Ion Mobility-Based Enrichment-Free N-Terminomics Analysis Reveals Novel Legumain Substrates in Murine Spleen

Mol Cell Proteomics. 2024 Jan 8;23(2):100714. doi: 10.1016/j.mcpro.2024.100714. Online ahead of print.ABSTRACTAberrant levels of the asparaginyl endopeptidase legumain have been linked to inflammation, neurodegeneration, and cancer, yet our understanding of this protease is incomplete. Systematic attempts to identify legumain substrates have been previously confined to in vitro studies, which fail to mirror physiological conditions and obscure biologically relevant cleavage events. Using high-field asymmetric waveform ion mobility spectrometry (FAIMS), we developed a streamlined approach for proteome and N-terminome analyses without the need for N-termini enrichment. Compared to unfractionated proteomic analysis, we demonstrate FAIMS fractionation improves N-termini identification by >2.5 fold, resulting in the identification of >2882 unique N-termini from limited sample amounts. In murine spleens, this approach identifies 6366 proteins and 2528 unique N-termini, with 235 cleavage events enriched in WT compared to legumain-deficient spleens. Among these, 119 neo-N-termini arose from asparaginyl endopeptidase activities, representing novel putative physiological legumain substrates. The direct cleavage of selected substrates by legumain was confirmed using in vitro assays, providing support for the existence of physiologically relevant extra-lysosomal legumain activity. Combined, these data shed critical light on the functions of legumain and demonstrate the utility of F...
Source: Molecular and Cellular Proteomics : MCP - Category: Molecular Biology Authors: Source Type: research