Mechanism of Formation of the Major Estradiol Product Ions Following Collisional Activation of the Molecular Anion in a Tandem Quadrupole Mass Spectrometer

The importance of the mass spectral product ion structure is highlighted in quantitative assays, which typically use multiple reaction monitoring (MRM), and in the discovery of novel metabolites. Estradiol is an important sex steroid whose quantitation and metabolite identification using tandem mass spectrometry has been widely employed in numerous clinical studies. Negative electrospray ionization tandem mass spectrometry of estradiol (E2) results in several product ions, including the abundant m/z 183 and 169. Although m/z 183 is one of the most abundant product ions used in many quantitative assays, the structure of m/z 183 has not been rigorously examined. We suggest a structure for m/z 183 and a mechanism of formation consistent with collision induced dissociation (CID) of E2 and several stable isotopes ([D₄]-E2, [¹³C₆]-E2, and [D₁]-E2). An additional product ion from E2, namely m/z 169, has also been examined. MS³ experiments indicated that both m/z 183 and m/z 169 originate from only E2 [M – H]^– m/z 271. These ions, m/z 183 and m/z 169, were also present in the collision induced decomposition mass spectra of other prominent estrogens, estrone (E1) and estriol (E3), indicating that these two product ions could be used to elucidate the estrogenic origin of novel metabolites. We propose two fragmentation schemes to explain the CID data and suggest a structure of m/z 183 and m/z 169 consistent with several isotopic variants and high resolution mass spectrometric measurements.      

Fragmentation Reactions of Methionine-Containing Protonated Octapeptides and Fragment Ions Therefrom: An Energy-Resolved Study

The fragmentation reactions of the MH⁺ ions as well as the b₇, a₇, and a₇* ions derived therefrom have been studied in detail for the octapeptides MAAAAAAA, AAMAAAAA, AAAAMAAA, and AAAAAAMA. Ionization was by electrospray using a QqToF mass spectrometer, which allowed a study of the evolution of the fragmentation channels as a function of the collision energy. Not surprisingly, the product ion mass spectra for the b₇ ions are independent of the original precursor sequence, indicating macrocyclization and reopening to the same mixture of protonated oxazolones prior to fragmentation. The results show that this sequence scrambling results in a distinct preference to place the Met residue in the C-terminal position of the protonated oxazolones. The a₇ and a₇* ions also produce product ion mass spectra independent of the original peptide sequence. The results for the a₇ ions indicate that fragmentation occurs primarily from an amide structure analogous to that observed for a₄ ions (Bythell et al. in J Am Chem Soc 132:14766–14779, 2010). Clearly, the rearrangement reaction they have proposed applies equally well to a_n ions as large as a₇. The major fragmentation modes of the MH⁺ ions at low collision energies produce b₇, b_6, and b₅ ions. As the collision energy is increased further fragmentation of these primary products produces, in part, non-direct sequence ions, which become prominent at lower m/z values, particularly for the peptides with the Met residue near the N-terminus.

Electron Capture Dissociation of Trivalent Metal Ion-Peptide Complexes

With electrospray ionization from aqueous solutions, trivalent metal ions readily adduct to small peptides resulting in formation of predominantly (peptide + M_T ? H)²⁺, where M_T = La, Tm, Lu, Sm, Ho, Yb, Pm, Tb, or Eu, for peptides with molecular weights below ~1000 Da, and predominantly (peptide + M_T)³⁺ for larger peptides. ECD of (peptide + M_T ? H)²⁺ results in extensive fragmentation from which nearly complete sequence information can be obtained, even for peptides for which only singly protonated ions are formed in the absence of the metal ions. ECD of these doubly charged complexes containing M_T results in significantly higher electron capture efficiency and sequence coverage than peptide-divalent metal ion complexes that have the same net charge. Formation of salt-bridge structures in which the metal ion coordinates to a carboxylate group are favored even for (peptide + M_T)³⁺. ECD of these latter complexes for large peptides results in electron capture by the protonation site located remotely from the metal ion and predominantly c/z fragments for all metals, except Eu³⁺, which undergoes a one electron reduction and only loss of small neutral molecules and b/y fragments are formed. These results indicate that solvation of the metal ion in these complexes is extensive, which results in the electrochemical properties of these metal ions being similar in both the peptide environment and in bulk water.      

