Electron capture dissociation of polypeptides using a 3 Tesla Fourier transform ion cyclotron resonance mass spectrometer

Electron capture dissociation (ECD) of polypeptides has been demonstrated using a commercially available 3 Tesla Fourier transform ion cyclotron resonance (FTICR) instrument. A conventional rhenium filament, designed for high-energy electron impact ionisation, was used to effect ECD of substance P, bee venom melittin and bovine insulin, oxidised B chain. A retarding field analysis of the effective electron kinetic energy distribution entering the ICR cell suggests that one of the most important parameters governing ECD for this particular instrument is the need to employ low trapping plate voltages. This is shown to maximise the abundance of low-energy electrons. The demonstration of ECD at this relatively low magnetic field strength could offer the prospect of more routine ECD analysis for the wider research community, given the reduced cost of such magnets and (at least theoretically) the greater ease of electron/ion cloud overlap at lower field.     

Electron capture dissociation distinguishes a single D-amino acid in a protein and probes the tertiary structure

First results are reported on the application of ECD in analysis of 2+ and 3+ ions of stereoisomers of Trp-cage (NLYIQWLKDGGPSSGRPPPS), the smallest and fastest-folding protein, which exhibits a tightly folded tertiary structure in solution. The chiral recognition based on the ratios of the abundances of z(18) and z(19) fragments in ECD of 2+ ions was excellent even for a single amino acid (Tyr) D-substitution (R(chiral) = 8.6). The chiral effect decreased with an increase of temperature at the electrospray ion source, as well as at a higher degree of ionization, 3+ ions (R(chiral) = 1.5). A general approach is suggested for charge localization in n+ ions by analysis of ECD mass spectra of (n + 1)+ ions. Application of this approach to 3+ Trp-cage ions revealed the protonation probability order in 2+ ions: Arg(16) > Gln(5) > approximately N-terminus. The ECD results for native form of the 2+ ions favor the preservation of the solution-phase tertiary structure, and chiral recognition through the interaction between the charges and the neutral bond network. Conversely, ECD of 3+ ions supports the dominance of ionic hydrogen bonding which determines a different gas-phase structure than found in solution. Vibrational activation of 2+ ions indicated greater stability of the native form, but the fragmentation patterns did not provide stereoisomer differentiation, thus underlying the special position of ECD among other MS/MS fragmentation techniques. Further ECD studies should yield more structural information as well as quantitative single-amino acid D/L content measurements in proteins.     

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.     

Improved low-energy electron injection systems for high rate electron capture dissociation in Fourier transform ion cyclotron resonance mass spectrometry

New low-energy electron injection systems based on indirectly heated dispenser cathodes facilitate electron capture dissociation (ECD) in Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. In this joint report, details are presented of the design and performance of these systems on two commercial FTICR instruments, 9.4 T Bruker BioAPEX in Uppsala and 4.7 T IonSpec Ultima in Odense. New results include obtaining meaningful one-scan MS/MS data for isolated precursor ions with millisecond irradiation times. The ECD rate improvement is not only due to the larger total electron current, but the larger emitting area as well.     

Comparison of electron capture dissociation and collisionally activated dissociation of polycations of peptide nucleic acids

Electron capture dissociation (ECD) in Fourier transform ion cyclotron resonance mass spectrometry coupled with electrospray ionization enhances the sequence elucidation of peptide nucleic acids compared with conventional low-energy collisionally activated dissociation (CAD). Examples are shown where ECD produced complete or extensive sequence coverage in PNAs six to ten nucleobases long. However, facile base losses from the reduced species and low abundances of backbone ECD fragments presented a significant problem. This was rationalized through the lower degree of charge solvation on the backbone compared to polypeptides. Combination of both CAD and ECD data is advantageous, as these techniques produce cleavages at different sites.     

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 capture dissociation of singly and multiply phosphorylated peptides

Analysis of phosphotyrosine and phosphoserine containing peptides by nano-electrospray Fourier transform ion cyclotron resonance (FTICR) mass spectrometry established electron capture dissociation (ECD) as a viable method for phosphopeptide sequencing. In general, ECD spectra of synthetic and native phosphopeptides appeared less complex than conventional collision activated dissociation (CAD) mass spectra of these species. ECD of multiply protonated phosphopeptide ions generated mainly c- and z(.)-type peptide fragment ion series. No loss of water, phosphate groups or phosphoric acid from intact phosphopeptide ions nor from the c and z(.) fragment ion products was observed in the ECD spectra. ECD enabled complete or near-complete amino acid sequencing of phosphopeptides for the assignment of up to four phosphorylation sites in peptides in the mass range 1400 to 3500 Da. Nano-scale Fe(III)-affinity chromatography combined with nano-electrospray FTMS/ECD facilitated phosphopeptide analysis and amino acid sequencing from crude proteolytic peptide mixtures.     

Electron capture dissociation of weakly bound polypeptide polycationic complexes

We have previously reported that, in electron capture dissociation (ECD), rupture of strong intramolecular bonds in weakly bound supramolecular aggregates can proceed without dissociation of weak intermolecular bonds. This is now illustrated on a series of non-specific peptide-peptide dimers as well as specific complexes of modified glycopeptide antibiotics with their target peptide. The weak nature of bonding is substantiated by blackbody infrared dissociation, low-energy collisional excitation and force-field simulations. The results are consistent with a non-ergodic ECD cleavage mechanism.     

Intramolecular hydrogen atom transfer in hydrogen-deficient polypeptide radical cations

Irradiation of protonated polypeptides NH₂–RH⁺–COOH by >10 eV electrons leads to further ionization and fast intramolecular charge transfer to the free N-terminus. The resulting species may undergo further hydrogen atom rearrangement to form distonic ions N⁺H₃–RH⁺–COO√. Such transfer is exothermic but can involve an appreciable barrier, e.g., 2.3±0.5 eV for MH^2+√ ions of the peptide ACTH 1–10. Radical polypeptide dications can, therefore, be viewed as hydrogen atom wires. Subsequent capture of low energy electrons results in fragmentation. The pattern of this electronic excitation dissociation (EED) is consistent with hydrogen transfer prior to electron capture.