Experimental and theoretical investigations of the loss of amino acid side chains in electron capture dissociation of model peptides

Loss of side chains from different amino acid residues in a model peptide framework of RGGGXGGGR under electron capture dissociation conditions were systematically investigated, where X represents one of the twenty common amino acid residues. The alpha-carbon radical cations initially formed by N-Calpha cleavage of peptide ions were shown to undergo secondary dissociation through losses of even-electron and/or odd-electron side-chain moieties. Among the twenty common amino acid residues studied, thirteen of them were found to lose their characteristic side chains in terms of odd-electron neutral fragments, and nine of them were found to lose even-electron neutral side chains. Several generalized dissociation pathways were proposed and were evaluated theoretically with truncated leucine-containing models using ab initio calculations at B3-PMP2/6-311++G(3df,2p)//B3LYP/6-31++G(d,p) level. Elimination of odd-electron side chain was associated with the initial abstraction of the hydrogen from the alpha-carbon bearing the side chain by the N-terminal alpha-carbon radical. Subsequent formation of alpha-beta carbon-carbon double bond leads to the elimination of the odd-electron side chain. The energy barrier for this reaction pathway was 89 kJmol-1. This reaction pathway was 111 kJmol-1 more favorable than the previously proposed pathway involving the formation of cyclic lactam. Elimination of even-electron side chain was associated with the initial abstraction of the gamma-hydrogen from the side chain by the N-terminal alpha-carbon radical. Subsequent formation of beta-gamma carbon-carbon double bond leads to the elimination of the even-electron side chain and the migration of the radical center to the alpha-carbon. The energy barrier for this fragmentation reaction was found to be 50 kJmol-1.     

Influence of charge state and amino acid composition on hydrogen transfer in electron capture dissociation of peptides

Although conventional N-Cα bond cleavage in electron capture dissociation (ECD) of multiply-charged peptides generates a complementary c′ and z′ fragment pair, the N-Cα cleavage followed by hydrogen transfer from c′ to z′ fragments produces other fragments, namely c′ and z′. In this study, the influence of charge state and amino acid composition on hydrogen transfer in ECD is described using sets of peptides. Hydrogen transferred ionic species such as c′ and z′ were observed in ECD spectra of doubly-protonated peptides, while the triply-protonated form did not demonstrate hydrogen transfer. The extent of hydrogen transfer in ECD of doubly-protonated peptides was dependent on constituent amino acids. The ECD of doubly-protonated peptides possessing numerous basic sites showed extensive hydrogen transfer compared with ECD of less basic peptides. The extent of hydrogen transfer is discussed from the viewpoints of the structure of peptide ions, the possibility of internal hydrogen bonding and intermediate lifetime of complex [c′+z′].     

Characterization of phosphate-containing metabolites by calcium adduction and electron capture dissociation

Several phosphate-containing metabolites, including nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), adenosine 5'-diphosphate ribose (ADP-r), adenosine 5'-triphosphate (ATP), and guanosine 5'-triphosphate (GTP), have been characterized with electron capture dissociation (ECD) and sustained off-resonance irradiation collision-activated dissociation (SORI-CAD) tandem mass spectrometry (MS/MS) in positive-ion mode. Calcium complexation was used to successfully produce abundant doubly charged cationic precursor ions with or without hydration. This approach enabled application of ECD to acidic metabolites for the first time. Fragmentation pathways observed in ECD and SORI-CAD of calcium-adducted phosphate-containing metabolites were complementary. Unique fragmentation was observed in ECD compared to SORI-CAD MS/MS, including ribose cross-ring cleavage for NAD and NADP, and generation of hydrated product ions, including cross-ring fragments, for hydrated ATP and GTP. A combination of ECD and CAD appears promising for maximizing structural information about metabolites.     

