Employing complementary fractionation‐based N‐terminomics approaches enhances the identification of legumain cleavage events in naïve and inflamed colon tissue

The mammalian lysosomal protease legumain is often dysregulated in pathophysiological conditions including inflammation, neurodegeneration, and cancer, yet its proteolytic targets are poorly defined. To profile protease substrates, degradomics techniques typically employ enrichment strategies to select for sub-stoichiometric and low-abundance peptides generated by proteolytic cleavage. However, recent advancements in degradomics techniques have revealed N-termini enrichment can be circumvented if peptide-based fractionation is employed, enabling simultaneous proteome and N-terminome analysis. Herein, we compare the previously published enrichment-free N-terminomics approach using high-field asymmetric waveform ion mobility spectrometry (FAIMS) to offline basic reverse-phase (bRP) fractionation to assess the complementarity of these fractionation methods for simultaneous proteomic and degradomic analyses. While at the protein level FAIMS and bRP provide access to overlapping proteomic coverage, at the N-terminus level each fractionation technique reveals unique cleavage information. Combining data from the two fractionation approaches revealed 6499 N-terminal peptides with N-terminal TMTpro labeling, allowing the identification of cleavage events modulated in the context of legumain deficiency in naïve murine colons and during dextran sulfate sodium (DSS)-induced colitis. Among these N-termini, we identify 35 putative legumain substrates in naïve and 41 in the DSS-treated colons, supporting a role for legumain in both pro-inflammatory and physiological conditions. Use of an additional negative selection method, High-efficiency Undecanal-based N-Termini EnRichment (HUNTER), further supplements this list of identified legumain substrates. Combined, this study identifies multiple putative substrates of legumain in healthy and inflamed murine colons as well as demonstrates the utility of using complementary fractionation approaches for degradomics studies.