Profiling of Protein N-Termini and Their Modifications in Complex Samples
| Autor: | Jayachandran N. Kizhakkedathu, Stefan Niedermaier, Fatih Demir, Pitter F. Huesgen |
|---|---|
| Jazyk: | angličtina |
| Předmět: |
0106 biological sciences
0301 basic medicine Protease medicine.diagnostic_test Chemistry Proteolysis medicine.medical_treatment Proteomics Proteolytic Regulation 01 natural sciences Mass spectrometric 03 medical and health sciences Negative selection 030104 developmental biology Biochemistry Proteome medicine 010606 plant biology & botany Translation initiation sites |
| Zdroj: | Methods in Molecular Biology Methods in Molecular Biology-Protein Terminal Profiling Methods in Molecular Biology ISBN: 9781493968497 |
| ISSN: | 1064-3745 1940-6029 |
| DOI: | 10.1007/978-1-4939-6850-3_4 |
| Popis: | Protein N termini are a unique window to the functional state of the proteome, revealing translation initiation sites, co-translation truncation and modification, posttranslational maturation, and further proteolytic processing into different proteoforms with distinct functions. As a direct readout of proteolytic activity, protein N termini further reveal proteolytic regulation of diverse biological processes and provide a route to determine specific substrates and hence the physiological functions for any protease of interest. Here, we describe our current protocol of the successful Terminal Amine Isotope Labeling of Substrates (TAILS) technique, which enriches protein N-terminal peptides from complex proteome samples by negative selection. Genome-encoded N termini, protease-generated neo-N termini, and endogenously modified N termini are all enriched simultaneously. Subsequent mass spectrometric analysis therefore profiles all protein N termini and their modifications present in a complex sample in a single experiment. We further provide a detailed protocol for the TAILS-compatible proteome preparation from plant material and discuss specific considerations for N terminome data analysis and annotation. |
| Databáze: | OpenAIRE |
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