The effect of histidine oxidation on the dissociation patterns of peptide ions

Oxidative modifications to amino acid side chains can change the dissociation pathways of peptide ions, although these variations are most commonly observed when cysteine and methionine residues are oxidized. In this work we describe the very noticeable effect that oxidation of histidine residues can have on the dissociation patterns of peptide ions containing this residue. A common product ion spectral feature of doubly charged tryptic peptides is enhanced cleavage at the C-terminal side of histidine residues. This preferential cleavage arises as a result of the unique acid/base character of the imidazole side chain that initiates cleavage of a proximal peptide bond for ions in which the number of protons does not exceed the number of basic residues. We demonstrate here that this enhanced cleavage is eliminated when histidine is oxidized to 2-oxo-histidine because the proton affinity and nucleophilicity of the imidazole side chain are lowered. Furthermore, we find that oxidation of histidine to 2-oxo-histidine can cause the misassignment of oxidized residues when more than one oxidized isomer is simultaneously subjected to tandem mass spectrometry (MS/MS). These spectral misinterpretations can usually be avoided by using multiple stages of MS/MS (MS(n)) or by specially optimized liquid chromatographic separation conditions. When these approaches are not accessible or do not work, N-terminal derivatization with sulfobenzoic acid avoids the problem of mistakenly assigning oxidized residues.     

The effect of phosphorylation on the electron capture dissociation of peptide ions

The effect of site and frequency of phosphorylation on the electron capture dissociation of peptide ions has been investigated. The ECD of a suite of synthetic peptides (APLSFRGSLPKSYVK; one unmodified, three singly-phosphorylated, three-doubly phosphorylated, and one triply-phosphorylated); two tryptic phosphopeptides (YKVPQLEIVPN(p)SAEER, alpha-casein and FQ(p)SEEQQQTEDELQDK, beta-casein) and their unmodified counterparts, were determined over a range of ECD cathode potentials. The results show that, for doubly-charged precursor ions, the presence of phosphorylation has a deleterious effect on ECD sequence coverage. The fragmentation patterns observed suggest that for peptides with multiple basic residues, the phospho-groups exist in their deprotonated form and form salt-bridges with protonated amino acid side chains. The fragmentation observed for the acidic tryptic peptides suggested the presence of noncovalent interactions, which were perturbed on phosphorylation. Increasing the ECD electron energy significantly improves sequence coverage. Alternatively, improved sequence coverage can be achieved by performing ECD on triply-charged precursor ions. The findings are important for the understanding of gas-phase fragmentation of phosphopeptides.     

Surface-induced dissociation of peptide ions in Fourier-transform mass spectrometry

Peptide molecular ion species up to m/z 3055 introduced into a Fourier-transform mass spectrometer can be made to undergo extensive fragmentation by electrically floating the ion cell. The proportion of ions dissociated increases with increasing voltage, with 48 eV producing the highest absolute abundance of fragment ions above m/z 200. At this energy, spectra closely resemble those from photodissociation at 193 nm, indicating an internal energy deposition of 6–7 eV; change of product abundances with kinetic energy resembles a conventional breakdown curve. The precursor ions apparently are electrostatically attracted to strike screen wires across the ion cell entrance, producing daughter ions of low kinetic energy.

     

Effect of the sarcosine residue on sequence scrambling in peptide b5 ions

The effect of N‐methylation on sequence scrambling in the fragmentation of b₅ ions has been investigated by studying a variety of peptides containing sarcosine (N‐methylglycine). The product ion mass spectra for the b₅ ions derived from Sar‐A‐A‐A‐Y‐A and Sar‐A‐A‐Y‐A‐A show only minor signals for non‐direct sequence ions the major fragmentation reactions occurring from the unrearranged structures. This is in contrast to the b₅ ions where the Sar residue is replaced by Ala and sequence scrambling occurs. The b₅ ion derived from Y‐Sar‐A‐A‐A‐A shows a product ion mass spectrum essentially identical to the spectrum of the b₅ ion derived from Sar‐A‐A‐A‐Y‐A, indicating that in the former case macrocyclization has occurred but the macrocyclic form shows a strong preference to reopen to put the Sar residue in the N‐terminal position. Similar results were obtained in the comparison of b₅ ions derived from A‐Sar‐A‐A‐Y‐A and Sar‐A‐A‐Y‐A‐A. The product ion mass spectra of the MH⁺ ions of Y‐Sar‐A‐A‐A‐A and A‐Sar‐A‐A‐Y‐A show substantial signals for non‐direct sequence ions indicating that fragmentation of the MH⁺ ions channels extensively through the respective b₅ ions and further fragmentation of these species. Copyright © 2014 John Wiley & Sons, Ltd.     

