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Scott A. Mcluckey - One of the best experts on this subject based on the ideXlab platform.
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ion ion charge inversion attachment in conjunction with dipolar dc Collisional Activation as a selective screen for sulfo and phosphopeptides
International Journal of Mass Spectrometry, 2019Co-Authors: Mack Shih, Scott A. McluckeyAbstract:Abstract We describe a gas-phase approach for the rapid screening of polypeptide anions for phosphorylation or sulfonation based on binding strengths to guanidinium-containing reagent ions. The approach relies on the generation of a complex via reaction of mixtures of deprotonated polypeptide anions with dicationic guanidinium-containing reagent ions and subsequent dipolar DC Collisional Activation of the complexes. The relative strengths of the electrostatic interactions of guanidinium with deprotonated acidic sites follows the order carboxylate
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Dipolar DC induced Collisional Activation of non-dissociated electron-transfer products.
Journal of mass spectrometry : JMS, 2019Co-Authors: Sarju Adhikari, Eric T. Dziekonski, Frank A. Londry, Scott A. McluckeyAbstract:The application of electron transfer and dipolar direct current induced Collisional Activation (ET-DDC) for enhanced sequence coverage of peptide/protein cations is described. A DDC potential is applied across one pair of opposing rods in the high-pressure collision cell of a hybrid quadrupole/time-of-flight tandem mass spectrometer (QqTOF) to induce Collisional Activation, in conjunction with electron transfer reactions. As a broadband technique, DDC can be employed for the simultaneous Collisional Activation of all the first-generation charge-reduced precursor ions (eg, electron transfer no-dissociation or ETnoD products) from electron transfer reactions over a relatively broad mass-to-charge range. A systematic study of ET-DDC induced collision Activation on peptide/protein cations revealed an increase in the variety (and abundances) of sequence informative fragment ions, mainly c- and z-type fragment ions, relative to products derived directly via electron transfer dissociation (ETD). Compared with ETD, which has low dissociation efficiency for low-charge-state precursor ions, ET-DDC also showed marked improvement, providing a sequence coverage of 80% to 85% for all the charge states of ubiquitin. Overall, this method provides a simple means for the broadband Collisional Activation of ETnoD ions in the same collision cell in which they are generated for improved structural characterization of polypeptide and protein cations subjected to ETD.
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rapidly alternating transmission mode electron transfer dissociation and Collisional Activation for the characterization of polypeptide ions
Analytical Chemistry, 2008Co-Authors: Hongling Han, Yu Xia, Min Yang, Scott A. McluckeyAbstract:Cation transmission/electron-transfer reagent anion storage mode electron-transfer ion/ion reactions and beam-type Collisional Activation of the polypeptide ions are performed in rapid succession in the high-pressure collision cell (Q2) of a quadrupole/time-of-flight tandem mass spectrometer (QqTOF), where the electron-transfer reagent anions are accumulated. Duty cycles for both electron-transfer dissociation (ETD) and collision-induced dissociation (CID) experiments are improved relative to ion trapping approaches since there are no discrete ion storage and reaction steps for ETD experiments and no discrete ion storage step and frequency tuning for CID experiments. For this technique, moderately high resolution and mass accuracy are also obtained due to mass analysis via the TOF analyzer. This relatively simple approach has been demonstrated with a triply charged tryptic peptide, a triply charged tryptic phosphopeptide, and a triply charged tryptic N-linked glycopeptide. For the tryptic peptide, the sequence is identified with more certainty than would be available from a single method alone due to the complementary information provided by these two dissociation methods. Because of the complementary information derived from both ETD and CID dissociation methods, peptide sequence and post-translational modification (PTM) sites for the phosphopeptide are identified. This combined ETD and CID approach is particularly useful for characterizing glycopeptides because ETD generates information about both peptide sequence and locations of the glycosylation sites, whereas CID provides information about the glycan structure.
