Tandem mass spectrometry

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 abilities to analyse chemical samples. A common use of tandem MS is the analysis of biomolecules, such as proteins and peptides. The molecules of a given sample are ionized and the first spectrometer (designated MS1) separates these ions by their mass-to-charge ratio (often given as m/z or m/Q). Ions of a particular m/z-ratio coming from MS1 are selected and then made to split into smaller fragment ions, e.g. by collision-induced dissociation, ion-molecule reaction, or photodissociation. These fragments are then introduced into the second mass spectrometer (MS2), which in turn separates the fragments by their m/z-ratio and detects them. The fragmentation step makes it possible to identify and separate ions that have very similar m/z-ratios in regular mass spectrometers. Structure

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

Ion coalescence in Fourier transform mass spectrometry: should we worry about this in shotgun proteomics?

Luca Fornelli

Coupling of motion of the ion clouds with close m/z values is a well-established phenomenon for ion-trapping mass analyzers. In Fourier transform ion cyclotron resonance mass spectrometry it is known as ion coalescence. Recently, ion coalescence was demonstrated and semiquantitatively characterized for the Orbitrap mass analyzer as well. When it occurs, the coalescence negatively affects the basic characteristics of a mass analyzer. Specifically, the dynamic range available for the high resolving power mass measurements reduces. In shotgun proteomics, another potentially adverse effect of ion coalescence is interference of the isotopic envelopes for the coeluting-precursor ions of close m/z values, subjected to isolation before fragmentation. In this work we characterize coalescence events for synthetic-peptide mixtures with fully and partially overlapping C-13-isotope envelopes, including pairs of peptides with glutamine-deamidation. Furthermore, we demonstrate that fragmentation of the otherwise coalesced peptide ion clouds may remove the locking between them owing to the total charge redistribution between more ion species in the mass spectrum. Finally, we estimated the possible-scale of the coalescence phenomenon for shotgun proteomics by considering the fraction of coeluted peptide pairs with the close masses using literature data for the yeast proteome. It was found that up to one-tenth of all peptide identifications with the relative mass differences of 20 ppm or less in the corresponding pairs may potentially experience the coalescence of the C-13-isotopic envelopes. However, sample complexity in a real proteomics experiment and precursor ion-signal splitting between many channels in tandem mass spectrometry drastically increase the threshold for coalescence, thus leading to practically coalescence-free proteomics based on Fourier transform mass spectrometry.

Im Publications2015

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

Aleksey Vorobyev

EPFL2012

Ion coalescence in Fourier transform mass spectrometry: should we worry about this in shotgun proteomics?

Luca Fornelli

Im Publications2015

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

Characteristics of isobaric tagging-based peptide quantification by high resolution tandem-in-time mass spectrometry

Ion coalescence in Fourier transform mass spectrometry: should we worry about this in shotgun proteomics?

Characteristics of isobaric tagging-based peptide quantification by high resolution tandem-in-time mass spectrometry

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

Ion coalescence in Fourier transform mass spectrometry: should we worry about this in shotgun proteomics?