Central Institute for Engineering, Electronics and Analytics, Forschungszentrum Jülich, Jülich, Germany.
Department of Biomedicine, Aarhus University, Aarhus, Denmark.
Methods Mol Biol. 2022;2447:139-158. doi: 10.1007/978-1-0716-2079-3_12.
Protein N-termini provide unique and distinguishing information on proteolytically processed or N-terminally modified proteoforms. Also splicing, use of alternative translation initiation sites, and a variety of co- and post-translational N-terminal modifications generate distinct proteoforms that are unambiguously identified by their N-termini. However, N-terminal peptides are only a small fraction among all peptides generated in a shotgun proteome digest, are often of low stoichiometric abundance, and therefore require enrichment. Various protocols for enrichment of N-terminal peptides have been established and successfully been used for protease substrate discovery and profiling of N-terminal modification, but often require large amounts of proteome. We have recently established the High-efficiency Undecanal-based N-Termini EnRichment (HUNTER) as a fast and sensitive method to enable enrichment of protein N-termini from limited sample sources with as little as a few microgram proteome. Here we present our current HUNTER protocol for sensitive plant N-terminome profiling, including sample preparation, enrichment of N-terminal peptides, and mass spectrometry data analysis.
蛋白质 N 末端提供了有关蛋白水解处理或 N 末端修饰的蛋白形式的独特和有区别的信息。此外，剪接、使用替代翻译起始位点以及各种共翻译和翻译后 N 末端修饰会产生不同的蛋白形式，这些蛋白形式可以通过其 N 末端明确识别。然而，与在鸟枪法蛋白质组消化中产生的所有肽相比，N 末端肽只是一小部分，通常丰度较低，因此需要富集。已经建立了各种用于富集 N 末端肽的方案，并成功用于蛋白酶底物发现和 N 末端修饰的分析，但通常需要大量的蛋白质组。我们最近建立了高效十一醛基 N 末端富集（HUNTER）作为一种快速和敏感的方法，可从少量几微克蛋白质组的有限样本来源中富集蛋白质 N 末端。在这里，我们介绍了我们目前用于敏感植物 N 末端组学分析的 HUNTER 方案，包括样品制备、N 末端肽的富集以及质谱数据分析。
