LAUSR

Characterization and monitoring of post-translational modifications (PTMs) are key analytical requirements during the development of biologics. Top and middle-down (MD) approaches aim at capturing a direct snapshot of all proteoforms with their combinatorial distribution. However, classical MD data analysis is predominantly limited to the interpretation of terminal ion series and PTMs matched by mass. In this study, time-resolved deconvolution (TRD) maps were produced to detect variants and impurities in Fd, Fc/2 and LC subunits of an IgG1 consistently across multiple samples. Classical MD analysis retrieved terminal ions suggesting a deamidation at a NN motif for a LC+1Da species, and inconclusive information for a LC+40Da species. Additionally, we performed differential analysis of all MS2 ions across unmodified and variant subunit spectra to focus data analysis on spectral differences and reveal diagnostic ions (present, absent, enriched, or depleted ions) before fragment assignment. This sensitive methodology was able to detect diagnostic ions in a chimeric spectrum pointing at a Proline to Histidine sequence variant (+40Da) missed by classical MD analysis. This methodology was pivotal to unravel relevant terminal ions and internal fragments N-terminal to Proline as diagnostic ions to confirm the deamidation site. Moreover, different cleavage propensities were revealed at the deamidated DN site compared to the native NN motif for terminal and internal fragments, which may be tracked as a diagnostic behavior. Differential analysis may refine the detection of novel diagnostic ions and leverage the sequence information of internal fragments for the characterization of product-related variants and impurities by MD mass spectrometry.