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.
Highlights
Protein N termini reveal fundamental regulatory mechanisms and their perturbation in disease
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
Workflow Optimization—The most sensitive protocols established to date enrich protein N termini by negative selection, where protein amines are blocked with amine-reactive reagents before proteome digestion [24]
Summary
Protein N termini reveal fundamental regulatory mechanisms and their perturbation in disease. To overcome this challenge, dedicated methods for selective enrichment and unbiased identification of N-terminal peptides from complex proteomes have been developed (8 –12) Such N-terminome profiling has greatly advanced our understanding of apoptosis [10, 13], revealed novel proteolytic proteoforms in human tissues [14, 15] and animal models of disease [16], identified protease substrates underlying common disease and rare genetic disorders [17, 18] and enabled characterization of alternative protein translation initiation sites [19, 20] and protein N-terminal modifications [21].
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