Loss of Internal Backbone Carbonyls: Additional Evidence for Sequence-Scrambling in Collision-Induced Dissociation of y-Type Ions

It is shown that y-type ions, after losing C-terminal H₂O or NH₃, can lose an internal backbone carbonyl (CO) from different peptide positions and yield structurally different product fragment ions upon collision-induced dissociation (CID). Such CO losses from internal peptide backbones of y-fragment ions are not unique to a single peptide and were observed in four of five model peptides studied herein. Experimental details on examples of CO losses from y-type fragment ions for an isotopically labeled AAAAHAA-NH₂ heptapeptide and des-acetylated-α-melanocyte-stimulating hormone (dα-MSH) (SYSMEHFRWGKPV-NH₂) are reported. Results from isotope labeling, tandem mass spectrometry (MSⁿ), and ion mobility-mass spectrometry (IM-MS) confirm that CO losses from different amino acids of m/z-isolated y-type ions yield structurally different ions. It is shown that losses of internal backbone carbonyls (as CID products of m/z-isolated y-type ions) are among intermediate steps towards formation of rearranged or permutated product fragment ions. Possible mechanisms for generation of the observed sequence-scrambled a-“like” ions, as intermediates in sequence-scrambling pathways of y-type ions, are proposed and discussed. ?      

Gas-Phase Synthesis of Singly and Multiply Charged Polyoxovanadate Anions Employing Electrospray Ionization and Collision Induced Dissociation

Electrospray ionization mass spectrometry (ESI-MS) combined with in-source fragmentation and tandem mass spectrometry (MS/MS) experiments were used to generate a wide range of singly and multiply charged vanadium oxide cluster anions including V_xO_y ^n– and V_xO_yCl^n– ions (x = 1–14, y = 2–36, n = 1–3), protonated clusters, and ligand-bound polyoxovanadate anions. The cluster anions were produced by electrospraying a solution of tetradecavanadate, V₁₄O₃₆Cl(L)₅ (L = Et₄N⁺, tetraethylammonium), in acetonitrile. Under mild source conditions, ESI-MS generates a distribution of doubly and triply charged V_xO_yCl^n– and V_xO_yCl(L)^(n–1)– clusters predominantly containing 14 vanadium atoms as well as their protonated analogs. Accurate mass measurement using a high-resolution LTQ/Orbitrap mass spectrometer (m/Δm = 60,000 at m/z 410) enabled unambiguous assignment of the elemental composition of the majority of peaks in the ESI-MS spectrum. In addition, high-sensitivity mass spectrometry allowed the charge state of the cluster ions to be assigned based on the separation of the major from the much less abundant minor isotope of vanadium. In-source fragmentation resulted in facile formation of smaller V_xO_yCl^(1–2)– and V_xO_y ^(1–2)– anions. Collision-induced dissociation (CID) experiments enabled systematic study of the gas-phase fragmentation pathways of the cluster anions originating from solution and from in-source CID. Surprisingly simple fragmentation patterns were obtained for all singly and doubly charged V_xO_yCl and V_xO_y species generated through multiple MS/MS experiments. In contrast, cluster anions originating directly from solution produced comparatively complex CID spectra. These results are consistent with the formation of more stable structures of V_xO_yCl and V_xO_y anions through low-energy CID. Furthermore, our results demonstrate that solution-phase synthesis of one precursor cluster anion combined with gas-phase CID is an efficient approach for the top-down synthesis of a wide range of singly and multiply charged gas-phase metal oxide cluster anions for subsequent investigations of structure and reactivity using mass spectrometry and ion spectroscopy techniques.      

