Repeatability and reproducibility of product ion abundances in electron capture dissociation mass spectrometry of peptides

Hisham Ben Hamidane, Yury Tsybin, Aleksey Vorobyev
2011
Journal paper

Abstract

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.

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Hisham Ben Hamidane, Yury Tsybin, Aleksey Vorobyev
2011
Journal paper

Abstract

Official source

https://infoscience.epfl.ch/record/169697?ln=en

About this result

Related concepts (13)

Tandem mass spectrometry, also known as MS/MS or MS2, is a technique in instrumental analysis where two or more mass analyzers are coupled together using an additional reaction step to increase their

Peptides are short chains of amino acids linked by peptide bonds. A polypeptide is a longer, continuous, unbranched peptide chain. Polypeptides which have a molecular mass of 10,000 Da or more are c

Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. The results are presented as a mass spectrum, a plot of intensity as a function of the mass

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Novel Approaches in Tandem Mass Spectrometry for Peptide and Protein Structure Analysis

Aleksey Vorobyev

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

Principles of electron capture and transfer dissociation mass spectrometry applied to peptide and protein structure analysis

Luca Fornelli, Unige Anna Laskay, Yury Tsybin, Matthew Wodrich, Konstantin Zhurov

This tutorial review describes the principles and practices of electron capture and transfer dissociation (ECD/ETD or ExD) mass spectrometry (MS) employed for peptide and protein structure analysis. ExD MS relies on interactions between gas phase peptide or protein ions carrying multiple positive charges with either free low-energy (similar to 1 eV) electrons (ECD), or with reagent radical anions possessing an electron available for transfer (ETD). As a result of recent implementation on sensitive, high resolution, high mass accuracy, and liquid chromatography timescale-compatible mass spectrometers, ExD, more specifically, ETD MS has received particular interest in life science research. In addition to describing the fundamental aspects of ExD radical ion chemistry, this tutorial provides practical guidelines for peptide de novo sequencing with ExD MS, as well as reviews some of the current capabilities and limitations of these techniques. The merits of ExD MS are discussed primarily within the context of life science research.

Royal Soc Chemistry2013

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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.

Im Publications2015

Novel Approaches in Tandem Mass Spectrometry for Peptide and Protein Structure Analysis

Aleksey Vorobyev

EPFL2012