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bioRxiv · 10.1101/2023.07.18.549248

FAIMS-enabled N-terminomics analysis reveals novel legumain substrates in murine spleen

Ziegler, A. R.·Dufour, A.·Scott, N. E.·Edgington-Mitchell, L. E.
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Abstract

Aberrant 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 previously been 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 sensitive and streamlined approach for proteome and N-terminome analyses in a single analytical method without the need for N-termini enrichment. Compared to unfractionated proteomic analysis, we demonstrate FAIMS fractionation improves neo-N- termini identification by >2.5 fold, resulting in identification of >2,882 unique neo-N-termini from limited sample amounts. Within murine spleens, this approach identifies 6,366 proteins and 2,528 unique neo-N-termini, with 235 cleavage events enriched in wild-type 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 demonstrates the utility of FAIMS as an accessible method to improve depth and quality of N- terminomics studies.

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Ziegler, A. R., Dufour, A., Scott, N. E., Edgington-Mitchell, L. E.. 2023-07-19. FAIMS-enabled N-terminomics analysis reveals novel legumain substrates in murine spleen. https://doi.org/10.1101/2023.07.18.549248

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