Energy Resolved Interplay between Preferential Backbone Cleavage and PTM Lability in Top-Down Mass Spectrometry

A mechanistic understanding of collision induced dissociation is well established for fragmentation propensities of peptides in unmodified and modified forms. Major limitations in the characterization of unmodified peptides and peptides with labile posttranslational modifications (PTMs) include preferential cleavage N-terminal to proline, C-terminal to acidic residues as well as loss of PTMs, such as phosphorylation and glycosylation. However, the relative contributions of certain fragmentation pathways are not completely understood at the intact protein level. Here, we evaluated the top-down fragmentation propensities of α-casein, β-casein, ribonuclease B, and ovalbumin under denatured and native conditions using energy resolved higher energy collisional dissociation (HCD). Relative product ion abundance, residue fragmentation propensity, and sequence coverage were used to compare cleavages N-terminal to Pro and C-terminal to Asp/Glu, phosphoric acid neutral loss, and glycan dissociation. Pro-directed cleavage dominated α- and β-casein under both conditions, with limited phosphoric acid loss. Glycoproteins exhibited condition and energy dependent redistribution. Denatured RNase B shifted from Pro directed cleavage toward oxonium ion formation, whereas native RNase B shifted from sequential mannose loss toward Pro directed cleavage. Native ovalbumin shifted from Pro directed to acidic residue directed fragmentation. High relative abundance reflects fragmentation at fewer sites, supporting complementary measurements of ion abundance and fragmentation propensity. These results demonstrate that top-down proteoform characterization is not significantly limited by the neutral loss of labile PTMs as would be predicted by peptide fragmentation. Rather, sequence coverage and thus proteoform characterization are largely limited by sequence specific preferential cleavages, nevertheless highlighting the need for alternative electron and photon-based ion activation methods.