Proteomics

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

ABSTRACT: Legumain is cysteine protease primarily localised to the endo-lysosomal system. Upregulated legumain activity is associated with inflammation, neurodegeneration, and tumorigenesis. Whilst inhibiting legumain in mouse models has demonstrated therapeutic benefit, the proteolytic mechanisms underpinning its various functions are not well known and thus, further characterisation of its physiological substrates is required. Here, we developed FAIMS-enabled N-terminomics for sensitive and streamlined identification of both protein abundance changes and N-termini in complex samples. Comparison of wildtype and legumain-deficient murine spleens identified 6,366 proteins and 2,528 N-termini, of which 235 were enriched in wildtype spleens. These included 119 with asparaginyl cleavages corresponding to 110 proteins, indicating legumain-specific activity. Surprisingly, many of these localised to the nucleus and cytoplasm, hinting at novel extra-lysosomal roles of legumain. We further confirmed the direct processing of selected substrates by legumain in vitro; together, validating FAIMS-enabled N-terminomics for protease substrate detection in an unbiased and systematic manner.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Alexander Ziegler  

LAB HEAD: Laura E. Edgington-Mitchell

PROVIDER: PXD047733 | Pride | 2024-05-23

REPOSITORIES: Pride

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Publications

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

Ziegler Alexander R AR   Dufour Antoine A   Scott Nichollas E NE   Edgington-Mitchell Laura E LE  

Molecular & cellular proteomics : MCP 20240108 2

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 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 an  ...[more]

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