Naive and DSS Murine Colon TMTpro bRP fractionation N-terminomics

ABSTRACT: 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 abundant peptides generated from 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 analysis. While at the protein levels FAIMS and bRP provide access to overlapping proteomic coverage, at the N-terminal level each fractionation technique reveals unique cleavage information. Combining fractionation approaches we identify 6,499 novel N-terminal peptides with N-terminal TMTpro labelling allowing the identification of cleavage events modulated in the context of Dextran Sulfate Sodium (DSS) induced colitis within wild type and legumain-deficient colons. Among these N-termini, we identify 35 putative legumain substrates in naïve and 41 in the DSS colons supporting legumain’s role in both pro-inflammatory and physiological conditions. Use of an additional negative selection method, High-efficiency Undecanal-based N-Termini EnRichment (HUNTER), further validated and supplemented this list of identified legumain substrates. Combined, this study identifies multiple putative substrates of legumain in healthy and inflamed murine colons as well as the utility of using complementary fractionation approaches for degradomics studies.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Colon

DISEASE(S): Colitis

SUBMITTER: Alexander Ziegler  

LAB HEAD: Laura Edgington-Mitchell

PROVIDER: PXD060852 | Pride | 2025-09-29

REPOSITORIES: Pride