Difference of Electron Capture and Transfer Dissociation Mass Spectrometry on Ni<Superscript>2+</Superscript>-, Cu<Superscript>2+</Superscript>-, and Zn<Superscript>2+</Superscript>-Polyhistidine Complexes in the Absence of Remote Protons

Electron capture dissociation (ECD) and electron transfer dissociation (ETD) in metal-peptide complexes are dependent on the metal cation in the complex. The divalent transition metals Ni²⁺, Cu²⁺, and Zn²⁺ were used as charge carriers to produce metal-polyhistidine complexes in the absence of remote protons, since these metal cations strongly bind to neutral histidine residues in peptides. In the case of the ECD and ETD of Cu²⁺-polyhistidine complexes, the metal cation in the complex was reduced and the recombination energy was redistributed throughout the peptide to lead a zwitterionic peptide form having a protonated histidine residue and a deprotonated amide nitrogen. The zwitterion then underwent peptide bond cleavage, producing a and b fragment ions. In contrast, ECD and ETD induced different fragmentation processes in Zn²⁺-polyhistidine complexes. Although the N–C_α bond in the Zn²⁺-polyhistidine complex was cleaved by ETD, ECD of Zn²⁺-polyhistidine induced peptide bond cleavage accompanied with hydrogen atom release. The different fragmentation modes by ECD and ETD originated from the different electronic states of the charge-reduced complexes resulting from these processes. The details of the fragmentation processes were investigated by density functional theory.

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Charge Reversal Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

We report the first charge reversal experiments performed by tandem-in-time rather than tandem-in-space MS/MS. Precursor odd-electron anions from fullerene C₆₀, and even-electron ions from 2,7-di-tert-butylfluorene-9-carboxylic acid and 3,3′-bicarbazole were converted into positive product ions (^–CR⁺) inside the magnet of a Fourier transform ion cyclotron resonance mass spectrometer. Charge reversal was activated by irradiating precursor ions with high energy electrons or UV photons: the first reported use of those activation methods for charge reversal. We suggest that high energy electrons achieve charge reversal in one step as double electron transfer, whereas UV-activated ^–CR⁺ takes place stepwise through two single electron transfers and formally corresponds to a neutralization-reionization (^–NR⁺) experiment.      

N-Protonated Isomers and Coulombic Barriers to Dissociation of Doubly Protonated Ala<Subscript>8</Subscript>Arg

Collision-induced dissociation (or tandem mass spectrometry, MS/MS) of a protonated peptide results in a spectrum of fragment ions that is useful for inferring amino acid sequence. This is now commonplace and a foundation of proteomics. The underlying chemical and physical processes are believed to be those familiar from physical organic chemistry and chemical kinetics. However, first-principles predictions remain intractable because of the conflicting necessities for high accuracy (to achieve qualitatively correct kinetics) and computational speed (to compensate for the high cost of reliable calculations on such large molecules). To make progress, shortcuts are needed. Inspired by the popular mobile proton model, we have previously proposed a simplified theoretical model in which the gas-phase fragmentation pattern of protonated peptides reflects the relative stabilities of N-protonated isomers, thus avoiding the need for transition-state information. For singly protonated Ala _n (n = 3–11), the resulting predictions were in qualitative agreement with the results from low-energy MS/MS experiments. Here, the comparison is extended to a model tryptic peptide, doubly protonated Ala₈Arg. This is of interest because doubly protonated tryptic peptides are the most important in proteomics. In comparison with experimental results, our model seriously overpredicts the degree of backbone fragmentation at N₉. We offer an improved model that corrects this deficiency. The principal change is to include Coulombic barriers, which hinder the separation of the product cations from each other. Coulombic barriers may be equally important in MS/MS of all multiply charged peptide ions.

