TitleDirect Determination of Antibody Chain Pairing by Top-down and Middle-down Mass Spectrometry Using Electron Capture Dissociation and Ultraviolet Photodissociation.
Publication TypeJournal Article
Year of Publication2020
AuthorsShaw JB, Liu W, Ev YVVasil, Bracken CC, Malhan N, Guthals A, Beckman JS, Voinov VG
JournalAnal Chem
Volume92
Issue1
Pagination766-773
Date Published2020 01 07
ISSN1520-6882
Abstract

One challenge associated with the discovery and development of monoclonal antibody (mAb) therapeutics is the determination of heavy chain and light chain pairing. Advances in MS instrumentation and MS/MS methods have greatly enhanced capabilities for the analysis of large intact proteins yielding much more detailed and accurate proteoform characterization. Consequently, direct interrogation of intact antibodies or F(ab')2 and Fab fragments has the potential to significantly streamline therapeutic mAb discovery processes. Here, we demonstrate for the first time the ability to efficiently cleave disulfide bonds linking heavy and light chains of mAbs using electron capture dissociation (ECD) and 157 nm ultraviolet photodissociation (UVPD). The combination of intact mAb, Fab, or F(ab')2 mass, intact LC and Fd masses, and CDR3 sequence coverage enabled determination of heavy chain and light chain pairing from a single experiment and experimental condition. These results demonstrate the potential of top-down and middle-down proteomics to significantly streamline therapeutic antibody discovery.

DOI10.1021/acs.analchem.9b03129
PubMed ID31769659
Grant ListR43 GM122131 / GM / NIGMS NIH HHS / United States
R44 GM122131 / GM / NIGMS NIH HHS / United States