Site-specific amide hydrogen exchange in melittin probed by electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry

Electron capture dissociation (ECD) has been proposed to be a non-ergodic process, i.e. to provide backbone dissociation of gas-phase peptides faster than randomization of the imparted energy. One potential consequence could be that ECD can fragment deuterated peptides without causing hydrogen scrambling and thereby provide amino acid residue-specific amide hydrogen exchange rates. Such a feature would improve the resolution of approaches involving solution-phase amide hydrogen exchange combined with mass spectrometry for protein structural characterization. Here, we explore this hypothesis using melittin, a haemolytic polypeptide from bee venom, as our model system. Exchange rates in methanol calculated from consecutive c-type ion pairs show some correlation with previous NMR data: the amide hydrogens of leucine 13 and alanine 15, located at the unstructured kink surrounding proline 14 in the melittin structure adopted in methanol, appear as fast exchangers and the amide hydrogens of leucine 16 and lysine 23, buried within the helical regions of melittin, appear as slow exchangers. However, calculations based on c-type ions for other amide hydrogens do not correlate well with NMR data, and evidence for deuterium scrambling in ECD was obtained from z*-type ions.     

Solution-phase deuterium/hydrogen exchange at a specific residue using nozzle-skimmer and electron capture dissociation mass spectrometry

Information about protein conformation can be obtained with hydrogen/deuterium exchange (HDX) mass spectrometry. The isotopic solution-phase exchange of specific amide hydrogen atoms can be followed using low-vacuum nozzle-skimmer collision-induced dissociation (CID). In this study, the nozzle-skimmer technique was complemented by electron capture dissociation (ECD) Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS). The solution-phase exchange at a specific residue is monitored by comparing isotopic distributions of two consecutive b- or c-type ions. While nozzle-skimmer fragmentation takes place in the low-vacuum region of the mass spectrometer, ECD occurs at ultra-high vacuum within the mass analyzer cell of the FTICR mass spectrometer. The dissociations take place at 10(-4) and 10(-9) mbar, respectively. Low-vacuum nozzle-skimmer fragmentation can result in intramolecular exchange between product ions and solvent molecules in the gas phase. Consequently, the solution-phase information about protein or peptide conformation is lost. It was not possible to monitor isotopic solution-phase exchange at the eighth residue in substance P, (Phe)8, with nozzle-skimmer CID. By using the in-cell ECD fragmentation method, the solution-phase exchange at the (Phe)8 residue was preserved during mass spectrometric analysis. This result shows the complementary aspects of applying fragmentation at low and at high vacuum, when studying isotopic exchange in solution at specific residues using FTICRMS.     

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.     

Study of the dissociation of a charge-reduced phosphopeptide formed by electron transfer from an alkali metal target

Doubly protonated phosphopeptide (YGGMHRQET(p)VDC) ions obtained by electrospray ionization were collided with Xe and Cs targets to give singly and doubly charged positive ions via collision-induced dissociation (CID). The resulting ions were analyzed and detected by using an electrostatic analyzer (ESA). Whereas doubly charged fragment ions resulting from collisionally activated dissociation (CAD) were dominant in the CID spectrum with the Xe target, singly charged fragment ions resulting from electron transfer dissociation (ETD) were dominant in the CID spectrum with the Cs target. The most intense peak resulting from ETD was estimated to be associated with the charge-reduced ion with H2 lost from the precursor. Five c-type fragment ions with amino acid residues detached consecutively from the C-terminal were clearly observed without a loss of the phosphate group. These ions must be formed by N--Calpha bond cleavage, in a manner similar to the cases of electron capture dissociation (ECD) and ETD from negative ions. Although the accuracy in m/z of the CID spectra was about +/-1 Th because of the mass analysis using the ESA, it is supposed from the m/z values of the c-type ions that these ions were accompanied by the loss of a hydrogen atom. Four z-type (or y--NH3, or y--H2O) ions analogously detached consecutively from the N-terminal were also observed. The fragmentation processes took place within the time scale of 4.5 micros in the high-energy collision. The present results demonstrated that high-energy ETD with the alkali metal target allowed determination of the position of phosphorylation and the amino acid sequence of post-translational peptides.     

