Spectrométrie de masse à trappe orbitale

La spectrométrie de masse à trappe orbitale consiste en l'utilisation d'un Orbitrap (nom commercial) qui permet le piégeage des ions par un champ électrostatique. Ces ions possèdent une fréquence d'oscillation axiale variable en fonction de leur ratio masse sur charge, ce qui permet de les séparer. L'Orbitrap est composée d'une électrode centrale ainsi qu'une électrode externe en forme de cylindre. L'électrode centrale coaxiale à l'axe interne piège les ions dans un mouvement orbitale. Un spectre de masse peut être généré à partir du détecteur qui convertit l'image du courant induit des ions piégés en spectre de fréquence en utilisant la transformée de Fourier. Historique En 1923, Kingdon développait le principe de la trappe orbitale où des ions étaient piégés par des champs électrostatiques. Ce piège se compose d’un fil central muni d’une électrode cylindrique externe. Knight ajoute en 1981 une électrode externe modifiée, soit un terme qua

Top-down analysis of immunoglobulin G isotypes 1 and 2 with electron transfer dissociation on a high-field Orbitrap mass spectrometer

Daniel Ayoub, Luca Fornelli, Yury Tsybin

The increasing importance of immunoglobulins G (IgGs) as biotherapeutics calls for improved structural characterization methods designed for these large (similar to 150 kDa) macromolecules. Analysis workflows have to be rapid, robust, and require minimal sample preparation. In a previous work we showed the potential of Orbitrap Fourier transform mass spectrometry (FTMS) combined with electron transfer dissociation (ETD) for the top-down investigation of an intact IgG1, resulting in-30% sequence coverage. Here, we describe a top-down analysis of two IgGs1 (adalimumab and trastuzumab) and one IgG2 (panitumumab) performed with ETD on a mass spectrometer equipped with a high-field Orbitrap mass analyzer. For the IgGs1, sequence coverage comparable to the previous results was achieved in a two-fold reduced number of summed transients, which corresponds, taken together with the significantly increased spectra acquisition rate, to-six-fold improvement in analysis time. Furthermore, we studied the influence of ion-ion interaction times on ETD product ions for IgGs1, and the differences in fragmentation behavior between IgGs1 and IgG2, which present structural differences. Overall, these results reinforce the hypothesis that gas phase dissociation using both energy threshold-based and radical driven ion activations is directed to specific regions of the polypeptide chains mostly by the location of disulfide bonds. Significance of the study: Compared with our previous report, the results presented herein demonstrate the power of technological advances of the next generation Orbitrap (TM) platform, including the use of a high-field compact (i.e., D20) Orbitrap mass analyzer, and a dedicated manipulation strategy for large protein ions (via their trapping in the HCD collision cell along with reduction of the pressure in the cell). Notably, these important developments became recently commercially available in the top-end Orbitrap platforms under the name of "Protein Mode". Furthermore, we continued exploring the advantages offered by the summation (averaging) of transients (time-domain data) for improving the signal-to-noise ratio of top-down mass spectra. Finally, for the first time we report the application of the hybrid ion activation technique that combines electron transfer dissociation and higher energy collisional dissociation, known as EThcD, on intact monoclonal antibodies. Under these specific instrumental parameters, EThcD produces a partially complementary fragmentation pattern compared to ETD, increasing the overall sequence coverage especially at the protein termini. (C) 2017 Elsevier B.V. All rights reserved.

Elsevier Science Bv2017

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

Drug-to-Antibody Ratio Estimation via Proteoform Peak Integration in the Analysis of Antibody-Oligonucleotide Conjugates with Orbitrap Fourier Transform Mass Spectrometry

Natalia Gasilova, Anton Kozhinov, Laure Menin, Konstantin Nagornov, Yury Tsybin

The therapeutic efficacy and pharmacokinetics of antibody-drug conjugates (ADCs) in general, and antibody-oligonucleotide conjugates (AOCs) in particular, depend on the drug-to-antibody ratio (DAR) distribution and average value. The DAR is considered a critical quality attribute, and information pertaining to it needs to be gathered during ADC/AOC development, production, and storage. However, because of the high structural complexity of ADC/AOC samples, particularly in the initial drug-development stages, the application of the current state-of-the-art mass spectrometric approaches can be limited for DAR analysis. Here, we demonstrate a novel approach for the analysis of complex ADC/AOC samples, following native size-exclusion chromatography Orbitrap Fourier transform mass spectrometry (FTMS). The approach is based on the integration of the proteoform-level mass spectral peaks in order to provide an estimate of the DAR distribution and its average value with less than 10% error. The peak integration is performed via a truncation of the Orbitrap's unreduced time-domain ion signals (transients) before mass spectra generation via FT processing. Transient recording and processing are undertaken using an external data acquisition system, FTMS Booster X2, coupled to a Q Exactive HF Orbitrap FTMS instrument. This approach has been applied to the analysis of whole and subunit-level trastuzumab conjugates with oligonucleotides. The obtained results indicate that ADC/AOC sample purification or simplification procedures, for example, deglycosylation, could be omitted or minimized prior to the DAR analysis, streamlining the drug-development process.