title: Analysis of intact monoclonal antibody IgG1 by electron transfer dissociation Orbitrap FTMS.
abstract: The primary structural information of proteins employed as biotherapeutics is essential if one wishes to understand their structure-function relationship, as well as in the rational design of new therapeutics and for quality control. Given both the large size (around 150 kDa) and the structural complexity of intact immunoglobulin G (IgG), which includes a variable number of disulfide bridges, its extensive fragmentation and subsequent sequence determination by means of tandem mass spectrometry (MS) are challenging. Here, we applied electron transfer dissociation (ETD), implemented on a hybrid Orbitrap Fourier transform mass spectrometer (FTMS), to analyze a commercial recombinant IgG in a liquid chromatography (LC)-tandem mass spectrometry (MS/MS) top-down experiment. The lack of sensitivity typically observed during the top-down MS of large proteins was addressed by averaging time-domain transients recorded in different LC-MS/MS experiments before performing Fourier transform signal processing. The results demonstrate that an improved signal-to-noise ratio, along with the higher resolution and mass accuracy provided by Orbitrap FTMS (relative to previous applications of top-down ETD-based proteomics on IgG), is essential for comprehensive analysis. Specifically, ETD on Orbitrap FTMS produced about 33% sequence coverage of an intact IgG, signifying an almost 2-fold increase in IgG sequence coverage relative to prior ETD-based analysis of intact monoclonal antibodies of a similar subclass. These results suggest the potential application of the developed methodology to other classes of large proteins and biomolecules.
authors.affiliation: Biomolecular Mass Spectrometry Laboratory, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland.
authors.forename: Luca
authors.initials: L
authors.lastname: Fornelli
authors.forename: Eugen
authors.initials: E
authors.lastname: Damoc
authors.forename: Paul M
authors.initials: PM
authors.lastname: Thomas
authors.forename: Neil L
authors.initials: NL
authors.lastname: Kelleher
authors.forename: Konstantin
authors.initials: K
authors.lastname: Aizikov
authors.forename: Eduard
authors.initials: E
authors.lastname: Denisov
authors.forename: Alexander
authors.initials: A
authors.lastname: Makarov
authors.forename: Yury O
authors.initials: YO
authors.lastname: Tsybin
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000032:analysis*; D002853:Chromatography, Liquid; D005583:Fourier Analysis; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis*; D040901:Proteomics; D059629:Signal-To-Noise Ratio; D013329:Structure-Activity Relationship
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D010446:Peptide Fragments