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Tandem Mass Spectrometry and Ion Mobility Reveals Structural Insight into Eicosanoid Product Ion Formation

Ion mobility measurements of product ions were used to characterize the collisional cross section (CCS) of various complex lipid [M-H]^? ions using traveling wave ion mobility mass spectrometry (TWIMS). TWIMS analysis of various product ions derived after collisional activation of mono- and dihydroxy arachidonate metabolites was found to be more complex than the analysis of intact molecular ions and provided some insight into molecular mechanisms involved in product ion formation. The CCS observed for the molecular ion [M-H]^? and certain product ions were consistent with a folded ion structure, the latter predicted by the proposed mechanisms of product ion formation. Unexpectedly, product ions from [M-H-H₂O-CO₂]^? and [M-H-H₂O]^? displayed complex ion mobility profiles suggesting multiple mechanisms of ion formation. The [M-H-H₂O]^? ion from LTB₄ was studied in more detail using both nitrogen and helium as the drift gas in the ion mobility cell. One population of [M-H-H₂O]^? product ions from LTB₄ was consistent with formation of covalent ring structures, while the ions displaying a higher CCS were consistent with a more open-chain structure. Using molecular dynamics and theoretical CCS calculations, energy minimized structures of those product ions with the open-chain structures were found to have a higher CCS than a folded molecular ion structure. The measurement of product ion mobility can be an additional and unique signature of eicosanoids measured by LC-MS/MS techniques.

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Non-Direct Sequence Ions in the Tandem Mass Spectrometry of Protonated Peptide Amides—an Energy-Resolved Study

The fragmentation reactions of the MH⁺ ions of Leu-enkephalin amide and a variety of heptapeptide amides have been studied in detail as a function of collision energy using a QqToF beam type mass spectrometer. The initial fragmentation of the protonated amides involves primarily formation of b_n ions, including significant loss of NH₃ from the MH⁺ ions. Further fragmentation of these b_n ions occurs following macrocyclization/ring opening leading in many cases to b_n ions with permuted sequences and, thus, to formation of non-direct sequence ions. The importance of these non-direct sequence ions increases markedly with increasing collision energy, making peptide sequence determination difficult, if not impossible, at higher collision energies.

Quantification of Competing H3PO4 Versus HPO3 + H2O Neutral Losses from Regioselective 18O-Labeled Phosphopeptides

Abundant neutral losses of 98 Da are often observed upon ion trap CID-MS/MS of protonated phosphopeptide ions. Two competing fragmentation pathways are involved in this process, namely, the direct loss of H₃PO₄ from the phosphorylated residue and the combined losses of HPO₃ and H₂O from the phosphorylation site and from an additional site within the peptide, respectively. These competing pathways produce product ions with different structures but the same m/z values, potentially limiting the utility of CID-MS³ for phosphorylation site localization. To quantify the relative contributions of these pathways and to determine the conditions under which each pathway predominates, we have examined the ion trap CID-MS/MS fragmentation of a series of regioselective ¹⁸O-phosphate ester labeled phosphopeptides prepared using novel solution-phase amino acid synthesis and solid-phase peptide synthesis methodologies. By comparing the intensity of the –100 Da (–H₃PO₃ ¹⁸O) versus –98 Da (–[HPO₃ + H₂O]) neutral loss product ions formed upon MS/MS, quantification of the two pathways was achieved. Factors that affect the extent of formation of the competing neutral losses were investigated, with the combined loss pathway predominantly occurring under conditions of limited proton mobility, and with increased combined losses observed for phosphothreonine compared with phosphoserine-containing peptides. The combined loss pathway was found to be less dominant under ion activation conditions associated with HCD-MS/MS. Finally, the contribution of carboxylic acid functional groups and backbone amide bonds to the water loss in the combined loss fragmentation pathway was determined via methyl esterification and by examination of a phosphopeptide lacking side-chain hydroxyl groups.