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ion trap Collisional Activation of c and z ions formed via gas phase ion ion electron transfer dissociation
Journal of Proteome Research, 2007Co-Authors: Hongling Han, Yu Xia, Scott A. McluckeyAbstract:A series of c- and z•-type product ions formed via gas-phase electron-transfer ion/ion reactions between protonated polypeptides with azobenzene radical anions are subjected to ion trap collision Activation in a linear ion trap. Fragment ions including a-, b-, y-type and ammonia-loss ions are typically observed in collision induced dissociation (CID) of c ions, showing almost identical CID patterns as those of the C-terminal amidated peptides consisting of the same sequences. Collisional Activation of z• species mainly gives rise to side-chain losses and peptide backbone cleavages resulting in a-, b-, c-, x-, y-, and z-type ions. Most of the fragmentation pathways of z• species upon ion trap CID can be accounted for by radical driven processes. The side-chain losses from z• species are different from the small losses observed from the charge-reduced peptide molecular species in electron-transfer dissociation (ETD), which indicates rearrangement of the radical species. Characteristic side-chain losses are ...
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beam type Collisional Activation of polypeptide cations that survive ion ion electron transfer
Rapid Communications in Mass Spectrometry, 2007Co-Authors: Hongling Han, Yu Xia, Scott A. McluckeyAbstract:Doubly protonated peptides that undergo an electron transfer reaction without dissociation in a linear ion trap can be subjected to beam-type Collisional Activation upon transfer from the linear ion trap into an adjacent mass analyzer, as demonstrated here with a hybrid triple quadrupole/linear ion trap system. The Activation can be promoted by use of a DC offset difference between the ion trap used for reaction and the ion trap into which the products are injected of 12–16 V, which gives rise to energetic collisions between the transferred ions and the collision/bath gas employed in the linear ion trap used for ion/ion reactions. Such a process can be executed routinely on hybrid linear ion trap/triple quadrupole tandem mass spectrometers and is demonstrated here with several model peptides as well as a few dozen tryptic peptides. Collisional Activation of the peptide precursor ions that survive electron transfer frequently provides structural information that is absent from the precursor ions that fragment spontaneously upon electron transfer. The degree to which additional structural information is obtained by Collisional Activation of the surviving singly charged peptide ions depends upon peptide size. Little or no additional structural information is obtained from small peptides (<8 residues) due to the high electron transfer dissociation (ETD) efficiencies noted for these peptides as well as the extensive sequence information that tends to be forthcoming from ETD of such species. Collisional Activation of the surviving electron transfer products provided greatest benefit for peptides of 8–15 residues. Copyright © 2007 John Wiley & Sons, Ltd.
Minh Tho Nguyen - One of the best experts on this subject based on the ideXlab platform.
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Collisional Activation of protonated C-halogenopyrazoles
Chemical Physics Letters, 2001Co-Authors: Robert Flammang, Monique Barbieux-flammang, Pascal Gerbaux, José Elguero, Minh Tho NguyenAbstract:Abstract Collisional Activation of protonated 3-halogenopyrazoles (X–Pz, X=Cl, Br and I) in the high or low translational energy regime induced an intense loss of X giving C 3 H 4 N 2 + radical cations whose structure depends on the nature of the halogen. Protonated 3-I–Pz generated thus ionized pyrazole a , whereas protonated 3-Cl–Pz was a precursor of an isomeric species ascribed to a dehydropyrazolium distonic structure b . A mixture of C 3 H 4 N 2 + ions was formed in protonated 3-Br–Pz. B3LYP/6-31++G(d,p) computations confirmed a regiospecific N 2 -protonation, and a low energy content of the distonic ions b or c (50 kJ mol −1 above a and lying in deep energy wells). Two competitive C–H and C–X bond cleavages were invoked to explain the contrasting behaviour of various protonated X–Pz under dehalogenation conditions.
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High‐energy Collisional Activation of the molecular ions of thiophene‐2‐one with different target gases
Journal of mass spectrometry : JMS, 2001Co-Authors: Pascal Gerbaux, Robert Flammang, Valérie Sciamanna, Minh Tho NguyenAbstract:Collisional Activation of keV thiophene-2-one radical cations 1 + with O2 or NO as the target gas leads to a desulfuration reaction. This peculiar reaction is insignificant or absent with other targets such as helium, argon, methane or nitrogen. The radical cations produced in this desulfuration reaction are most probably vinylketene ions, as indicated by a triple mass spectrometric (MS/MS/MS) experiment performed on a ‘hybrid’ tandem mass spectrometer of sector‐quadrupole‐sector configuration. Tentatively, it is proposed that population of an excited state accounts for the non-ergodic behavior of 1 + upon collision with oxygen or nitric oxide. Ab initio molecular orbital calculations using molecular orbital theory (UMP2, UCCSD(T)) and density functional theory (B3LYP) with 6‐31G(d,p) and 6‐311++G(d,p) basis sets were used to evaluate the relative energy of the excited quartet state of 1 + radical cations. This quartet state is calculated to lie about 3.6 eV above the 2 A 00 ground state and 0.9 eV above the C4H4O + + S dissociation products. It is proposed that the quartet ion serves as the precursor for the spontaneous desulfuration. Copyright © 2001 John Wiley & Sons, Ltd.