Characterization of neurohistone variants and post-translational modifications by electron capture dissociation mass spectrometry

Post-translational modifications (PTMs) of histones are intimately involved in chromatin structure and thus have roles in cellular processes through their impact on gene activation or repression. At the forefront in histone PTM analysis are mass spectrometry-based techniques, which have capabilities to produce improved views of processes affected by chromatin remodeling via histone modifications. In this report, we take the first mass spectrometric look at histone variant expression and post-translational modifications from histones isolated from rat brain tissue. Analyses of whole rat brain identified specific histone H2A and H2B gene family members and several H4 and H3 post-translational modification sites by electron capture dissociation (ECD) mass spectrometry. We subsequently compared these results to selected rat brain regions. Major differences in the expression profiles of H2A and H2B gene family members or in the post-translational modifications on histone H4 were not observed from the different brain regions using a Top Down approach. However, “Middle Down” mass spectrometry facilitating improved characterization of the histone H3 tail (1–50 residues), revealed an enrichment of trimethylation on Lys9 from cerebellum tissue compared to H3 extracted from whole brain, cerebral cortex or hypothalamus tissue. We forward this study in honor of Professor Donald F. Hunt, whose pioneering efforts in protein and PTM analyses have spawned new eras and numerous careers, many exemplified in this special issue.     

Charge remote fragmentation in electron capture and electron transfer dissociation

Secondary fragmentations of three synthetic peptides (human αA crystallin peptide 1-11, the deamidated form of human βB2 crystallin peptide 4-14, and amyloid β peptide 25-35) were studied in both electron capture dissociation (ECD) and electron-transfer dissociation (ETD) mode. In ECD, in addition to c and z· ion formations, charge remote fragmentations (CRF) of z· ions were abundant, resulting in internal fragment formation or partial/entire side-chain losses from amino acids, sometimes several residues away from the backbone cleavage site, and to some extent multiple side-chain losses. The internal fragments were observed in peptides with basic residues located in the middle of the sequences, which was different from most tryptic peptides with basic residues located at the C-terminus. These secondary cleavages were initiated by hydrogen abstraction at the α-, β-, or γ-position of the amino acid side chain. In comparison, ETD generates fewer CRF fragments than ECD. This secondary cleavage study will facilitate ECD/ETD spectra interpretation, and help de novo sequencing and database searching.     

Dipole and Coulomb forces in electron capture dissociation and electron transfer dissociation mass spectroscopy

Ab initio electronic structure calculations were performed on a doubly charged polypeptide model H(+)-Lys(Ala)(19)-CO-CH(NH(2))-CH(2)-SS-CH(2)-(NH(2))CH-CO-(Ala)(19)-Lys-H(+) consisting of a C-terminal protonated Lys followed by a 19-Ala α-helix with a 20th Ala-like unit whose side chain is linked by a disulfide bond to a corresponding Ala-like unit connected to a second 19-Ala α-helix terminated by a second C-terminal-protonated Lys. The Coulomb potentials arising from the two charged Lys residues and dipole potentials arising from the two oppositely directed 72 D dipoles of the α-helices act to stabilize the SS bond's σ* orbital. The Coulomb potentials provide stabilization of 1 eV, while the two large dipoles generate an additional 4 eV. Such stabilization allows the SS σ* orbital to attach an electron and thereby generate disulfide bond cleavage products. Although calculations are performed only on SS bond cleavage, discussion of N-C(α) bond cleavage caused by electron attachment to amide π* orbitals is also presented. The magnitudes of the stabilization energies as well as the fact that they arise from Coulomb and dipole potentials are supported by results on a small model system consisting of a H(3)C-SS-CH(3) molecule with positive and negative fractional point charges to its left and right designed to represent (i) two positive charges ca. 32 ? distant (i.e., the two charged Lys sites of the peptide model) and (ii) two 72 D dipoles (i.e., the two α-helices). Earlier workers suggested that internal dipole forces in polypeptides could act to guide incoming free electrons (i.e., in electron capture dissociation (ECD)) toward the positive end of the dipole and thus affect the branching ratios for cleaving various bonds. Those workers argued that, because of the huge mass difference between an anion donor and a free electron, internal dipole forces would have a far smaller influence over the trajectory of a donor (i.e., in electron transfer dissociation (ETD)). The present findings suggest that, in addition to their effects on guiding electron or donor trajectories, dipole potentials (in combination with Coulomb potentials) also alter the energies of SS σ* and amide π* orbitals, which then affects the ability of these orbitals to bind an electron. Thus, both by trajectory-guiding and by orbital energy stabilization, Coulomb and dipole potentials can have significant influences on the branching ratios of ECD and ETC in which disulfide or N-C(α) bonds are cleaved.     