Electron capture dissociation of peptides metalated with alkaline-earth metal ions

The possible use of divalent alkaline-earth metal ions, including Mg2+, Ca2+, Sr2+, and Ba2+, as charge carrier for electron capture dissociation of peptides was investigated. Model peptides of RGGGVGGGR and NGGGWGGGN were used to simplify the interpretation of spectral information. It was demonstrated that useful electron capture dissociation (ECD) tandem mass spectra of these metalated peptides could be generated. Interestingly, peptides metalated with different alkaline-earth metal ions generated very similar ECD tandem mass spectra. Metalated c-ions and z-ions were the predominant fragment ions. Only Mg2+-metalated peptides gave somewhat different results. Some nonmetalated c-ions were observed from ECD of [RGGGVGGGR + Mg]2+ but not from [NGGGWGGGN + Mg]2+. Together with some ab initio calculations, it was established that the bound metal ions might activate the acidity of the amide hydrogen. With the presence of high proton affinity moiety, such as N-terminal amino group and/or side chain of the arginine residues, the metalated peptide ions could exist predominantly in their zwitterion forms, in which one or two backbone amide group(s) was deprotonated and the high proton affinity functional group(s) was protonated. It was believed that electron capture leads primarily to the reduction of the mobile proton rather than the metal ions. With this zwitterion model, the formation of nonmetalated c-fragments and the generation of similar ECD spectra for peptides metalated with various alkaline-earth metal ions could readily to be explained. Another interesting observation in the ECD mass spectra of metalated peptides is related to the enhanced formation of the minor ECD products, i.e., (c - 1)(+*) and (z + 1)+ ions. Together with ab initio calculations using a truncated peptide model, various possible reaction mechanisms for the formation of these minor ECD products were evaluated. It was concluded that hydrogen transfer between the initiated formed c and z(.) species plays an important role in the formation (c - 1)(+*) and (z + 1)+ ions. Although peptides metalated with these metal ions do not have better ECD efficiency compared to the multiply-protonated peptides, it provides practical accessibility of ECD methods to analyze small peptides with no basic amino acid residues.     

Electron capture dissociation and infrared multiphoton dissociation of oligodeoxynucleotide dications

We report electron capture dissociation (ECD) and infrared multiphoton dissociation (IRMPD) of doubly protonated and protonated/alkali metal ionized oligodeoxynucleotides. Mass spectra following ECD of the homodeoxynucleotides polydC, polydG, and polydA contain w or d "sequence" ions. For polydC and polydA, the observed fragments are even-electron ions, whereas radical w/d ions are observed for polydG. Base loss is seen for polydG and polydA but is a minor fragmentation pathway in ECD of polydC. We also observe fragment ions corresponding to w/d plus water in the spectra of polydC and d(GCATGC). Although the structure of these ions is not clear, they are suggested to proceed through a pentavalent phosphorane intermediate. The major fragment in ECD of d(GCATGC) is a d ion. Radical a- or z-type fragment ions are observed in most cases. IRMPD primarily results in base loss, but backbone fragmentation is also observed. IRMPD provides more sequence information than ECD, but the spectra are more complex due to extensive base and water losses. It is proposed that the smaller degree of sequence coverage in ECD, with fragmentation mostly occurring close to the ends of the molecules, is a consequence of a mechanism in which the electron is captured at a P=O bond, resulting in a negatively charged phosphate group. Consequently, at least two protons (or alkali metal cations) must be present to observe a w or d fragment ion, a requirement that is less likely for small fragments.     

Divalent metal ion-peptide interactions probed by electron capture dissociation of trications

Electron capture dissociation (ECD) of the peptide Substance P (SubP) complexed with divalent metals has been investigated. ECD of [SubP + H + M]3+ (M2+ = Mg2+ -Ba2+ and Mn2+ -Zn2+) allowed observation of a larger number of product ions than previous investigations of doubly charged metal-containing peptides. ECD of Mg-Ba, Mn, Fe, and Zn-containing complexes resulted in product ions with and without the metal from cleavage of backbone amine bonds (c' and z* -type ions). By contrast, ECD of Co and Ni-containing complexes yielded major bond cleavages within the C-terminal methionine residue (likely to be the metal ion binding site). Cu-containing complexes displayed yet another behavior: amide bond cleavage (b and y'-type ions). We believe some results can be rationalized both within the hot hydrogen atom mechanism and mechanisms involving electron capture into excited states, such as the recently proposed amide superbase mechanism. However, some behavior, including formation of (cn 'M - H)+ ions for Ca-Ba, is best explained within the latter mechanisms with initial electron capture at the metal. In addition, the ECD behavior appears to correlate with the metal second ionization energy (IE2). Co and Ni (displaying sequestered fragmentation) have IE2s of 17.1 and 18.2 eV, respectively, whereas IE2s for Mg-Ba, Mn, and Fe (yielding random cleavage) are 10.0 to 16.2 eV. This behavior is difficult to explain within the hot hydrogen atom mechanism because hydrogen transfer should not be influenced by IE2s. However, the drastically different fragmentation patterns for Co, Ni, and Cu compared to the other metals can also be explained by their higher propensity for nitrogen (as opposed to oxygen) binding. Nevertheless, these results imply that directed fragmentation can be accomplished via careful selection of the cationizing agent.     