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high energy Collisional Activation of the molecular ions of thiophene 2 one with different target gases
Journal of Mass Spectrometry, 2001Co-Authors: Pascal Gerbaux, Valérie Sciamanna, R Flammang, Minh Tho NguyenAbstract:Collisional Activation of keV thiophene-2-one radical cations 1 + with O2 or NO as the target gas leads to a desulfuration reaction. This peculiar reaction is insignificant or absent with other targets such as helium, argon, methane or nitrogen. The radical cations produced in this desulfuration reaction are most probably vinylketene ions, as indicated by a triple mass spectrometric (MS/MS/MS) experiment performed on a ‘hybrid’ tandem mass spectrometer of sector‐quadrupole‐sector configuration. Tentatively, it is proposed that population of an excited state accounts for the non-ergodic behavior of 1 + upon collision with oxygen or nitric oxide. Ab initio molecular orbital calculations using molecular orbital theory (UMP2, UCCSD(T)) and density functional theory (B3LYP) with 6‐31G(d,p) and 6‐311++G(d,p) basis sets were used to evaluate the relative energy of the excited quartet state of 1 + radical cations. This quartet state is calculated to lie about 3.6 eV above the 2 A 00 ground state and 0.9 eV above the C4H4O + + S dissociation products. It is proposed that the quartet ion serves as the precursor for the spontaneous desulfuration. Copyright © 2001 John Wiley & Sons, Ltd.
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Collisional Activation of protonated halogeno-pyridines: different behaviour of target gases
Chemical Physics Letters, 2000Co-Authors: Pascal Gerbaux, Robert Flammang, Minh Tho NguyenAbstract:Abstract Protonated 2-Cl and 2-Br-pyridines undergo facile dehalogenation upon high energy (8 keV range) Collisional Activation provided the target is NO, or even better O 2 , instead of He or Ar. In the low energy regime (20–30 eV range), debromination occurs more readily than dechlorination, but the peculiar behaviour of O 2 in favouring an X-loss over a HX-elimination, is no longer detected (using a MS 3 instrument). B3LYP/6-31G(d) calculations on X-pyridines with X=F, Cl and Br on positions 2, 3 and 4, suggest that, if a protonated pyridine is formed in the lower-lying triplet state following strong interaction with O 2 or NO having higher spin, the loss of Cl or Br becomes almost spontaneous (F-loss is more difficult). Proton affinity PA(pyridine)=940±15 kJ/mol, is decreased by 5–15 kJ/mol upon halogenation.
Jean H. Futrell - One of the best experts on this subject based on the ideXlab platform.
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On the efficiency of energy transfer in Collisional Activation of small peptides
The Journal of Chemical Physics, 2002Co-Authors: Julia Laskin, Jean H. FutrellAbstract:We present a study of the efficiency of the internal excitation of small peptide ions upon multiple-collision Activation with Ar and ion-surface interaction with self-assembled monolayers of fluorinated alkylthiol on gold. Internal energy distributions are extracted from RRKM modeling of collision energy-resolved fragmentation efficiency curves for protonated tri-, tetra-, and penta-alanine, and polyltetra-alanine. The efficiency of T→V transfer in surface Collisional Activation decreases for larger peptides. This is readily rationalized by the corresponding decrease in the center-of-mass collisions energy. For all peptides except protonated dialanine, energy transfer upon multiple-collision Activation is rather insensitive to the peptide size and composition. The average energy deposited into protonated dialanine is substantially lower than the excitation level achieved for other peptides. Master equation modeling revealed that energy-transfer efficiency in peptide collisions with Ar is the same for all ...
