title: Top-down structural analysis of an intact monoclonal antibody by electron capture dissociation-Fourier transform ion cyclotron resonance-mass spectrometry.
abstract: Top-down electron capture dissociation (ECD) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry was performed for structural analysis of an intact monoclonal antibody (IgG1kappa (κ) isotype, ~148 kDa). Simultaneous ECD for all charge states (42+ to 58+) generates more extensive cleavages than ECD for an isolated single charge state. The cleavages are mainly localized in the variable domains of both heavy and light chains, the respective regions between the variable and constant domains in both chains, the region between heavy-chain constant domains CH2 and CH3, and the disulfide bond (S-S)-linked heavy-chain constant domain CH3. The light chain yields mainly N-terminal fragment ions due to the protection of the interchain disulfide bond between light and heavy chain, and limited cleavage sites are observed in the variable domains for each chain, where the S-S spans the polypeptide backbone. Only a few cleavages in the S-S-linked light-chain constant domain, hinge region, and heavy-chain constant domains CH1 and CH2 are observed, leaving glycosylation uncharacterized. Top-down ECD with a custom-built 9.4 T FTICR mass spectrometer provides more extensive sequence coverage for structural characterization of IgG1κ than does top-down collision-induced dissociation (CID) and electron transfer dissociation (ETD) with hybrid quadrupole time-of-flight instruments and comparable sequence coverage for top-down ETD with orbitrap mass analyzers.
authors.affiliation: Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32303, United States.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Mao
authors.forename: Santosh G
authors.initials: SG
authors.lastname: Valeja
authors.forename: Jason C
authors.initials: JC
authors.lastname: Rouse
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Hendrickson
authors.forename: Alan G
authors.initials: AG
authors.lastname: Marshall
mesh_terms: D000911:Antibodies, Monoclonal / Q000032:analysis*; D004583:Electrons; D005583:Fourier Analysis; D013058:Mass Spectrometry; D011487:Protein Conformation
chemical_list: D000911:Antibodies, Monoclonal