biostudies-literatureZiegler ARNational Health and Medical Research CouncilAustralian Research Councile70186https://www.ebi.ac.uk/biostudies/studies/S-EPMC12447244biostudies-literatureUnknown34(10)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) fractionaProtein science : a publication of the Protein SocietyEmploying complementary fractionation-based N-terminomics approaches enhances the identification of legumain cleavage events in naive and inflamed colon tissue.PMC1244724420189802011119FT200100270DP210100362Ziegler ARScott NEParker BLEdgington-Mitchell LEfalseEmploying complementary fractionation-based N-terminomics approaches enhances the identification of legumain cleavage events in naive 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) fractiona2025-01-01T00:00:00Z2025 Oct2026-06-03T15:37:05.295Z2026-05-30T03:07:25.77ZS-EPMC124472444097044310.1002/pro.70186