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Tandem mass spectrometry: dissociation of ions by Collisional Activation
Journal of mass spectrometry : JMS, 2000Co-Authors: Anil K. Shukla, Jean H. FutrellAbstract:This review presents a brief historical introduction to the development of tandem mass spectrometry and its principal applications. It is placed in the context of the general principles underlying mass spectrometry, particularly the relationships between internal energy and fragmentation kinetics. The center-of-mass framework is presented as a convenient means of applying conservation of momentum to the energy transfer problem in tandem mass spectrometry as a means of deducing energy transfer in the Collisional Activation step and kinetic energy release as activated ions dissociate into fragment ions and neutrals. The principles of molecular beam methods are summarized and illustrative examples are given for which definitive information on reaction dynamics is available. The importance of scattering—very little appreciated in early discussions of tandem mass spectrometry—is shown to be the natural consequence of impulsive collisions, which appears to be a general mechanism for energy exchange in Collisional Activation. It is shown that the average energy transferred in single collisions is much less than the theoretical maximum given by the center-of-mass collision energy and the Massey criterion is presented as a simplistic rationale for understanding the essentially exponential decline in the energy transfer function above and below the relative velocity at which the probability for energy transfer is maximized. The issues of energy transfer in collisions of large molecular ions with low-mass neutrals are reviewed and a general description of energy transfer in multiple collisions is presented. It is shown that the center-of-mass and Massey criterion limitations are pragmatically overcome by multiple collision Activation in ion traps. Surface-induced dissociation is presented as a viable alternative to multiple collision Activation which is especially attractive for Activation of large molecular ions. Finally, a few of the emerging dynamics principles governing energy transfer and dissociation of peptides are summarized. Copyright © 2000 John Wiley & Sons, Ltd.
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SPECIAL FEATURE: PERSPECTIVE Tandem mass spectrometry: dissociation of ions by Collisional Activation
2000Co-Authors: Anil K. Shukla, Jean H. FutrellAbstract:This review presents a brief historical introduction to the development of tandem mass spectrometry and its principal applications. It is placed in the context of the general principles underlying mass spectrometry, particularly the relationships between internal energy and fragmentation kinetics. The center-of-mass framework is presented as a convenient means of applying conservation of momentum to the energy transfer problem in tandem mass spectrometry as a means of deducing energy transfer in the Collisional Activation step and kinetic energy release as activated ions dissociate into fragment ions and neutrals. The principles of molecular beam methods are summarized and illustrative examples are given for which definitive information on reaction dynamics is available. The importance of scattering—very little appreciated in early discussions of tandem mass spectrometry— is shown to be the natural consequence of impulsive collisions, which appears to be a general mechanism for energy exchange in Collisional Activation. It is shown that the average energy transferred in single collisions is much less than the theoretical maximum given by the center-of-mass collision energy and the Massey criterion is presented as a simplistic rationale for understanding the essentially exponential decline in the energy transfer function above and below the relative velocity at which the probability for energy transfer is maximized. The issues of energy transfer in collisions of large molecular ions with low-mass neutrals are reviewed and a general description of energy transfer in multiple collisions is presented. It is shown that the center-of-mass and Massey criterion limitations are pragmatically overcome by multiple collision Activation in ion traps. Surface-induced dissociation is presented as a viable alternative to multiple collision Activation which is especially attractive for Activation of large molecular ions. Finally, a few of the emerging dynamics principles governing energy transfer and dissociation of peptides are summarized. Copyright 2000 John Wiley & Sons, Ltd.
Robert Flammang - One of the best experts on this subject based on the ideXlab platform.
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Collisional Activation of protonated C-halogenopyrazoles
Chemical Physics Letters, 2001Co-Authors: Robert Flammang, Monique Barbieux-flammang, Pascal Gerbaux, José Elguero, Minh Tho NguyenAbstract:Abstract Collisional Activation of protonated 3-halogenopyrazoles (X–Pz, X=Cl, Br and I) in the high or low translational energy regime induced an intense loss of X giving C 3 H 4 N 2 + radical cations whose structure depends on the nature of the halogen. Protonated 3-I–Pz generated thus ionized pyrazole a , whereas protonated 3-Cl–Pz was a precursor of an isomeric species ascribed to a dehydropyrazolium distonic structure b . A mixture of C 3 H 4 N 2 + ions was formed in protonated 3-Br–Pz. B3LYP/6-31++G(d,p) computations confirmed a regiospecific N 2 -protonation, and a low energy content of the distonic ions b or c (50 kJ mol −1 above a and lying in deep energy wells). Two competitive C–H and C–X bond cleavages were invoked to explain the contrasting behaviour of various protonated X–Pz under dehalogenation conditions.
