A Data Processing Workflow Combining Orthogonal MS/MS Fragmentation Data for Comprehensive Middle-down Analysis

September 10, 2024

Middle-down protein sequencing of intact or partially digested therapeutic antibodies offers a faster and less complex alternative to characterizing antibodies by peptide mapping and provides more information than intact mass analysis. By utilizing novel enzymes that cleave antibodies to generate well-defined fragments that are amenable to characterization by LC-MS/MS, middle-down protein sequencing facilitates validation of modifications detected at the peptide level. A key requirement of these analyses is high-confidence identification of the ions used for sequencing. Combining two orthogonal dissociation methods (collision-induced [CID] and electron-activated [EAD] dissociation) provides complementary fragmentation patterns that increase overall sequence coverage and increased confidence in each fragment ion identification.