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Tandem Native Mass-Spectrometry on Antibody–Drug Conjugates and Submillion Da Antibody–Antigen Protein Assemblies on an Orbitrap EMR Equipped with a High-Mass Quadrupole Mass Selector
journal contribution
posted on 2015-06-16, 00:00 authored by Andrey Dyachenko, Guanbo Wang, Mike Belov, Alexander Makarov, Rob N. de Jong, Ewald T. J. van
den Bremer, Paul W. H. I. Parren, Albert J. R. HeckNative mass spectrometry is emerging
as a powerful tool for the
characterization of intact antibodies and antibody-based therapeutics.
Here, we demonstrate new possibilities provided by the implementation
of a high mass quadrupole mass selector on the recently introduced
Orbitrap Exactive EMR mass spectrometer. This configuration allows
precursor ion selection, and thus tandem mass spectrometry experiments,
even on analytes with masses in the hundreds of kilodaltons. We apply
tandem mass spectrometry to localize the drug molecules in the therapeutic
antibody–drug conjugate brentuximab vedotin, which displays
a heterogeneous drug load. Our tandem MS data reveal that drug conjugation
takes place nonhomogeneously to cysteine residues both on the light
and heavy chains. Next, we analyzed how many antigens bind to IgG
hexamers, based on a recently described antibody mutant IgG1-RGY that
forms hexamers and activates complement in solution. The fully saturated
IgG1–RGY–antigen complexes displayed a stoichiometry
of IgG:CD38 of 6:12, possessing a molecular weight of about 1.26 MDa
and demonstrating that IgG assembly does not hamper antigen binding.
Through tandem MS experiments, we retrieve information about the spatial
arrangement and stoichiometry of the subunits within this complex.
These examples underscore the potential of this further modified Orbitrap-EMR
instrument especially for the in-depth characterization by native
tandem mass spectrometry of antibodies and antibody-based constructs.
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IgG assemblyOrbitrap Exactive EMR mass spectrometerplace nonhomogeneouslyantigens bindtandem mass spectrometrydrug moleculesprecursor ion selectionmass quadrupole mass selectortandem mass spectrometry experimentstandem MS datadrug conjugationforms hexamersOrbitrap EMRtandem MS experimentsantibodyantigen bindingIgG hexamersactivates complement1.26 MDadrug loadcysteine residues
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