Fragmentation of Protonated <Emphasis Type="Italic">N</Emphasis>-(3-Aminophenyl)Benzamide and Its Derivatives in Gas Phase

An ion of m/z 110.06036 (ion formula [C₆H₈NO]⁺; error: 0.32 mDa) was observed in the collision induced dissociation tandem mass spectrometry experiments of protonated N-(3-aminophenyl)benzamide, which is a rearrangement product ion purportedly through nitrogen-oxygen (N–O) exchange. The N–O exchange rearrangement was confirmed by the MS/MS spectrum of protonated N-(3-aminophenyl)-O ¹⁸ -benzamide, where the rearranged ion, [C₆H₈NO ¹⁸ ]⁺ of m/z 112 was available because of the presence of O ¹⁸ . Theoretical calculations using Density Functional Theory (DFT) at B3LYP/6-31 g(d) level suggest that an ion-neutral complex containing a water molecule and a nitrilium ion was formed via a transition state (TS-1), followed by the water molecule migrating to the anilide ring, eventually leading to the formation of the rearranged ion of m/z 110. The rearrangement can be generalized to other protonated amide compounds with electron-donating groups at the meta position, such as, –OH, –CH₃, –OCH_3, –NH(CH₃)₂, –NH-Ph, and –NHCOCH₃, all of which show the corresponding rearranged ions in MS/MS spectra. However, the protonated amide compounds containing electron-withdrawing groups, including –Cl, –Br, –CN, –NO₂, and –CF₃, at the meta position did not display this type of rearrangement during dissociation. Additionally, effects of various acyl groups on the rearrangement were investigated. It was found that the rearrangement can be enhanced by substitution on the ring of the benzoyl with electron-withdrawing groups.

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Effects of Metal Ion Adduction on the Gas-Phase Conformations of Protein Ions

Changes in protein ion conformation as a result of nonspecific adduction of metal ions to the protein during electrospray ionization (ESI) from aqueous solutions were investigated using traveling wave ion mobility spectrometry (TWIMS). For all proteins examined, protein cations (and in most cases anions) with nonspecific metal ion adducts are more compact than the fully protonated (or deprotonated) ions with the same charge state. Compaction of protein cations upon nonspecific metal ion binding is most significant for intermediate charge state ions, and there is a greater reduction in collisional cross section with increasing number of metal ion adducts and increasing ion valency, consistent with an electrostatic interaction between the ions and the protein. Protein cations with the greatest number of adducted metal ions are no more compact than the lowest protonated ions formed from aqueous solutions. These results show that smaller collisional cross sections for metal-attached protein ions are not a good indicator of a specific metal–protein interaction in solution because nonspecific metal ion adduction also results in smaller gaseous protein cation cross sections. In contrast, the collisional cross section of α-lactalbumin, which specifically binds one Ca²⁺, is larger for the holo-form compared with the apo-form, in agreement with solution-phase measurements. Because compaction of protein cations occurs when metal ion adduction is nonspecific, elongation of a protein cation may be a more reliable indicator that a specific metal ion–protein interaction occurs in solution.      

Di- and tetranuclear heterometallic CuII-LnIII complexes (Ln = Gd and Dy): Synthesis, structure and magnetic properties

Four 3d-4f heterometallic complexes, [CuⅡ LnⅢ (bpt) 2 (NO 3 ) 3 (MeOH)] (Ln = Gd, 1; Dy, 2; bptH = 3,5-bis(pyrid-2-yl)-1,2,4- triazole), [CuⅡ 2 LnⅢ 2 (μ-OH) 2 (bpt) 4 Cl 4 (H 2 O) 2 ]·6H 2 O (Ln = Gd, 3; Dy, 4), have been synthesized under solvothermal conditions. X-ray structural analyses reveal that 1 and 2 are isostructural while 3 and 4 are isostructural. In each complex, the copper and gadolinium or dysprosium ions are linked by two triazolate bridges and form a CuⅡ -LnⅢ dinuclear unit. The intramolecular Cu-Ln distances are 4.542, 4.525, 4.545 and 4.538 for 1, 2, 3 and 4, respectively. Two dinuclear CuLn units are bridged by two OH- groups into the zig-zag tetranuclear {CuⅡ 2 LnⅢ 2 } structures with the Ln(Ⅲ) Ln(Ⅲ) distances of 3.742 and 3.684 for 3 and 4, respectively. Magnetic studies show that the antiferromagnetic CuⅡ-LnⅢ interactions occur in 1 (J CuGd = 0.21 cm-1 ) and 2. The antiferromagnetic interaction occurs in complex 3 with J CuGd = 0.82 cm-1 and J GdGd = 0.065 cm-1 , while dominant ferromagnetic interaction occurs in complex 4.     

