møller−Plesset (MP2, frozen core)25 calculations using the larger 6-311++G(2d,p) and aug-cc-pVTZ basis sets.26 Additional energies were obtained with coupled-cluster single-point calculations27 with single, double, and perturbational triple excitations (CCSD(T),28 using the 6-31G(d) basis set, and these were expanded to effective CCSD(T)/aug-cc-pVTZ energies using the standard linear formula, E[CCSD(T)/aug-cc-pVTZ] ≈ E[CCSD(T)/6-31G(d)] + E[MP2/aug-cc-pVTZ] − E[MP2/6-31G-
Scheme 2
The z2(LR) and z2(LK) ions, denoted by A+, undergo uniform dissociations by loss of C3H7 radicals from the Leu side chain forming w2 ions (B+), see also Figure S1, Supporting Information. The dissociation is characterized by rate constant k1. Competing with dissociation, ions A+ undergo cooling collisions with the He gas in the ion trap, forming a fraction of nonreactive ions (A′+). The cooling is characterized by rate constant kcool. In addition, A+, A′+, and B+ are depleted by neutralizing ion−ion reactions with fluoranthene anion radicals that are characterized by the respective rate constants k2N and k3N for pseudo-first order reactions. The reaction kinetics in Scheme 2 is described by eq 3−5 that have closed-form solutions (eq 6−8), where K = k1 + k2N + kcool: dA+ = −(k1 + k 2N + kcool)A+ dt
(3)
dA′+ = kcool A+ − k 2N A′+ dt
(4)
dB+ = k1 A+ − k 3N B+ dt
(5)
A+ = A +0 e−(k1+ k 2N + kcool)t = A +0 e−Kt
(6)
A′+ =
B+ =
A +0 kcool −k 2Nt [e − e−Kt ] K − k 2N
A +0 k1 [e−k3Nt − e−Kt ] K − k 3N
(7)
(8) C
DOI: 10.1021/jp510244d J. Phys. Chem. B XXXX, XXX, XXX−XXX
The Journal of Physical Chemistry B
Article
(d)].29 The MP2 energies were corrected for contamination by higher spin states using the standard spin projection procedure30,31 and are labeled as PMP2. All reported energies include zero-point vibrational energy (ZPVE) corrections. The structure and energy data are compiled in Tables S13−S29 of the Supporting Information. The TS energies, scaled harmonic frequencies and principal moments of inertia were used for Rice-Ramsperger-Kassel-Marcus theory (RRKM) calculations.32 These were performed with the QCPE program33 that was recompiled for Windows XP and Windows 7, as described previously.34
■
RESULTS Kinetic Measurements. The z2 ions generated by ETD from AnLK and AnLR peptide ions undergo a single channel dissociation by loss of C3H7 radicals from the leucine side chain, forming the pertinent w2 fragment ions. This was established by generating the z2 ions by ETD, selecting the stable fraction by mass, and collisionally activating them at different levels of excitation (Figure S1, Supporting Information). These experiments established that the z 2 ion dissociation was dominated by a single reaction channel, and its kinetics depended on a single rate constant (k1). Timedependent ETD measurements were carried out for z2(LR) ions generated from doubly charged AALR, AAALR, and AAAALR ions and for analogous z2(LK) ions generated from doubly charged AAALK and AAAALK ions to produce timeresolved ion intensity profiles analogous to breakdown diagrams. These are illustrated with the breakdown diagram for the z2 and w2 ions from AAALK (Figure 2); the others are given in Figures S2−S4 in the Supporting Information. Figure 2 graphs show that the time−resolved breakdown diagram for the z2 and w2 ions consists of two components. The first component (bottom panel in Figure 2) represents a fast depletion of the z2 ions and formation of the w2 ions which occurs on the time scale of