The effect of charge state and the localization of charge on the collision-induced dissociation of peptide ions

The effect that charge state has on the collision-induced dissociation (CID) of peptide ions is examined in detail for several representative peptides under high-energy collision conditions. The CID spectra of singly and doubly charged precursor ions (generated by fast-atom bombardment and electrospray ionization, respectively) are compared for several peptides with similar primary structure. It is shown that for peptides that contain highly basic amino acids, the dissociation of doubly charged ions is strongly influenced by the position of these residues within the peptide and the general observations reported concerning the dissociation of singly charged ions can be extended to precursors with higher charge states. Based on the dissociation behavior of the doubly charged ions of these peptides, it is demonstrated that two charges can reside in close proximity in the precursor ions, overcoming possible repulsion effects, when favored by a high concentration of basic sites. In addition)’ this work illustrates that in the case of doubly charged ions..the charge state of some fragment ions can be determined directly from the mass-to-charge ratio assignments of the CID spectrum.     

Use of 157-nm photodissociation to probe structures of y- and b-type ions produced in collision-induced dissociation of peptide ions

y- and b-type fragment ions produced in the collisional dissociation of arginine-terminated peptide ions are photodissociated with 157-nm light in a linear trap. y-type ions are shown to have the same structure as that of intact peptides of the same sequence with the ionizing proton located at the most basic residue(s). For generic b-type ions, the ionizing proton is shown to be sequestered at the N-terminal arginine, which is consistent with the proposed oxazolone structure.     

Radical-driven dissociation of odd-electron peptide radical ions produced in 157 nm photodissociation

Odd-electron a+1 radical ions generated in the 157 nm photodissociation of peptide ions were investigated in an ion trap mass spectrometer. To localize the radical, peptide backbone amide hydrogens were replaced with deuterium. When the resulting radical ions underwent hydrogen elimination, no H/D scrambling was obvious, suggesting that without collisional activation, the radical resides on the terminal α-carbon. Upon collisional excitation, odd-electron radical ions dissociate through two favored pathways: the production of a-type ions at aromatic amino acids via homolytic cleavage of backbone C_α-C(O) bonds and side-chain losses at nonaromatic amino acids. When aromatic residues are not present, nonaromatic residues can also lead to a-type ions. In addition to a-type ions, serine and threonine yield c _n−1 and a _n−1+1 ions where n denotes the position of the serine or threonine. All of these fragments appear to be directed by the radical and they strongly depend on the amino acid side-chain structure. In addition, thermal fragments are also occasionally observed following cleavage of labile Xxx-Pro bonds and their formation appears to be kinetically competitive with radical migration.     

An investigation of the energetics of peptide ion dissociation by laser desorption chemical ionization fourier transform mass spectrometry