Radical conversion and migration in electron capture dissociation

Electron capture dissociation (ECD) is an important analytical technique which is used frequently in proteomics experiments to reveal information about both primary sequence and post-translational modifications. Although the utility of ECD is unquestioned, the underlying chemistry which leads to the observed fragmentation is still under debate. Backbone dissociation is frequently the exclusive focus when mechanistic questions about ECD are posed, despite the fact that numerous other abundant dissociation channels exist. Herein, the focus is shifted to side chain loss and other dissociation channels which offer clues about the underlying mechanism(s). It is found that the initially formed hydrogen abundant radicals in ECD can convert quickly to hydrogen deficient radicals via a variety of pathways. Dissociation which occurs subsequent to this conversion is mediated by hydrogen deficient radical chemistry, which has been the subject of extensive study in experiments which are independent from ECD. Statistical analysis of fragments observed in ECD is in excellent agreement with predictions made by an understanding of hydrogen deficient radical chemistry. Furthermore, hydrogen deficient radical mediated dissociation likely contributes to observed ECD fragmentation patterns in unexpected ways, such as the selective dissociation observed at disulfide bonds. Many aspects of dissociation observed in ECD are easily reproduced in well-controlled experiments examining hydrogen deficient radicals generated by non-ECD methods. All of these observations indicate that when considering the means by which electron capture leads to dissociation, hydrogen deficient radical chemistry must be given careful consideration.     

Identification and localization of the fatty acid modification in ghrelin by electron capture dissociation

Electron capture dissociation (ECD) has been demonstrated to be an effective fragmentation technique for characterizing the site and structure of the fatty acid modification in ghrelin, a 28-residue growth-hormone-releasing peptide that has an unusual ester-linked n-octanoyl (C8:0) modification at Ser-3. ECD cleaves 21 of 23 possible backbone amine bonds, with the product ions (c and z· ions) covering a greater amino acid sequence than those obtained by collisionally activated dissociation (CAD). Consistent with the ECD nonergodic mechanism, the ester-linked octanoyl group is retained on all backbone cleavage product ions, allowing for direct localization of this labile modification. In addition, ECD also induces the ester bond cleavage to cause the loss of octanoic acid from the ghrelin molecular ion; the elimination process is initiated by the capture of an electron at the protonated ester group, which is followed by the radical-site-initiated reaction known as -cleavage. The chemical composition of the attached fatty acid can be directly obtained from the accurate Fourier transform ion cyclotron resonance (FTICR) mass measurement of the ester bond cleavage product ions.     

Toward a general mechanism of electron capture dissociation

The effects of positive charge on the properties of ammonium and amide radicals were investigated by ab initio and density functional theory calculations with the goal of elucidating the energetics of electron capture dissociation (ECD) of multiply charged peptide ions. The electronic properties of the amide group in N-methylacetamide (NMA) are greatly affected by the presence of a remote charge in the form of a point charge, methylammonium, or guanidinium cations. The common effect of the remote charge is an increase of the electron affinity of the amide group, resulting in exothermic electron capture. The N-Calpha bond dissociation and transition state energies in charge-stabilized NMA anions are 20-50 kJ mol(-1) greater than in the hydrogen atom adduct. The zwitterions formed by electron capture have proton affinities that were calculated as 1030-1350 kJ mol(-1), and are sufficiently basic for the amide carbonyl to exothermically abstract a proton from the ammonium, guanidinium and imidazolium groups in protonated lysine, arginine, and histidine residues, respectively. A new mechanism is proposed for ECD of multiply charged peptide and protein cations in which the electron enters a charge-stabilized electronic state delocalized over the amide group, which is a superbase that abstracts a proton from a sterically proximate amino acid residue to form a labile aminoketyl radical that dissociates by N-Calpha bond cleavage. This mechanism explains the low selectivity of N-Calpha bond dissociations induced by electron capture, and is applicable to dissociations of peptide ions in which the charge carriers are metal ions or quaternary ammonium groups. The new amide superbase and the previously proposed mechanisms of ECD can be uniformly viewed as being triggered by intramolecular proton transfer in charge-reduced amide cation-radicals. In contrast, remote charge affects N-H bond dissociation in weakly bound ground electronic states of hypervalent ammonium radicals, as represented by methylammonium, CH3NH3*, but has a negligible effect on the N-H bond dissociation in the strongly bound excited electronic states. This refutes previous speculations that loss of "hot hydrogen" can occur from an excited state of an ammonium radical.     