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High‐energy Collisional Activation of the molecular ions of thiophene‐2‐one with different target gases
Journal of mass spectrometry : JMS, 2001Co-Authors: Pascal Gerbaux, Robert Flammang, Valérie Sciamanna, Minh Tho NguyenAbstract:Collisional Activation of keV thiophene-2-one radical cations 1 + with O2 or NO as the target gas leads to a desulfuration reaction. This peculiar reaction is insignificant or absent with other targets such as helium, argon, methane or nitrogen. The radical cations produced in this desulfuration reaction are most probably vinylketene ions, as indicated by a triple mass spectrometric (MS/MS/MS) experiment performed on a ‘hybrid’ tandem mass spectrometer of sector‐quadrupole‐sector configuration. Tentatively, it is proposed that population of an excited state accounts for the non-ergodic behavior of 1 + upon collision with oxygen or nitric oxide. Ab initio molecular orbital calculations using molecular orbital theory (UMP2, UCCSD(T)) and density functional theory (B3LYP) with 6‐31G(d,p) and 6‐311++G(d,p) basis sets were used to evaluate the relative energy of the excited quartet state of 1 + radical cations. This quartet state is calculated to lie about 3.6 eV above the 2 A 00 ground state and 0.9 eV above the C4H4O + + S dissociation products. It is proposed that the quartet ion serves as the precursor for the spontaneous desulfuration. Copyright © 2001 John Wiley & Sons, Ltd.
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Collisional Activation of protonated halogeno-pyridines: different behaviour of target gases
Chemical Physics Letters, 2000Co-Authors: Pascal Gerbaux, Robert Flammang, Minh Tho NguyenAbstract:Abstract Protonated 2-Cl and 2-Br-pyridines undergo facile dehalogenation upon high energy (8 keV range) Collisional Activation provided the target is NO, or even better O 2 , instead of He or Ar. In the low energy regime (20–30 eV range), debromination occurs more readily than dechlorination, but the peculiar behaviour of O 2 in favouring an X-loss over a HX-elimination, is no longer detected (using a MS 3 instrument). B3LYP/6-31G(d) calculations on X-pyridines with X=F, Cl and Br on positions 2, 3 and 4, suggest that, if a protonated pyridine is formed in the lower-lying triplet state following strong interaction with O 2 or NO having higher spin, the loss of Cl or Br becomes almost spontaneous (F-loss is more difficult). Proton affinity PA(pyridine)=940±15 kJ/mol, is decreased by 5–15 kJ/mol upon halogenation.
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an unexpected effect of the nature of the collision gas in Collisional Activation mass spectrometry
Rapid Communications in Mass Spectrometry, 1996Co-Authors: Robert Flammang, Laurence Gallez, Yves Van Haverbeke, Ming Wah Wong, Curt WentrupAbstract:Radical-cations a-c with the structure HN=C=C=X (X=O, NH, S respectively) have been studied by Collisional-Activation (CA) mass spectrometry using different target gases (helium, oxygen and nitrogen). An unusual effect of the nature of the collision gas has been noted for oxygen which induces a very intense loss of a nitrogen atom for ions a and b, but not for ion c. The replacement of the NH hydrogen by a methyl group suppresses this effect Oxygen therefore appears to induce an isomerization of ions a and b into nitrene isomers a′ and b′ (N - CH=C=X). The use of nitrogen, which gives essentially the same CA spectra as helium, eliminates the possibility of an effect based on the center-of-mass collision energy. G2(MP2) calculations indicate that the nitrene radical-cations lie close in energy to the cumulene ions when X=O or NH.
Pascal Gerbaux - One of the best experts on this subject based on the ideXlab platform.
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Collisional Activation of protonated C-halogenopyrazoles
Chemical Physics Letters, 2001Co-Authors: Robert Flammang, Monique Barbieux-flammang, Pascal Gerbaux, José Elguero, Minh Tho NguyenAbstract:Abstract Collisional Activation of protonated 3-halogenopyrazoles (X–Pz, X=Cl, Br and I) in the high or low translational energy regime induced an intense loss of X giving C 3 H 4 N 2 + radical cations whose structure depends on the nature of the halogen. Protonated 3-I–Pz generated thus ionized pyrazole a , whereas protonated 3-Cl–Pz was a precursor of an isomeric species ascribed to a dehydropyrazolium distonic structure b . A mixture of C 3 H 4 N 2 + ions was formed in protonated 3-Br–Pz. B3LYP/6-31++G(d,p) computations confirmed a regiospecific N 2 -protonation, and a low energy content of the distonic ions b or c (50 kJ mol −1 above a and lying in deep energy wells). Two competitive C–H and C–X bond cleavages were invoked to explain the contrasting behaviour of various protonated X–Pz under dehalogenation conditions.
