Madzharova Elizabeta, Sabino Fabio, Auf dem Keller Ulrich
Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark.
Methods Mol Biol. 2019;1944:115-126. doi: 10.1007/978-1-4939-9095-5_8.
Global characterization of protein N termini provides valuable information on proteome dynamics and diversity in health and disease. Driven by the progress in mass spectrometry-based proteomics, novel approaches for the dedicated investigation of protein N termini and protease substrates have been recently developed. Terminal amine isotopic labeling of substrates (TAILS) is a quantitative proteomics approach suitable for high-throughput and system-wide profiling of protein N termini in complex biological matrices. TAILS employs isotopic labeling of primary amines of intact proteins in combination with an amine-reactive high molecular weight polymer (HPG-ALD) for depletion of internal tryptic peptides and high enrichment of protein N termini by negative selection. Thereby, TAILS allows simultaneous identification of the natural N termini, protease-generated neo-N termini, and endogenously modified (e.g., acetylated) N termini. In this chapter, we provide a protocol for tandem mass tag (TMT)-TAILS analysis and further discuss specific considerations regarding N-terminome data interpretation using Proteome Discoverer™ software.
蛋白质N端的全局表征为健康和疾病状态下蛋白质组的动力学和多样性提供了有价值的信息。在基于质谱的蛋白质组学取得进展的推动下，最近开发了用于专门研究蛋白质N端和蛋白酶底物的新方法。底物末端胺同位素标记法（TAILS）是一种定量蛋白质组学方法，适用于在复杂生物基质中对蛋白质N端进行高通量和全系统分析。TAILS将完整蛋白质的一级胺进行同位素标记，同时结合一种胺反应性高分子量聚合物（HPG-ALD），用于去除内部胰蛋白酶肽段，并通过阴性选择高度富集蛋白质N端。因此，TAILS能够同时鉴定天然N端、蛋白酶产生的新N端以及内源性修饰（如乙酰化）的N端。在本章中，我们提供了串联质量标签（TMT）-TAILS分析的方案，并进一步讨论了使用Proteome Discoverer™软件解释N端蛋白质组数据的具体注意事项。