Sequence analysis of peptide:oligonucleotide heteroconjugates by electron capture dissociation and electron transfer dissociation

Mass spectrometry analysis of protein-nucleic acid cross-links is challenging due to the dramatically different chemical properties of the two components. Identifying specific sites of attachment between proteins and nucleic acids requires methods that enable sequencing of both the peptide and oligonucleotide component of the heteroconjugate cross-link. While collision-induced dissociation (CID) has previously been used for sequencing such heteroconjugates, CID generates fragmentation along the phosphodiester backbone of the oligonucleotide preferentially. The result is a reduction in peptide fragmentation within the heteroconjugate. In this work, we have examined the effectiveness of electron capture dissociation (ECD) and electron-transfer dissociation (ETD) for sequencing heteroconjugates. Both methods were found to yield preferential fragmentation of the peptide component of a peptide:oligonucleotide heteroconjugate, with minimal differences in sequence coverage between these two electron-induced dissociation methods. Sequence coverage was found to increase with increasing charge state of the heteroconjugate, but decreases with increasing size of the oligonucleotide component. To overcome potential intermolecular interactions between the two components of the heteroconjugate, supplemental activation with ETD was explored. The addition of a supplemental activation step was found to increase peptide sequence coverage over ETD alone, suggesting that electrostatic interactions between the peptide and oligonucleotide components are one limiting factor in sequence coverage by these two approaches. These results show that ECD/ETD methods can be used for the tandem mass spectrometry sequencing of peptide:oligonucleotide heteroconjugates, and these methods are complementary to existing CID methods already used for sequencing of protein-nucleic acid cross-links.     

Supramolecular control of self-assembling terthiophene-peptide conjugates through the amino acid side chain

The self-assembly of oligothiophene-peptide conjugates can be directed through the systematic variation of the peptide sequence into different nanostructures, including flat spicules, nanotubes, spiral sheets, and giant, flat sheets. Furthermore, the assembly of these molecules is not controlled by steric interactions between the amino acid side chains.     

Electron capture dissociation product ion abundances at the X amino acid in RAAAA-X-AAAAK peptides correlate with amino acid polarity and radical stability

We present mechanistic studies aimed at improving the understanding of the product ion formation rules in electron capture dissociation (ECD) of peptides and proteins in Fourier transform ion cyclotron resonance mass spectrometry. In particular, we attempted to quantify the recently reported general correlation of ECD product ion abundance (PIA) with amino acid hydrophobicity. The results obtained on a series of model H-RAAAAXAAAAK-OH peptides confirm a direct correlation of ECD PIA with X amino acid hydrophobicity and polarity. The correlation factor (R) exceeds 0.9 for 12 amino acids (Ile, Val, His, Asn, Asp, Glu, Gln, Ser, Thr, Gly, Cys, and Ala). The deviation of ECD PIA for seven outliers (Pro is not taken into consideration) is explained by their specific radical stabilization properties (Phe, Trp, Tyr, Met, and Leu) and amino acid basicity (Lys, Arg). Phosphorylation of Ser, Thr, and Tyr decreases the efficiency of ECD around phosphorylated residues, as expected. The systematic arrangement of amino acids reported here indicates a possible route toward development of a predictive model for quantitative electron capture/transfer dissociation tandem mass spectrometry, with possible applications in proteomics.     

Charge location directs electron capture dissociation of peptide dications

The effect of peptide dication charge location on electron capture dissociation (ECD) fragmentation pattern is investigated. ECD fragmentation patterns are compared for peptides with amide and free acid C-terminal groups. ECD of free acid compared with C-terminally amidated peptides with basic residues near the N-terminus demonstrates increased formation of a-type ions. Similarly, ECD of free acid compared with C-terminally amidated peptides with basic residues near the C-terminus exhibits increased formation of y-type ions. Alteration of the peptide sequence to inhibit the formation of charged side chains (i.e., amino acid substitution and acetylation) provides further evidence for charge location effect on ECD. We propose that formation of zwitterionic peptide structures increases the likelihood of amide nitrogen protonation (versus basic side chains), which is responsible for the increase in a- and y-type ion formation.     