Long-lived electron capture dissociation product ions experience radical migration via hydrogen abstraction

To explore the mechanism of electron capture dissociation (ECD) of linear peptides, a set of 16-mer peptides were synthesized with deuterium labeled on the alpha-carbon position of four glycines. The ECD spectra of these peptides showed that such peptides exhibit a preference for the radical to migrate to the alpha-carbon position on glycine via hydrogen (or deuterium) abstraction before the final cleavage and generation of the detected product ions. The data show c-type fragment ions, ions corresponding to the radical cation of the c-type fragments, c*, and they also show c*-1 peaks in the deuterated peptides only. The presence of the c*-1 peaks is best explained by radical-mediated scrambling of the deuterium atoms in the long-lived, metastable, radical intermediate complex formed by initial electron capture, followed by dissociation of the complex. These data suggest the presence of at least two mechanisms, one slow, one fast. The abundance of H* and -CO losses from the precursor ion changed upon deuterium labeling indicating the presence of a kinetic isotope effect, which suggests that the values reported here represent an underestimation of radical migration and H/D scrambling in the observed fragments.     

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.     

Electron capture dissociation proceeds with a low degree of intramolecular migration of peptide amide hydrogens

Hydrogen (1H/2H) exchange combined with mass spectrometry (HX-MS) has become a recognized method for the analysis of protein structural dynamics. Presently, the incorporated deuterons are typically localized by enzymatic cleavage of the labeled proteins and single residue resolution is normally only obtained for a few residues. Determination of site-specific deuterium levels by gas-phase fragmentation in tandem mass spectrometers would greatly increase the applicability of the HX-MS method. The biggest obstacle in achieving this goal is the intramolecular hydrogen migration (i.e., hydrogen scrambling) that occurs during vibrational excitation of gas-phase ions. Unlike traditional collisional ion activation, electron capture dissociation (ECD) is not associated with substantial vibrational excitation. We investigated the extent of intramolecular backbone amide hydrogen (1H/2H) migration upon ECD using peptides with a unique selective deuterium incorporation. Our results show that only limited amide hydrogen migration occurs upon ECD, provided that vibrational excitation prior to the electron capture event is minimized. Peptide ions that are excessively vibrationally excited in the electrospray ion source by, e.g., high declustering potentials or during precursor ion selection (via sideband excitation) in the external linear quadrupole ion trap undergo nearly complete hydrogen (1H/2H) scrambling. Similarly, collision-induced dissociation (CID) in the external linear quadrupole ion trap results in complete or extensive hydrogen (1H/2H) scrambling. This precludes the use of CID as a method to obtain site-specific information from proteins that are labeled in solution-phase 1H/2H exchange experiments. In contrast, the deuteration levels of the c- and z-fragment ions generated from ECD closely mimic the known solution deuteration pattern of the selectively labeled peptides. This excellent correlation between the results obtained from gas phase and solution suggests that ECD holds great promise as a general method to obtain single residue resolution in proteins from solution 1H/2H exchange experiments.     

Electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry in the electron energy range 0-50 eV

Electron capture dissociation (ECD) of polypeptide cations was obtained with pencil and hollow electron beams for both sidekick and gas-assisted dynamic ion trapping (GADT) using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) with an electrostatic ion transfer line. Increasing the number of trapped ions by multiple ICR trap loads using GADT improved the ECD sensitivity in comparison with sidekick ion trapping and ECD efficiency in comparison with single ion trap load by GADT. Furthermore, enhanced sensitivity made it possible to observe ECD in a wide range of electron energies (0-50 eV). The degree, rate and fragmentation characteristics of ECD FTICR-MS were investigated as functions of electron energy, electron irradiation time, electron flux and ion trapping parameters for this broad energy range. The results obtained show that the rate of ECD is higher for more energetic (>1 eV) electrons. Long electron irradiation time with energetic electrons reduces average fragment ion mass and decreases efficiency of formation of c- and z-type ions. The obtained dependencies suggest that the average fragment ion mass and the ECD efficiency are functions of the total fluence of the electron beam (electron energy multiplied by irradiation time). The measured electron energy distributions in low-energy ECD and hot ECD regimes are about 1 eV at full width half maximum in employed experimental configurations.     

Electron capture and collisionally activated dissociation mass spectrometry of doubly charged hyperbranched polyesteramides

Electron capture dissociation (ECD) of doubly protonated hyperbranched polyesteramide oligomers (1100-1900 Da) was examined and compared with the structural information obtained by low energy collisionally activated dissociation (CAD). Both the ester and amide bonds of the protonated species were cleaved easily upon ECD with the formation of odd electron (OE(.+)) or even electron (EE(+)) fragment ions. Several mechanistic schemes are proposed that describe the complex ECD fragmentation behavior of the multiply charged oligomers. In contrast to studies of biomolecules, the present results indicate that consecutive cleavages induced by intramolecular H-shifts are significant for ECD and of less importance for low energy CAD. The capture of an electron by the ionized species results in fragmentation associated with a redistribution of the excess internal energy over the products and the subsequent bond cleavage. Low energy, multiple collision CAD is found to be a more selective dissociation method than ECD in view of the observation that only amide bonds are cleaved for most of the hyperbranched polymers examined with CAD in this study. ECD appears not to provide complementary structural information compared to CAD in the study of hyperbranched polymers, even though a significantly more complex ECD fragmentation behavior is observed. ECD is shown to be of use for the structural characterization of large oligomers that may not dissociate upon low energy CAD. This is a direct result of the fact that ECD produces ionized hyperbranched oligomers with a relatively high internal energy.     

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.     

Abundant <Emphasis Type="Italic">b</Emphasis>-type ions produced in electron capture dissociation of peptides without basic amino acid residues

We have investigated electron capture dissociation (ECD) of doubly protonated peptides with few or no basic amino acid residues (BAARs). For peptides containing one His, abundant b-type ions were only found when His was located adjacent to the N-terminus. Interestingly, b-type ions, particularly b(5)(+), were found to be the dominant product ions in ECD of peptides without BAARs. Fragmentation patterns of luteinizing hormone releasing hormone (LHRH) and vasopressin (VP), containing one Arg and one His, respectively, were compared to those of Q(8)-LHRH and oxytocin (OT) in which the BAAR is replaced with a non-BAAR. More b-type ions were found for Q(8)-LHRH and OT than for LHRH and VP. We also performed ECD of melittin and found no b-type ions from ECD of the 4+ charge state; however, many low abundance b-type ions were produced in ECD of the 5+ charge state. Possible mechanisms for the formation of b-type ions are discussed and we propose that such ions are formed as a consequence of protons being located at backbone amide nitrogens.     

Optimization of experimental parameters for electron capture dissociation of peptides in a Fourier transform mass spectrometer

This paper describes our effort in optimizing the experimental parameters for electron capture dissociation (ECD) of peptides in a commercially available Fourier-transform mass spectrometer. Using a built-in electrically heated filament electron gun, it was demonstrated that good quality ECD spectra of peptides (MW < 2500) could be obtained by irradiating the isolated peptide molecule-ions with a short pulse (50 ms) of low-energy (3–6 eV) electrons. In addition, we have also demonstrated that pulsing of inert cooling gas (argon) could further improve the intensity of the ECD-induced fragment ions. Due presumably to the influence of the strong magnetic field on the trajectories of electrons, the distance between the electron gun and the trapped-ion cell (i.e., 108 mm versus 20 mm) was found to have little influence on the efficiency of the ECD process(es). From a systematic study on the impact of the filament heating current, filament bias voltage, and electron irradiation time on the intensities of precursor ions and various fragment ions, it was postulated that subsequent capture of electrons by the fragment ions, i.e., neutralization of the fragment ions, might be a significant event for limiting the intensity of the fragment ions.     

