All Ion Differential Analysis Refines the Detection of Terminal and Internal Diagnostic Fragment Ions for the Characterization of Biologics Product-Related Variants and Impurities by Middle-down Mass Spectrometry

Abstract

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 +1 Da species, and inconclusive information for a LC+40 Da species. Additionally, we performed differential analysis of all MS 2 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-tohistidine sequence variant (+40 Da) 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 on internal fragments for the characterization of product-related variants and impurities by MD mass spectrometry.

Ion Activation Methods for Peptides and Proteins

“…Recently, middle-down methods have also been extended to the analysis of antibody–drug conjugates (ADCs), showcasing the ability of ETD to characterize conjugation sites, occupancy, and microvariants at the subunit level . Advanced data processing via differential analysis of fragment ions was used to accurately distinguish antibody proteoforms by enhancing the assignment of both terminal and internal fragment ions, thereby allowing the localization of deamidation events and single point mutations …”

Internal Fragment Ions Disambiguate and Increase Identifications in Top-Down Proteomics

“…In this study, MetaMorpheus was allowed to search for internal fragments of any length; however, some users may prefer to raise the minimum internal fragment length to an arbitrary number of amino acids (e.g., 4 or 5) to reduce the chance of random false-positive assignments. 7 MetaMorpheus allows users to specify a minimum allowed internal fragment length (Figure S1), but we found that this parameter has a negligible impact on the results of the search.…”

“…Previous work suggests that a large fraction of these unidentified fragment ions can be attributed to internal fragments. − Unlike canonical terminal fragments, which contain either the original intact proteoform N- or C-terminus, internal fragments originate from proteoform molecules that have been fragmented multiple times and no longer possess either of the original termini. Several top-down analyses of individual proteins have been published and demonstrate the utility of internal fragments for understanding proteoform fragmentation spectra and improving sequence coverage. − Despite the demonstrated utility of internal fragments, − they are not typically used in top-down analyses.…”

Increasing Top-Down Mass Spectrometry Sequence Coverage by an Order of Magnitude through Optimized Internal Fragment Generation and Assignment

“…Kelleher and colleagues proposed methods for presenting internal fragmentation maps, which we employ and expand upon here. Notable applications of internal fragments include characterizing macromolecular complexes, intact disulfide bonds, , biotherapeutics, NF Kappa proteoform quantification, and two-dimensional “deep” top-down sequencing of calmodulin. These and additional studies demonstrated the generation of internal fragments by a variety of dissociation techniques, including activated ion-electron-transfer dissociation, ultraviolet photodissociation (UVPD), and high-energy electron capture dissociation .…”