On the use of electron capture rate constants to describe electron capture dissociation mass spectrometry of peptides

Unige Anna Laskay, Yury Tsybin, Aleksey Vorobyev, Konstantin Zhurov
Im Publications, 2015
Article

Résumé

Electron capture dissociation (ECD) tandem mass spectrometry (MS/MS) is a powerful analytical tool for peptide and protein structure analysis. The product ion abundance (PIA) distribution in ECD MS/MS is known to vary as a function of electron irradiation period. This variation complicates the development of a method of peptide identification by correlation of ECD MS/MS data with experimental and theoretical mass spectra. Here, we first develop a kinetic model to describe primary electron capture by peptide dications leading to product ion formation and secondary electron capture resulting in product ion neutralization. We apply the developed kinetic model to calculate product ion formation rate constants and electron capture rate constants of product ions from ECD mass spectra acquired using various electron irradiation periods. Contrary to ECD PIA distributions, the product ion formation rate constants are shown to be independent of electron irradiation period and, thus, may be employed to characterize ECD product ion formation more universally. The electron capture rate constants of product ions in ECD Fourier transform ion cyclotron resonance MS were found to correlate (with a correlation factor, R-2, of ca 0.9) with ion mobility cross sections of product ions in electron transfer dissociation. Finally, we demonstrate that the electron irradiation period influences the ratio of radical and even-electron c and z product ions.

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https://infoscience.epfl.ch/record/216187?ln=fr

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Unige Anna Laskay, Yury Tsybin, Aleksey Vorobyev, Konstantin Zhurov
Im Publications, 2015
Article

Résumé

Official source

https://infoscience.epfl.ch/record/216187?ln=fr

À propos de ce résultat

Concepts associés (14)

Concepts associés

Chargement

Publications associées (37)

Publications associées

Chargement

Results of the mechanistic studies of free-electron based fragmentation methods addressing peptide and protein structure analysis with mass spectrometry (MS), electron capture dissociation (ECD) and electron ionization dissociation (EID), are presented. Series of model peptides, H-RAAAA-X-AAAAK-OH and H-RGGGG-X-GGGGK-OH, were rationally designed and studied to understand the role of a single amino acid residue on ECD process. The results show a direct correlation of ECD product ion abundance (PIA) with amino acid hydrophobicity and radical stability of the central amino acid, X, in model peptides. Moreover, comparison of ECD PIA distributions for polyalanine- and polyglycine -based peptides, despite a substantial structure differences between them, demonstrates a high correlation (R=0.86) as well. Ion activation prior to ECD qualitatively preserves the PIA correlation with hydrophobicity, however redistributes the fragmentation channels. This redistribution can be rationalized in terms of: (i) further dissociation of product ions, due to the excess of ion internal energy; (ii) expansion of precursor ion conformational landscape reducing the conformational specificity in localization of unpaired electron and allowing competition of different reactive groups to induce the backbone cleavage. In particular, the competition between N-terminal R, C-terminal K and central X side chains to participate in formation of aminoketyl radicals was considered to explain the central residue specific formation of z5 and [z6-H] ions; and to explain the shift of branching ratio between c- and z-ions toward the formation of c-ions. Further studies were focused on the studying of free electron – ion interaction cross sections. The difference in low energy electron capture cross sections (CSs) of ECD product ions was found to correlate with ECD PIA pattern being a function of electron irradiation period in ECD. The developed experimental methodology and ECD kinetic model were applied to deduce the dissociation rate constants and electron capture CSs from ECD PIA values. The observed correlation between low energy electron capture CSs and ion mobility (IM) CSs provides interesting material for further studies. Finally, characterization of interaction between high energy (50-100 eV) electrons and protein ions in EID demonstrates that the EID cross sections are proportional (R = 0.98), and of the same order of magnitude, to the values of the IM CSs. The results support the feasibility of employing the EID MS for accurate estimation of the geometrical cross sections of gaseous biomolecular ions as a method of biomolecular structure analysis. To summarize, improved understanding in (i) radical chemistry beyond standard ECD parameters and (ii) electron – ion interactions with respect to the ion structure increases the quantity and quality of information possible to achieve by MS/MS, and, therefore, provides a strong basis for further improvements in peptide and protein structure characterization as well as understanding of energy absorption and relaxation process in biomolecules.