The energy dependence of competing fragmentation pathways of protonated peptide molecules is studied via laser desorption—chemical ionization in a Fourier transform ion cyclotron resonance spectrometer. Neutral peptide molecules are desorbed by the technique of substrate-assisted laser desorption, followed by post-ionization with a proton transfer reagent ion species. The chemical ionization reaction activates the protonated peptide molecules, which then fragment in accordance with the amount of excess energy that is deposited. Chemical ionization forms a protonated molecule with a narrower distribution of activation energy than can be formed by activation methods such as collision activated dissociation. Furthermore, the upper limit of the activation energy is well defined and is approximately given by the enthalpy of the chemical ionization reaction. Control over the fragmentation of peptide ions is demonstrated through reactions between desorbed peptide molecules with different reagent ion species. The fragmentation behavior of peptide ions with different internal energies is established by generation of a breakdown curve for the peptide under investigation. Breakdown curves are reported for the peptides Val-Pro, Val-Pro-Leu, Phe-Phe-Gly-Leu-Met NH₂, and Arg-Lys-Asp-Val-Tyr. The derived breakdown curve of Val-Pro has been fitted by using quasi-equilibrium Rice-Ramsperger-Kassel-Marcus theory to model the unimolecular dissociation of the protonated peptide to provide a better understanding of the mechanisms for the formation of fragment ions that originate from protonated peptides.     

Using accurate mass electrospray ionization-time-of-flight mass spectrometry with in-source collision-induced dissociation to sequence peptide mixtures

Although data-dependent LC-MS-MS with database searching has become au courant for identifying proteins, the technique is constrained by duty-cycle inefficiency and the inability of most tandem mass analyzers to accurately measure peptide product ion masses. In this work, a novel approach is presented for simultaneous peptide fragmentation and accurate mass measurement using in-source collision-induced dissociation (CID) on electrospray ionization (ESI)-time-of-flight (TOF) MS. By employing internal mass reference compounds, mass measurement accuracy within +/-5 ppm for tryptic peptide precursors and +/-10 ppm for most sequence-specific product ions was consistently achieved. Analysis of a complex solution containing several digested protein standards did not adversely affect instrument performance.     

Neutral fragment mass spectra via ambient thermal dissociation of peptide and protein ions

A novel method for the fragmentation of peptide and protein ions at atmospheric pressure outside the mass spectrometer is described. Peptide/protein ions generated by electrosonic spray ionization (ESSI) are carried through a heated coiled metal tube where they fragment. Fragment ions of types a, b, and y are observed for peptides such as angiotensin II and bradykinin. In the case of phosphopeptides, informative b and y ions which preserve the labile phosphate groups are observed in the negative ion mode, which is potentially useful in the location of phosphorylation sites in proteins through chemical analysis of phosphopeptides. The thermal dissociation method extends to proteins such as ubiquitin and myoglobin, giving rise to y-type and other fragment ions. The most important feature of this method is that it also allows characterization of the neutral fragments arising from thermal dissociation by use of on-line corona discharge ionization. This neutral re-ionization experiment is much easier to perform outside the mass spectrometer than as conventionally done, in vacuum. It yields increased structural information from the resulting mass spectra in both the positive and the negative ion modes.     

Examination of Ortho effects in the collisionally activated dissociation of closed-shell aromatic ions

The collisionally activated dissociation of a variety of isomeric disubstituted aromatic ions formed by ion–molecule reactions were examined in order to characterize ortho effects in closed-shell systems. Closed-shell ions of methoxyacetophenone, hydroxyacetophenone, methoxyphenol, anisaldehyde and hydroxybenzaldehyde were formed by proton transfer, methyl addition or methyne addition by using dimethyl ether or ethylene oxide as chemical ionization reagents, and then the structures of these adducts were studied by deuterium-labelling methods and by collisionally activated dissociation techniques in a triple quadrupole mass spectrometer or a quadrupole ion trap. Typically, the meta and para isomers have qualitatively similar dissociation spectra which reflect the types of dissociation reactions observed for the corresponding monosubstituted aromatic ions. The predominant dissociation pathways of the [M + H]⁺ and [M + 15]⁺ ions are directed by the electron-withdrawing substituents, whereas the major dissociation pathways of the [M + 13]⁺ ions are related to the electron-releasing substituent. In contrast, the dissociation routes of the corresponding ortho isomers are dramatically different. This is attributed to the opportunity for functional group interactions of the ortho isomers which facilitate alternative pathways.     

