Alex G. Harrison

Chemical ionization mass spectrometry

INTRODUCTION. PREFACE. MOLECULAR MASS DETERMINATION. STRUCTURE ELUCIDATION. IDENTIFICATION AND QUANTITATION. SCOPE OF THE PRESENT WORK. FUNDAMENTALS OF GAS PHASE ION CHEMISTRY. ION/MOLECULE COLLISION RATES. Langevin Ion-Induced Dipole Theory. Average Dipole Orientation (ADO) Theory. POSITIVE ION/MOLECULE REACTIONS. Charge Exchange. Proton and Hydrogen Atom Transfer. Negative Ion Transfer Reactions. Condensation Reactions. Clustering or Association Reactions. NEGATIVE ION/MOLECULE REACTIONS. Electron/Molecule Interactions. Associative Detachment Reactions. Displacement and Elimination Reactions. Proton Transfer Reactions. Charge Exchange Reactions. Association Reactions. THERMOCHEMICAL PROPERTIES OF GAS-PHASE IONS. Gas Phase Basicities: Proton Affinities. Hydride Ion Affinities. Electron Affinities. Gas Phase Acidities: Proton Affinities of Anions. INSTRUMENTATION FOR CHEMICAL IONIZATION. MEDIUM PRESSURE MASS SPECTROMETRY. ATMOSPHERIC PRESSURE IONIZATION (API) MASS SPECTROMETRY. FOURIER TRANSFORM MASS SPECTROMETRY (FTMS). QUADRUPOLE ION TRAP MASS SPECTROMETRY. PULSED POSITIVE ION-NEGATIVE ION CHEMICAL IONIZATION. SAMPLE INTRODUCTION IN CHEMICAL IONIZATION MASS SPECTROMETRY. INSTRUMENTATION FOR COLLISION-INDUCED DISSOCIATION (CID) STUDIES. CHEMICAL IONIZATION REAGENT SYSTEMS. POSITIVE ION REAGENT SYSTEMS. Bronsted Acid Reagent Systems. Hydride Ion Abstraction Reagent Systems. Charge Exchange Reagent Systems. Condensation Reaction Reagents. NO as a Positive Ion Reagent System. Tetramethylsilane Reagent System. Metal Ion Reagent Systems. Miscellaneous Positive Ion Reagent Systems. NEGATIVE ION REAGENT GAS SYSTEMS. Electron Capture Reagent Systems. Bronsted Base Reagent Systems. 0- as a Reagent Ion. 02- as a Reagent Ion. SENSITIVITY OF CHEMICAL IONIZATION. CHEMICAL IONIZATION MASS SPECTRA. ALKANES (CnH2n+2). ALKENES AND CYCLOALKANES (CnH2n). ALKYNES, ALKADIENES, AND CYCLOALKENES (CnH2n-2). AROMATIC HYDROCARBONS. ALCOHOLS. ETHERS. ALDEHYDES AND KETONES. CARBOXYLIC ACIDS AND ESTERS. AMINES. NITRO COMPOUNDS. HALOGENATED COMPOUNDS. AMINO ACIDS AND DERIVATIVES. CARBOHYDRATES AND DERIVATIVES. SELECTED TOPICS IN CHEMICAL IONIZATION MASS SPECTROMETRY. ISOTOPE EXCHANGE REACTIONS IN CHEMICAL IONIZATION STUDIES. STEREOCHEMICAL EFFECTS IN CHEMICAL IONIZATION MASS SPECTRA. TANDEM MASS SPECTROMETRY AND CHEMICAL IONIZATION. REACTIVE COLLISIONS IN QUADRUPOLE COLLISION CELLS.

WHY ARE B IONS STABLE SPECIES IN PEPTIDE SPECTRA

Protonated amino acids and derivatives RCH(NH2)C(+O)X · H+ (X = OH, NH2, OCH3) do not form stable acylium ions on loss of HX, but rather the acylium ion eliminates CO to form the immonium ion RCH = NH2+. By contrast, protonated dipeptide derivatives H2NCH(R)C(+O)NHCH(R′)C(+O)X · H+ [X = OH, OCH3, NH2, NHCH(R″)COOH] form stable B2 ions by elimination of HX. These B2 ions fragment on the metastable ion time scale by elimination of CO with substantial kinetic energy release (T1/2 = 0.3–0.5 eV). Similarly, protonated N-acetyl amino acid derivatives CH3C(+O)NHCH(R′)C(+O)X · H+ [X = OH, OCH3, NH2, NHCH(R″)COOH] form stable B ions by loss of HX. These B ions also fragment unimolecularly by loss of CO with T1/2 values of ∼ 0.5 eV. These large kinetic energy releases indicate that a stable configuration of the B ions fragments by way of activation to a reacting configuration that is higher in energy than the products, and some of the fragmentation exothermicity of the final step is partitioned into kinetic energy of the separating fragments. We conclude that the stable configuration is a protonated oxazolone, which is formed by interaction of the developing charge (as HX is lost) with the N-terminus carbonyl group and that the reacting configuration is the acyclic acylium ion. This conclusion is supported by the similar fragmentation behavior of protonated 2-phenyl-5-oxazolone and the B ion derived by loss of H-Gly-OH from protonated C6H5C(+O)-Gly-Gly-OH. In addition, ab initio calculations on the simplest B ion, nominally HC(+O)NHCH2CO+, show that the lowest energy structure is the protonated oxazolone. The acyclic acylium isomer is 1.49 eV higher in energy than the protonated oxazolone and 0.88 eV higher in energy than the fragmentation products, HC(+O)N+H = CH2 + CO, which is consistent with the kinetic energy releases measured.