Investigation of the presence of <Emphasis Type="Italic">b</Emphasis> ions in electron capture dissociation mass spectra

Previously, we have indicated (Cooper, H.J., et al. Int. J. Mass Spectrom., 2003, 228, 723-728) that electron capture dissociation (ECD) of the doubly protonated peptides, Leu(4)-Sar-Leu(3)-Lys-OH, Leu(4)-Ala-Leu(3)-Lys-OH, Gly(4)-Sar-Gly(3)-Lys-NH(2), and Gly(3)-Pro-Sar-Gly(3)-Lys-NH(2), results in abundant b ions, which derive from fragmentation of backbone amide bonds, a nonstandard fragmentation channel in ECD. The instrumental conditions were such that the possibility that collision-induced dissociation processes were contributing to the observed spectra was eliminated. In a separate study (Fung, Y.M.E., et al. Eur. J. Mass Spectrom., 2004, 10, 449-457. ECD of peptides Arg-(Gly)(n)-Xxx-(Gly)(n)-Arg, where Xxx is the amino acid of interest, did not result in b ions. The variation in ECD observed for strikingly similar peptides suggests that the nature of the charge carrier (Arg or Lys) is instrumental in governing the fragmentation channels. Here, we describe the ECD behavior of a suite of model peptides designed such that the nature and position of the charge carrier could be probed. The results suggest that the presence of b ions in ECD spectra is a consequence of both charge carrier and peptide structure. Possible mechanisms for the formation of b ions following electron capture are discussed.     

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.     

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.     

Combined infrared multiphoton dissociation and electron capture dissociation with a hollow electron beam in Fourier transform ion cyclotron resonance mass spectrometry

An electron injection system based on an indirectly heated ring-shaped dispenser cathode has been developed and installed in a 7 Tesla Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. This new hardware design allows high-rate electron capture dissociation (ECD) to be carried out by a hollow electron beam coaxial with the ion cyclotron resonance (ICR) trap. Infrared multiphoton dissociation (IRMPD) can also be performed with an on-axis IR-laser beam passing through a hole at the centre of the dispenser cathode. Electron and photon irradiation times of the order of 100 ms are required for efficient ECD and IRMPD, respectively. As ECD and IRMPD generate fragments of different types (mostly c, z and b, y, respectively), complementary structural information that improves the characterization of peptides and proteins by FTICR mass spectrometry can be obtained. The developed technique enables the consecutive or simultaneous use of the ECD and IRMPD methods within a single FTICR experimental sequence and on the same ensemble of trapped ions in multistage tandem (MS/MS/MS or MS(n)) mass spectrometry. Flexible changing between ECD and IRMPD should present advantages for the analysis of protein digests separated by liquid chromatography prior to FTICRMS. Furthermore, ion activation by either electron or laser irradiation prior to, as well as after, dissociation by IRMPD or ECD increases the efficiency of ion fragmentation, including the w-type fragment ion formation, and improves sequencing of peptides with multiple disulfide bridges. The developed instrumental configuration is essential for combined ECD and IRMPD on FTICR mass spectrometers with limited access into the ICR trap.     

Electron capture dissociation of b (2+) peptide fragments reveals the presence of the acylium ion structure

Electron capture dissociation (ECD) of peptides and their fragments has now been extended to b ( n) ( 2+) ions, where it also produced far more structural information than collisional activation. Interestingly, b ( n) ( 2+) ions revealed abundant loss of CO from radical monocations and the presence of c ((n - 1)) ( +.) fragments. The CO loss from peptide radical cations is unusual and was attributed to neutralization of the -C identical with O(+) group in the acylium ion structure, supported by the observation of c ( (n - 1)) ( +.) ions that can only be formed from an open-chain ion. These characteristic features were most prominent for b ( 12)( 2+) ions of renin substrate and least prominent for b ( n) ( 2+) ions of substance P (n = 9,10). Totally, out of seven b ( n) ( 2+) ions studied, CO loss above 3% level was present in all spectra as well as c ( (n - 1))( +.) fragments of three species, suggesting that the acylium ion structure plays a significant role for at least some b ( 2+) ions. This is an unexpected result in view of the literature data for small, singly charged b ions, for which the protonated oxazolone structure is favoured in ab initio calculations. Apparently, more studies are required before extrapolating the small molecule results to large species. The CO loss in ECD can be used for distinguishing between b and y ions in the MS/MS spectrum of larger molecules.