Even-electron ions: a systematic study of the neutral species lost in the dissociation of quasi-molecular ions

The collision-induced dissociations of the even-electron [M + H](+) and/or [M - H](-) ions of 121 model compounds (mainly small aromatic compounds with one to three functional groups) ionized by electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) have been studied using an ion trap instrument, and the results are compared with the literature data. While some functional groups (such as COOH, COOCH(3), SO(3)H in the negative ion mode, or NO(2) in both the positive and negative ion modes) generally promote the loss of neutrals that are characteristic as well as specific, other functional groups (such as COOH in the positive ion mode) give rise to the loss of neutrals that are characteristic, but not specific. Finally, functional groups such as OH and NH(2) in aromatic compounds do not lead to the loss of a neutral that reflects the presence of these substituents. In general, the dissociation of [M + H](+) and [M - H](-) ions generated from aliphatic compounds or compounds containing an aliphatic moiety obeys the even-electron rule (loss of a molecule), but deviations from this rule (loss of a radical) are sometimes observed for aromatic compounds, in particular for nitroaromatic compounds. Thermochemical data and ab initio calculations at the CBS-QB3 level of theory provide an explanation for these exceptions. When comparing the dissociation behaviour of the even-electron [M + H](+) and/or [M - H](-) ions (generated by ESI or APCI) with that of the corresponding odd-electron [M](+) ions (generated by electron ionization, EI), three cases may be distinguished: (1) the dissociation of the two ionic species differs completely; (2) the dissociation involves the loss of a common neutral, yielding product ions differing in mass by one Da, or (3) the dissociations lead to a common product ion.     

Time-dependence of the isotope effects in the unimolecular dissociation of tertiary amine molecular ions

The intramolecular secondary isotope effects on the α-cleavage of deuterium-labelled N-methyldipentylamine radical cations have been studied as a function of ion lifetime by field ionization kinetics. The isotope effects observed are all normal and increase in magnitude with increasing ion lifetime, with the exception of the δ-labelled compound which shows an inverse effect (predominant loss of the labelled radical) at times shorter than 10^?9 s, and a normal effect at longer times. The isotope effects reflect differences in zero-point energies of the transition states as well as the influence of slight reductions of isotope-dependent frequencies on the state sums–a statistical weight effect. The latter is particularly important at high ion energies and is the primary reason for the occurrence of the inverse isotope effect. The time dependence of the normal and inverse isotope effect is reproduced by QET/RRKM calculations.     

The influence of sialylation on glycan negative ion dissociation and energetics

For the analysis of native glycans using tandem mass spectrometry (MS), it is desirable to choose conditions whereby abundances of cross-ring cleavages indicative of branch positions are maximized. Recently, negative ion tandem mass spectrometry has been shown to produce significantly higher abundances of such ions in glycans compared to the positive ion mode. Much of this prior work has concerned fragmentation patterns in asialo glycans. The present work compares the abundances of critical cross-ring cleavage ions using negative mode tandem mass spectrometry for milk oligosaccharides and N-linked glycans. For comparison, product ion formation was studied for deprotonated and nitrated ions formed from asialo glycans and deprotonated ions from sialylated glycans. Breakdown profiles demonstrate clearly that more energy was required to fragment sialylated compounds to the same extent as either their asialo or nitrate adducted counterparts. The extraction of a proton from a ring hydroxyl group during the ionization process may be viewed, qualitatively, as imparting significantly more energy to the ion than would that from a molecule bearing an acidic group, so that acidic glycans are more stable in the gas phase, as the negative charge resides on the carboxyl group. These results have strong practical implications because a major portion of glycans released from mammalian proteins will be sialylated.     

Collision-induced dissociation of aligned ions

Laser light is used to prepare aligned beams of Na⁺₂ ions. The cross section for collision-induced dissociation of these ions is anisotropic.     

The energetics of shooting ions into the dodecahedrane cage

The activation barriers and energy profiles along the inclusion coordinate for the penetration of H+, He, Li+, Be+, Be2+, and Mg2+ into dodecahedrane 1 vary considerably with the nature of the projectile. Using Hartree-Fock and MP2 derived structures and energies, the overall process of creating endohedral X n+@1 complexes is examined in detail.