title: Top-down analysis of immunoglobulin G isotypes 1 and 2 with electron transfer dissociation on a high-field Orbitrap mass spectrometer.
abstract: The increasing importance of immunoglobulins G (IgGs) as biotherapeutics calls for improved structural characterization methods designed for these large (~150kDa) 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.
  
  
  
  Compared with our previous report, the results presented herein demonstrate the power of technological advances of the next generation Orbitrap™ 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.
authors.affiliation: Biomolecular Mass Spectrometry Laboratory, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland.
authors.forename: Luca
authors.initials: L
authors.lastname: Fornelli
authors.affiliation: Biomolecular Mass Spectrometry Laboratory, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland.
authors.forename: Daniel
authors.initials: D
authors.lastname: Ayoub
authors.affiliation: Thermo Fisher Scientific GmbH, 28199 Bremen, Germany.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Aizikov
authors.affiliation: Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, 46202 Indianapolis, IN, USA; Center for Computational Biology and Bioinformatics, Indiana University School of Medicine, 46202 Indianapolis, IN, USA.
authors.forename: Xiaowen
authors.initials: X
authors.lastname: Liu
authors.affiliation: Thermo Fisher Scientific GmbH, 28199 Bremen, Germany.
authors.forename: Eugen
authors.initials: E
authors.lastname: Damoc
authors.affiliation: Department of Computer Science and Engineering, University of California in San Diego, 92093 San Diego, CA, USA.
authors.forename: Pavel A
authors.initials: PA
authors.lastname: Pevzner
authors.affiliation: Thermo Fisher Scientific GmbH, 28199 Bremen, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Makarov
authors.affiliation: Centre d'Immunologie Pierre Fabre, 74160 St Julien-en-Genevois, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Beck
authors.affiliation: Biomolecular Mass Spectrometry Laboratory, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland; Spectroswiss Sàrl, EPFL Innovation Park, 1015 Lausanne, Switzerland. Electronic address: tsybin@spectroswiss.ch.
authors.forename: Yury O
authors.initials: YO
authors.lastname: Tsybin
mesh_terms: D000068879:Adalimumab / Q000737:chemistry*; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D004583:Electrons; D006801:Humans; D013058:Mass Spectrometry; D000077544:Panitumumab; D000068878:Trastuzumab / Q000737:chemistry*
chemical_list: D000911:Antibodies, Monoclonal; D000077544:Panitumumab; D000068879:Adalimumab; D000068878:Trastuzumab
keywords: Electron transfer dissociation, ETD; Immunoglobulin G, IgG; Orbitrap; Top-down