Low Mass MS/MS Fragments of Protonated Amino Acids Used for Distinction of Their 13C- Isotopomers in Metabolic Studies

Glu, Gln, Pro, and Ala are the main amino acids involved in ammonia detoxification in mosquitoes. In order to develop a tandem mass spectrometry method (MS²) to monitor each carbon of the above isotopically-labeled ¹³C-amino acids for metabolic studies, the compositions and origins of atoms in fragments of the protonated amino acid should be first elucidated. Thus, various electrospray (ESI)-based MS² tools were employed to study the fragmentation of these unlabeled and isotopically-labeled amino acids and better understand their dissociation pathways. A broad range of fragments, including previously-undescribed low m/z fragments was revealed. The formulae of the fragments (from m/z 130 down to m/z 27) were confirmed by their accurate masses. The structures and conformations of the larger fragments of Glu were also explored by ion mobility mass spectrometry (IM-MS) and gas-phase hydrogen/deuterium exchange (HDX) experiments. It was found that some low m/z fragments (m/z 27–30) are common to Glu, Gln, Pro, and Ala. The origins of carbons in these small fragments are discussed and additional collision induced dissociation (CID) MS² fragmentation pathways are proposed for them. It was also found that small fragments (≤m/z 84) of protonated, methylated Glu, and methylated Gln are the same as those of the underivatized Glu and Gln. Taken together, the new approach of utilizing low m/z fragments can be applied to distinguish, identify, and quantify ¹³C-amino acids labeled at various positions, either in the backbone or side chain.      

Implementation of Dipolar Resonant Excitation for Collision Induced Dissociation with Ion Mobility/Time-of-Flight MS

An ion mobility/time-of-flight mass spectrometer (IMS/TOF MS) platform that allows for resonant excitation collision induced dissociation (CID) is presented. Highly efficient, mass-resolved fragmentation without additional excitation of product ions was accomplished and over-fragmentation common in beam-type CID experiments was alleviated. A quadrupole ion guide was modified to apply a dipolar AC signal across a pair of rods for resonant excitation. The method was characterized with singly protonated methionine enkephalin and triply protonated peptide angiotensin I, yielding maximum CID efficiencies of 44 % and 84 %, respectively. The Mathieu q_x,y parameter was set at 0.707 for these experiments to maximize pseudopotential well depths and CID efficiencies. Resonant excitation CID was compared with beam-type CID for the peptide mixture. The ability to apply resonant waveforms in mobility-resolved windows is demonstrated with a peptide mixture yielding fragmentation over a range of mass-to-charge (m/z) ratios within a single IMS-MS analysis.

Electron Capture Dissociation Studies of the Fragmentation Patterns of Doubly Protonated and Mixed Protonated-Sodiated Peptoids

The fragmentation patterns of a group of doubly protonated ([P + 2H]²⁺) and mixed protonated-sodiated ([P + H + Na]²⁺) peptide-mimicking oligomers, known as peptoids, have been studied using electron capturing dissociation (ECD) tandem mass spectrometry techniques. For all the peptoids studied, the primary backbone fragmentation occurred at the N-C_α bonds. The N-terminal fragment ions, the C-ions (protonated) and the C′-ions (sodiated) were observed universally for all the peptoids regardless of the types of charge carrier. The C-terminal ions varied depending on the type of charge carrier. The doubly protonated peptoids with at least one basic residue located at a position other than the N-terminus fragmented by producing the Z^?-series of ions. In addition, most doubly protonated peptoids also produced the Y-series of ions with notable abundances. The mixed protonated-sodiated peptoids fragmented by yielding the Z^?′-series of ions in addition to the C′-series. Chelation between the sodium cation and the amide groups of the peptoid chain might be an important factor that could stabilize both the N-terminal and the C-terminal fragment ions. Regardless of the types of the charge carrier, one notable fragmentation for all the peptoids was the elimination of a benzylic radical from the odd-electron positive ions of the protonated peptoids ([P + 2H]^?+) and the sodiated peptoids ([P + H + Na]^?+). The study showed potential utility of using the ECD technique for sequencing of peptoid libraries generated by combinatorial chemistry.