EPFL2012

Chargement

Peptide and protein ion abundances in mass spectrometry (MS) and tandem mass spectrometry (MS/MS) may provide orthogonal, or complementary, information to their mass measurements. Collision induced dissociation (CID), the most commonly used MS/MS technique, demonstrates high variability of fragmentation pathways and their relative product abundances as a function of experimental parameters. Moreover, a reliable prediction of relative abundances of CID products is possible only for relatively small systems, but not for larger biomolecules. Recently developed radical-induced fragmentation techniques, e.g., electron capture and transfer dissociation (ECD/ETD), appear to demonstrate more repeatable and reproducible fragmentation patterns. However, it has been reported that ECD/ETD product ion abundance distribution for peptides is rather flat and extracting additional peptide sequence information is yet to be done. In the present work, an attempt is made to understand the peptide's ECD/ETD product ion abundance formation fundamentals and to reveal the possible implications of this source of information for improving peptide and protein structural characterization. Specifically, we have shown that ECD/ETD of peptides is sensitive to the chemical environment in terms of peptide primary structure (amino acid nature) and possibly higher order structure (hydrogen bond connectivity). After demonstrating the analytical validity of the ECD/ETD product ion abundance methodology, its parameter dependence and its applicability to different experimental setups, results will be presented and rationalized along two main axes of comprehension: gas phase peptide radical chemistry and structure. Whereas fundamental aspects of ECD/ETD (mechanism, thermodynamics) can be addressed with the former, additional ECD/ETD yield modulations were observed and presumably relate to structural properties of ions in the gas phase. ECD specificity will be demonstrated on the class of amphipathic peptides, where periodicity is revealed in product ion abundance and correlated to peptide primary and secondary structure. To decipher between both contributions, sequence variations of amphipathic peptides were probed by ECD and correlated to gas phase structures suggested by ion mobility mass spectrometry and supported by molecular dynamics computations. After probing amino acid side chain influence, unraveling the chemical environment specificity of ECD/ETD led us to investigate peptide backbone modifications. Peptides containing beta amino acids revealed two main effects in ECD and ETD. We demonstrate that individual substitutions yield c and z type fragments only for amino acids that provide sufficient z• radical stabilization, whereas multiple substitutions shift the preferential fragmentation pathway to the otherwise minor a-type ion formation pathway indicating possible change in peptide protonation. Finally, understanding the partitioning of radical ions between N- and C-terminal fragments led us to investigate ion internal energy dependence. The extent of hydrogen atom transfer, the [c+z] complex lifetime and secondary fragmentation pathways (w-type ion formation) are directly modulated by ion internal energy as demonstrated by decoupling ETD from charge reduced CID. To summarize, ECD/ETD product ion abundance methodology developed in the thesis advances biomolecule radical chemistry understanding and provides a potentially useful methodology for peptide and protein structural characterization.

EPFL2011

Chargement

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Site-specific reproducibility and repeatability of electron capture dissociation (ECD) in Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) are of fundamental importance for product ion abundance (PIA)-based peptide and protein structure analysis. However, despite the growing interest in ECD PIA-based applications, these parameters have not yet been investigated in a consistent manner. Here, we first provide a detailed description of the experimental parameters for ECD-based tandem mass spectrometry performed on a hybrid linear ion trap (LTQ) FT-ICR MS. In the following, we describe the evaluation and comparison of ECD and infrared multiphoton dissociation (IRMPD) PIA methodologies upon variation of a number of experimental parameters, for example, cathode potential (electron energy), laser power, electron and photon irradiation periods and pre-irradiation delays, as well as precursor ion number. Ranges of experimental parameters that yielded an average PIA variation below 5% and 15% were determined for ECD and IRMPD, respectively. We report cleavage site-dependent ECD PIA variation below 20% and correlation coefficients between fragmentation patterns superior to 0.95 for experiments performed on three FT-ICR MS instruments. Overall, the encouraging results obtained for ECD PIA reproducibility and repeatability support the use of ECD PIA as a complementary source of information to m/z data in radical-induced dissociation applied for peptide and protein structure analysis.

2011

Chargement

EPFL2012

EPFL2011