Electron attachment step in electron capture dissociation (ECD) and electron transfer dissociation (ETD)

We have made use of classical dynamics trajectory simultions and ab initio electronic structure calculations to estimate the cross sections with which electrons are attached (in electron capture dissociation (ECD)) or transferred (in electron transfer dissociation (ETD)) to a model system that contained both an S-S bond that is cleaved and a -NH(3)(+) positively charged site. We used a Landau-Zener-Stueckelberg curve-crossing approximation to estimate the ETD rates for electron transfer from a CH(3)(-) anion to the -NH(3)(+) Rydberg orbital or the S-S sigma* orbital. We draw conclusions about ECD from our ETD results and from known experimental electron-attachment cross sections for cations and sigma-bonds. We predict the cross section for ETD at the positive site of our model compound to be an order of magnitude larger than that for transfer to the Coulomb-stabilized S-S bond site. We also predict that, in ECD, the cross section for electron capture at the positive site will be up to 3 orders of magnitude larger than that for capture at the S-S bond site. These results seem to suggest that attachment to such positive sites should dominate in producing S-S bond cleavage in our compound. However, we also note that cleavage induced by capture at the positive site will be diminished by an amount that is related to the distance from the positive site to the S-S bond. This dimunition can render cleavage through Coulomb-assisted S-S sigma* attachment competitive for our model compound. Implications for ECD and ETD of peptides and proteins in which SS or N-C(alpha) bonds are cleaved are also discussed, and we explain that such events are most likely susceptible to Coulomb-assisted attachment, because the S-S sigma* and C=O pi* orbitals are the lowest-lying antibonding orbitals in most peptides and proteins.     

Nonergodicity in electron capture dissociation investigated using hydrated ion nanocalorimetry

Hydrated divalent magnesium and calcium clusters are used as nanocalorimeters to measure the internal energy deposited into size-selected clusters upon capture of a thermally generated electron. The infrared radiation emitted from the cell and vacuum chamber surfaces as well as from the heated cathode results in some activation of these clusters, but this activation is minimal. No measurable excitation due to inelastic collisions occurs with the low-energy electrons used under these conditions. Two different dissociation pathways are observed for the divalent clusters that capture an electron: loss of water molecules (Pathway I) and loss of an H atom and water molecules (Pathway II). For Ca(H(2)O)(n)(2+), Pathway I occurs exclusively for n >or= 30 whereas Pathway II occurs exclusively for n 相似文献   

Influence of amino acid side chain packing on Fe-methionine coordination in thermostable cytochrome C

Paramagnetic NMR and optical studies of the oxidized forms of mesophile Pseudomonas aeruginosa cytochrome c(551) and its quintuple mutant (F7A/V13M/F34Y/E43Y/V78I), and thermophile Hydrogenobacter thermophilus cytochrome c(552) demonstrated that the amino acid side chain packings in the protein interior influence the coordination bond between the heme iron and the axial methionine in the proteins. The strength of heme axial coordinations was found to correlate with the overall protein thermostability.     

The role of conformation on electron capture dissociation of ubiquitin

Effects of protein conformation on electron capture dissociation (ECD) were investigated using high-field asymmetric waveform ion mobility spectrometry (FAIMS) and Fourier-transform ion cyclotron resonance mass spectrometry. Under the conditions of these experiments, the electron capture efficiency of ubiquitin 6+ formed from three different solution compositions differs significantly, ranging from 51 +/- 7% for ions formed from an acidified water/methanol solution to 88 +/- 2% for ions formed from a buffered aqueous solution. This result clearly indicates that these protein ions retain a memory of their solution-phase structure and that conformational differences can be probed in an ECD experiment. Multiple conformers for the 7+ and 8+ charge states of ubiquitin were separated using FAIMS. ECD spectra of conformer selected ions of the same charge states differ both in electron capture efficiency and in the fragment ion intensities. Conformers of a given charge state that have smaller collisional cross sections can have either a larger or smaller electron capture efficiency. A greater electron capture efficiency was observed for ubiquitin 6+ that has the same collisional cross section as one ubiquitin 7+ conformer, despite the lower charge state. These results indicate that the shape of the molecule can have a greater effect on electron capture efficiency than either collisional cross section or charge state alone. The cleavage locations of different conformers of a given charge state were the same indicating that the presence of different conformers in the gas phase is not due to difference in where charges are located, but rather reflect conformational differences most likely originating from solution. Small neutral losses observed from the singly- and doubly-reduced ubiquitin 6+ do not show a temperature dependence to their formation, consistent with these ions being formed by nonergodic processes.     