Proton Mobility in b<Subscript>2</Subscript> Ion Formation and Fragmentation Reactions of Histidine-Containing Peptides

A detailed energy-resolved study of the fragmentation reactions of protonated histidine-containing peptides and their b₂ ions has been undertaken. Density functional theory calculations were utilized to predict how the fragmentation reactions occur so that we might discern why the mass spectra demonstrated particular energy dependencies. We compare our results to the current literature and to synthetic b₂ ion standards. We show that the position of the His residue does affect the identity of the subsequent b₂ ion (diketopiperazine versus oxazolone versus lactam) and that energy-resolved CID can distinguish these isomeric products based on their fragmentation energetics. The histidine side chain facilitates every major transformation except trans-cis isomerization of the first amide bond, a necessary prerequisite to diketopiperazine b₂ ion formation. Despite this lack of catalyzation, trans-cis isomerization is predicted to be facile. Concomitantly, the subsequent amide bond cleavage reaction is rate-limiting.

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The Power of Accurate Energetics (or Thermochemistry: What is it Good for?)

The utility of measuring the energetics of ion-molecule reactions is discussed. After distinguishing between the terms of thermodynamics (macroscopic, equilibrium quantities) and energetics (microscopic and kinetically relevant quantities), the potential energy surfaces for ion-molecule reactions are reviewed and their implications discussed. Equations describing the kinetic energy dependence of ion-molecule reactions are introduced and the effects of entropy on reaction rates and branching ratios are discussed. Several case histories allow an exploration of the utility of accurate thermochemical information and probe how accurate such energetic information must be to be predictive. These case studies include decomposition of hydrated metal dications, the reaction of FeO⁺ with H₂, and fragmentation of a small protonated peptide (GG). These illustrate a range of interesting systems for which accurate energetic information has been influential in understanding the observed reactivity. Comparisons with theory demonstrate that experimental information is still required for truly predictive capability.      

Fragmentation Reactions of b<Subscript>5</Subscript> and a<Subscript>5</Subscript> Ions Containing Proline—The Structures of a<Subscript>5</Subscript> Ions

A detailed study has been made of the b₅ and a₅ ions derived from the amides H-Ala-Ala-Ala-Ala-Pro-NH₂, H-Ala-Ala-Ala-Pro-Ala-NH₂, and H-Ala-Ala-Pro-Ala-Ala-NH₂. From quasi-MS³ experiments it is shown that the product ion mass spectra of the three b₅ ions are essentially identical, indicating macrocyclization/reopening to produce a common mixture of intermediates prior to fragmentation. This is in agreement with numerous recent studies of sequence scrambling in b ions. By contrast, the product ion mass spectra for the a₅ ions show substantial differences, indicating significant differences in the mixture of structures undergoing fragmentation for these three species. The results are interpreted in terms of a mixture of classical substituted iminium ions as well as protonated C-terminal amides formed by cyclization/rearrangement as reported recently for a₄ ions (Bythell, Maître , Paizs, J . Am. Chem. Soc. 2010, 132, 14761–14779). Novel fragment ions observed upon fragmentation of the a₅ ions are protonated H-Pro-NH₂ and H-Pro-Ala-NH₂ which arise by fragmentation of the amides. The observation of these products provides strong experimental evidence for the cyclization/rearrangement reaction to form amides and shows that it also applies to a₅ ions.