Electron capture dissociation (ECD) has emerged as a powerful mass spectrometry technique for biomolecular structural analysis. Unlike traditional collision-based fragmentation methods, ECD preserves labile post-translational modifications while providing extensive sequence coverage. This enables researchers to decode the details and dynamics of proteins, antibodies, and other therapeutically relevant molecules. The following research summaries showcase how protein characterization with ECD is driving breakthroughs in fundamental understandings of protein structure and function.
Top-Down MS: Rapid Characterization of Cell Death Proteins
Researchers have developed a fast and precise method for analyzing modified Bcl-xL proteins, which are crucial in cell death processes. Rachel Franklin from Agilent Technologies and her team used genetic code expansion and top-down mass spectrometry to analyze modified Bcl-xL variants, including those with engineered phosphoserine and phosphoserine mimics. Unlike traditional procedures that require extensive sample preparation, this new approach uses ECD to break down the intact proteins and achieve 85–90% sequence coverage within minutes. Successful localization of phosphorylation modifications makes top-down analysis with ECD a powerful tool for characterizing phosphorylated proteins.
Decoding Fragmentation from Intact Antibodies with New Software
ExDViewer software enables routine, parameter-free analysis of monoclonal antibody fragmentation data for effective top or middle-down sequencing. Prior work has shown that ECD can simultaneously fragment both heavy and light chains of an intact antibody, resulting in a mixture spectrum of ECD fragments. Stephanie Sturgeon at Agilent Technologies and co-workers have now enhanced ExDViewer to provide high-quality analysis and visualization of mixture spectra results within seconds. ExDViewer enables rapid processing of data to remove charge and isotope elements, producing clear fragments used for sequencing key antibody regions called CDR sequences. These improvements provide a user-friendly and powerful tool for the analysis of monoclonal antibodies using top-down ECD MS.
Protein Therapeutics: Antibody Characterization with ECD
Labile post-translational modifications are better preserved using ECD than conventional MS fragmentation techniques such as collision-induced dissociation (CID). Common modifications, such as glycosylation and phosphorylation, can impact the safety, efficacy, and binding activity of monoclonal antibodies but are challenging to identify. Stephen Sciuto from Agilent Technologies and colleagues analyzed tryptic digests of NIST mAb and Infliximab using an 6545XT Q-TOF mass spectrometer in ExD or CID mode. ECD provided higher sequence coverage than CID, reaching 100% for glycosylated peptides in Infliximab. The ability to pinpoint glycosylation sites using fragment ions demonstrates the power of ECD for characterizing glycopeptides.
Monoclonal Antibodies: Faster Analysis with Microdroplets
The first reported combination of microdroplet reactions with ECD fragmentation offers a quicker way to analyze monoclonal antibodies for disease treatments. Thomas Walker from Agilent Technologies and co-workers demonstrate the use of an Agilent Jet Stream electrospray ionization source to facilitate in-spray chemical reduction and protease digestion of monoclonal antibodies. Downstream fragmentation of the microdroplet reaction products with ECD provided rapid characterization of intact antibodies in minutes. Efficient ECD fragmentation yielded rich sequence information including evidence of disulfide bond connectivity and confirmation of key sequences called complementary determining regions. These results highlight this method’s potential for fast and cost-effective antibody characterization with minimal sample preparation.
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