A mechanistic study of the electron capture dissociation of oligonucleotides

Electron capture dissociation (ECD) of a series of custom-synthesized oligonucleotide pentamers was performed in a Fourier-transform mass spectrometer with a conventional filament-type electron gun. Dissociation of oligonucleotide ions by electron capture generates primarily w/d-type and z/a-type ions with and without the loss of a nucleobase fragment ions. Minor yields of radical [z/a + H]. fragment ions were also observed in many cases. It is interesting to note that some nucleoside-like fragment ions and protonated nucleobase ions (except thymine-related nucleobases and nucleoside-like fragments) were observed in most ECD spectra. The formation of these low-mass fragment ions was tentatively attributed to the secondary fragmentation of the radical [z + H]. fragment ions. From the ECD tandem mass spectra of a series of C/T based binary oligonucleotide ions, including d(CTCTC), d(CTTTC), d(TCCCT), d(CCCCT), and d(TCCCC), it was clearly demonstrated that the formation of many sequence ions was sensitive to the position of cytosine (or the position of charge carrier). The findings of this work support a notion that the ECD of protonated oligonucleotide molecules is charge-directed with the electron being captured by the protonated nucleobase.     

Detection and characterization of methionine oxidation in peptides by collision-induced dissociation and electron capture dissociation

Electron capture dissociation (ECD) and collision-induced dissociation (CID), the two complementary fragmentation techniques, are demonstrated to be effective in the detection and localization of the methionine sulfoxide [Met(O)] residues in peptides using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. The presence of Met(O) can be easily recognized in the low-energy CID spectrum showing the characteristic loss of methanesulfenic acid (CH(3)SOH, 64 Da) from the side chain of Met(O). The position of Met(O) can then be localized by ECD which is capable of providing extensive peptide backbone fragmentation without detaching the labile Met(O) side chain. We studied CID and ECD of several Met(O)-containing peptides that included the 44-residue human growth hormone-releasing factor (GRF) and the human atrial natriuretic peptide (ANP). The distinction and complementarity of the two fragmentation techniques were particularly remarkable in their effects on ANP, a disulfide bond-containing peptide. While the predominant fragmentation pathway in CID of ANP was the loss of CH(3)SOH (64 Da) from the molecular ion, ECD of ANP resulted in many sequence-informative products, including those from cleavages within the disulfide-bonded cyclic structure, to allow for the direct localization of Met(O) without the typical procedures for disulfide bond reduction followed by [bond]SH alkylation.     

The effect of fixed charge modifications on electron capture dissociation

Electron capture dissociation (ECD) studies of two modified amyloid beta peptides (20-29 and 25-35) were performed to investigate the role of H* radicals in the ECD of peptide ions and the free-radical cascade (FRC) mechanism. 2,4,6-Trimethylpyridinium (TMP) was used as the fixed charge tag, which is postulated to both trap the originally formed radical upon electron capture and inhibit the H* generation. It was found that both the number and locations of the fixed charge groups influenced the backbone and side-chain cleavages of these peptides in ECD. In general, the frequency and extent of backbone cleavages decreased and those of side-chain cleavages increased with the addition of fixed charge tags. A singly labeled peptide with the tag group farther away from the protonated site experienced a smaller abundance decrease in backbone cleavage fragments than the one with the tag group closer to the protonated site. Despite the nonprotonated nature of all charge carriers in doubly labeled peptide ions, several c and z* ions were still observed in their ECD spectra. Thus, although H* transfer may be important for the NC(alpha) bond cleavage, there also exist other pathways, which would require a radical migration via H* abstraction through space or via an amide superbase mechanism. Finally, internal fragment ions were observed in the ECD of these linear peptides, indicating that the important role of the FRC in backbone cleavages is not limited to the ECD of cyclic peptides.