Sensitive determination of proteolytic proteoforms in limited microscale proteome samples
| Autor: | Janice Tsui, Domenic Tuscher, Lorenz Nierves, Enes K. Ergin, Anuli Uzozie, Samuel S.H. Weng, Fatih Demir, Philipp F. Lange, Sabrina Dirnberger, Pitter F. Huesgen |
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| Rok vydání: | 2019 |
| Předmět: |
enrichment
substrate identification Proteome medicine.medical_treatment Mutant Arabidopsis subcellular analysis Biochemistry Analytical Chemistry Neoplasms cell sorting Child Microscale chemistry 0303 health sciences medicine.diagnostic_test biology Chemistry N termini 030302 biochemistry & molecular biology Technological Innovation and Resources Brain Cell sorting Mitochondria 3. Good health N-terminal modifications Proteases proteolysis Proteasome Endopeptidase Complex Proteolysis Computational biology 03 medical and health sciences Immune system Protein Domains post-translational modifications medicine Animals Humans ddc:610 Rats Wistar Molecular Biology 030304 developmental biology Protease Wild type biology.organism_classification Pediatric cancer Peptide Fragments Rats clinical proteomics Seedlings proteases |
| Zdroj: | Molecular & Cellular Proteomics : MCP Molecular & Cellular Proteomics Molecular & cellular proteomics 18(11), 2335-2347 (2019). doi:10.1074/mcp.TIR119.001560 |
| Popis: | Protein N termini reveal fundamental regulatory mechanisms and their perturbation in disease. Regulatory proteolysis is often spatially and temporally confined, thus accessible only in minimal specimen incompatible with established protocols. We developed a robust, sensitive, scalable and automatable method for system-wide identification of thousands of N termini from minute samples. Applications revealed distinct N-terminal profiles in sorted immune cells and mitochondria from pediatric cancer patient cells, protease substrates in Arabidopsis seedlings and effects of chemotherapy on proteolytic proteoforms in clinical liquid biopsies. Graphical Abstract Highlights Single-pot workflow for manual or automated enrichment of N-terminal peptides. Sensitive enrichment of protein N termini from 10,000 cells or 2 μg crude proteome. Data independent acquisition improves precision of peptide level quantification. First degradomic analyses of sorted immune cells, single seedlings, and mitochondria from patient cells. 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. |
| Databáze: | OpenAIRE |
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