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High‐energy Collisional Activation of the molecular ions of thiophene‐2‐one with different target gases
Journal of mass spectrometry : JMS, 2001Co-Authors: Pascal Gerbaux, Robert Flammang, Valérie Sciamanna, Minh Tho NguyenAbstract:Collisional Activation of keV thiophene-2-one radical cations 1 + with O2 or NO as the target gas leads to a desulfuration reaction. This peculiar reaction is insignificant or absent with other targets such as helium, argon, methane or nitrogen. The radical cations produced in this desulfuration reaction are most probably vinylketene ions, as indicated by a triple mass spectrometric (MS/MS/MS) experiment performed on a ‘hybrid’ tandem mass spectrometer of sector‐quadrupole‐sector configuration. Tentatively, it is proposed that population of an excited state accounts for the non-ergodic behavior of 1 + upon collision with oxygen or nitric oxide. Ab initio molecular orbital calculations using molecular orbital theory (UMP2, UCCSD(T)) and density functional theory (B3LYP) with 6‐31G(d,p) and 6‐311++G(d,p) basis sets were used to evaluate the relative energy of the excited quartet state of 1 + radical cations. This quartet state is calculated to lie about 3.6 eV above the 2 A 00 ground state and 0.9 eV above the C4H4O + + S dissociation products. It is proposed that the quartet ion serves as the precursor for the spontaneous desulfuration. Copyright © 2001 John Wiley & Sons, Ltd.
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high energy Collisional Activation of the molecular ions of thiophene 2 one with different target gases
Journal of Mass Spectrometry, 2001Co-Authors: Pascal Gerbaux, Valérie Sciamanna, R Flammang, Minh Tho NguyenAbstract:Collisional Activation of keV thiophene-2-one radical cations 1 + with O2 or NO as the target gas leads to a desulfuration reaction. This peculiar reaction is insignificant or absent with other targets such as helium, argon, methane or nitrogen. The radical cations produced in this desulfuration reaction are most probably vinylketene ions, as indicated by a triple mass spectrometric (MS/MS/MS) experiment performed on a ‘hybrid’ tandem mass spectrometer of sector‐quadrupole‐sector configuration. Tentatively, it is proposed that population of an excited state accounts for the non-ergodic behavior of 1 + upon collision with oxygen or nitric oxide. Ab initio molecular orbital calculations using molecular orbital theory (UMP2, UCCSD(T)) and density functional theory (B3LYP) with 6‐31G(d,p) and 6‐311++G(d,p) basis sets were used to evaluate the relative energy of the excited quartet state of 1 + radical cations. This quartet state is calculated to lie about 3.6 eV above the 2 A 00 ground state and 0.9 eV above the C4H4O + + S dissociation products. It is proposed that the quartet ion serves as the precursor for the spontaneous desulfuration. Copyright © 2001 John Wiley & Sons, Ltd.
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Collisional Activation of protonated halogeno-pyridines: different behaviour of target gases
Chemical Physics Letters, 2000Co-Authors: Pascal Gerbaux, Robert Flammang, Minh Tho NguyenAbstract:Abstract Protonated 2-Cl and 2-Br-pyridines undergo facile dehalogenation upon high energy (8 keV range) Collisional Activation provided the target is NO, or even better O 2 , instead of He or Ar. In the low energy regime (20–30 eV range), debromination occurs more readily than dechlorination, but the peculiar behaviour of O 2 in favouring an X-loss over a HX-elimination, is no longer detected (using a MS 3 instrument). B3LYP/6-31G(d) calculations on X-pyridines with X=F, Cl and Br on positions 2, 3 and 4, suggest that, if a protonated pyridine is formed in the lower-lying triplet state following strong interaction with O 2 or NO having higher spin, the loss of Cl or Br becomes almost spontaneous (F-loss is more difficult). Proton affinity PA(pyridine)=940±15 kJ/mol, is decreased by 5–15 kJ/mol upon halogenation.