The gas‐phase basicities and proton affinities of amino acids and peptides

The methods of establishing gas-phase basicities and proton affinities are reviewed, particularly as applied to thermally labile and involatile molecules. The literature on the basicities of amino acids is critically reviewed, and a consistent set of gas-phase basicities for α-amino acids is recommended. The problems associated with deriving proton affinities from these data are discussed. The literature on the gas-phase basicities of peptides is reviewed, and inconsistencies in the experimental results are discussed.

The structure and fragmentation of Bn (n ≥ 3) ions in peptide spectra

The unimolecular and low energy collision-induced fragmentation reactions of the MH+ ions of N-acetyl-tri-alanine, N-acetyl-tri-alanine methyl ester, N-acetyl-tetra-alanine, tetra-alanine, penta-alanine, hexa-glycine, and Leu-enkephalin have been studied with a particular emphasis on the formation and fragmentation of Bn (n=3,4,5) ions. In addition, the metastable ion fragmentation reactions of protonated tetra-glycine, penta-glycine, and Leu-enkephalin amide have been studied. Bn ions are prominent stable species in all spectra. The Bn ions fragment, in part, by elimination of CO to form An ions; this reaction occurs on the metastable ion time scale with a substantial release of kinetic energy (T1/2=0. 3–0. 5 eV) that indicates that a stable configuration of the Bn ion fragments by way of a reacting configuration that is higher in energy than the fragmentation products, An + CO. Ab initio calculations strongly suggest that the stable configuration of the B3 and B4 ions is a protonated oxazolone formed by interaction of the developing charge with the next-nearest carbonyl group as HX is lost from the protonated species H-(Yyy)n-X · H+. The higher Bn ions also fragment, in part, to form the next-lower B ion, presumably in its stable protonated oxazolone form. This reaction is rationalized in terms of the three-dimensional structure of the Bn ions and it is proposed that the neutral eliminated is an α-lactam.

Fragmentation Reactions of Protonated α‐Amino Acids

The metastable ion fragmentation reactions of protonated α-amino acids were recorded. In addition, the low-energy collision-induced dissociation (CID) reactions were studied as a function of collision energy and breakdown graphs, expressing the energy dependence of the fragmentation reactions, were established for a variety of protonated amino acids. The fragmentation reactions observed depend strongly on the identity of the R group in H 2 NCH(R)COOH. Protonated amino acids containing only alkyl groups in the side-chain fragment primarily by elimination of (H 2 O + CO) in both metastable and CID reactions. Hydroxylic and acidic amino acids show loss of H 2 O and loss of (H 2 O + CO) from MH + with the H 2 O loss occurring from the side-chain and (H 2 O + CO) loss occurring from the α-carbohydroxy group. Amidic amino acids show NH 3 loss from the side-chain and (H 2 O + CO) loss from the carbohydroxy group. Aromatic and sulfur-containing amino acids show loss of NH 3 from MH + , as does lysine. Protonated arginine shows a variety of fragmentation pathways, including elimination of NH 3 , elimination of neutral guanidine and formation of protonated guanidine. The energy-dependent breakdown graphs elucidate a variety of secondary fragmentation reactions of the primary fragment ions

A hybrid BEQQ mass spectrometer for studies in gaseous ion chemistry

Abstract A hybrid mass spectrometer of BEQQ geometry (B, magnetic sector; E, electric sector; Q, quadrupole mass filter), designed for fundamental and applied studies in gaseous ion chemistry, is described. The high-resolution (BE) stage is followed by a deceleration lens, which also shapes the ion beam, an r.f.-only quadrupole collision cell, and a quadrupole mass analyzer. This assembly allows collision processes to be studied over the laboratory energy range from 0 to 500 eV with selection of the reactant ion at high mass resolution. The instrument is also equipped with dual collision cells and a deflector electrode in the field-free region between B and E for the study of neutralization—reionization reactions as well as single collision processes at 1–8 keV collision energy. The use of the instrument is illustrated with a variety of examples involving charge stripping, charge inversion, fast neutral ionization, and neutralization—reionization processes at high collision energies, as well as collision-induced dissociation reactions at both high and low collision energies. In addition, a number of experiments involving simultaneous scanning of the electric sector and the QQ stage are reported.

