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Mol Cell Proteomics. 2019 Aug 30. pii: mcp.TIR119.001560. doi: 10.1074/mcp.TIR119.001560. [Epub ahead of print]

Sensitive determination of proteolytic proteoforms in limited microscale proteome samples.

Author information

1
University of British Columbia, Canada.
2
Forschungszentrum Jülich, Germany.
3
Forschungszentrum Juelich, Germany.
4
ZEA-3 Analytics, Forschungszentrum Juelich, Germany.
5
Pathology and Laboratory Medicine, University of British Columbia, Canada philipp.lange@ubc.ca.

Abstract

Protein N termini unambiguously identify truncated, alternatively translated or modified proteoforms with distinct functions and reveal perturbations in disease. Selective enrichment of N-terminal peptides is necessary to achieve proteome-wide coverage for unbiased identification of site-specific regulatory proteolytic processing and protease substrates. However, many proteolytic processes are strictly confined in time and space and therefore can only be analyzed in minute samples that provide insufficient starting material for current enrichment protocols. Here we present High-efficiency Undecanal-based N Termini EnRichment (HUNTER), a robust, sensitive and scalable method for the analysis of previously inaccessible microscale samples. HUNTER achieved identification of >1000 N termini from as little as 2 µg raw HeLa cell lysate. Broad applicability is demonstrated by the first N-terminome analysis of sorted human primary immune cells and enriched mitochondrial fractions from pediatric cancer patients, as well as protease substrate identification from individual Arabidopsis thaliana wild type and Vacuolar Processing Enzyme-deficient mutant seedlings. We further implemented the workflow on a liquid handling system and demonstrate the feasibility of clinical degradomics by automated processing of liquid biopsies from pediatric cancer patients.

KEYWORDS:

Cell sorting; Clinical proteomics; Enrichment; N termini; N-terminal modifications*; Post-translational modifications*; Proteases*; Proteolysis*; Subcellular analysis; Substrate identification

PMID:
31471496
DOI:
10.1074/mcp.TIR119.001560
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