Amide bond dissociation in protonated peptides. Structures of the N-terminal ionic and neutral fragments

Abstract Three-stage tandem mass spectrometry (MS3) and neutral fragment reionization (NfR) are utilized to investigate the structures of the N-terminal ionic (bi) and neutral backbone fragments, respectively, produced from break-up of the amide bond in protonated peptides that have been collisionally activated. The b-type ion from [M+H]+ of C6H5CO-GF, which is produced by loss of the C-terminal phenylalanine, has the structure of protonated 2-phenyl-5-oxazolone. Conversely, the neutral fragment accompanying the y-type ion (protonated phenylalanine) is 2-phenyl-5-oxazolone. The b2 ions arising from [M+H]+ of underivatized tripeptides are also found to be protonated oxazolones. On the other hand, the neutral fragments released from the N-terminus of the tripeptides upon formation of y1 are shown to be diketopiperazines and not oxazolones. The combined MS3 and NfR data help propose dissociation mechanisms that account for the observed structures of ionic and neutral backbone fragments.

To b or not to b: the ongoing saga of peptide b ions.

Modern soft ionization techniques readily produce protonated or multiply protonated peptides. Collision-induced dissociation (CID) of these protonated species is often used as a method to obtain sequence information. In many cases fragmentation occurs at amide bonds. When the charge resides on the C-terminal fragment so-called y ions are produced which are known to be protonated amino acids or truncated peptides. When the charge resides on the N-terminal fragment so-called b ions are produced. Often the sequence of y and b ions are essential for peptide sequencing. The b ions have many possible structures, a knowledge of which is useful in this sequencing. The structures of b ions are reviewed in the following with particular emphasis on the variation of structure with the number of amino acid residues in the b ion and the effect of peptide side chain on b ion structure. The recent discovery of full cyclization of larger b ions results in challenges in peptide sequencing. This aspect is discussed in detail.

Sequence-scrambling fragmentation pathways of protonated peptides.

The gas-phase structures and fragmentation pathways of the N-terminal b and a fragments of YAGFL-NH(2), AGLFY-NH(2), GFLYA-NH(2), FLYAG-NH(2), and LYAGF-NH(2) were investigated using collision-induced dissociation (CID) and detailed molecular mechanics and density functional theory (DFT) calculations. Our combined experimental and theoretical approach allows probing of the scrambling and rearrangement reactions that take place in CID of b and a ions. It is shown that low-energy CID of the b(5) fragments of the above peptides produces nearly the same dissociation patterns. Furthermore, CID of protonated cyclo-(YAGFL) generates the same fragments with nearly identical ion abundances when similar experimental conditions are applied. This suggests that rapid cyclization of the primarily linear b(5) ions takes place and that the CID spectrum is indeed determined by the fragmentation behavior of the cyclic isomer. This can open up at various amide bonds, and its fragmentation behavior can be understood only by assuming a multitude of fragmenting linear structures. Our computational results fully support this cyclization-reopening mechanism by showing that protonated cyclo-(YAGFL) is energetically favored over the linear b(5) isomers. Furthermore, the cyclization-reopening transition structures are energetically less demanding than those of conventional bond-breaking reactions, allowing fast interconversion among the cyclic and linear isomers. This chemistry can lead in principle to complete loss of sequence information upon CID, as documented for the b(5) ion of FLYAG-NH(2). CID of the a(5) ions of the above peptides produces fragment ion distributions that can be explained by assuming b-type scrambling of their parent population and a --> a*-type rearrangement pathways ( Vachet , R. W. , Bishop , B. M. , Erickson , B. W. , and Glish , G. L. J. Am. Chem. Soc. 1997, 119, 5481 ). While a ions easily undergo cyclization, the resulting macrocycle predominantly reopens to regenerate the original linear structure. Computational data indicate that the a --> a*-type rearrangement pathways of the linear a isomers involve post-cleavage proton-bound dimer intermediates in which the fragments reassociate and the originally C-terminal fragment is transferred to the N-terminus.

Proton mobility in protonated amino acids and peptides

The MD+ ions of a variety of amino acids and small peptides have been prepared using CD, and (CD&CO as chemical ionization reagents. Using tandem mass spectrometry the fragmentation reactions of these MD’ ions have been studied, both those occurring unimolecularly on the metastable ion time scale (CD, CI) and those occurring following collisional activation ((CD&CO CI). The results show that the added D+ has undergone extensive interchange (leading to H/D scrambling) with all labile hydrogens including carboxylic hydrogens, hydroxylic hydrogens, amidic hydrogens and amino hydrogens. The results indicate that the proton added to amino acids and simple peptides is very mobile and samples all positions bearing labile hydrogens prior to fragmentation of the protonated species. 0 1997 Elsevier Science B.V.