Department of Molecular Biology and Radiobiology, Mendel University in Brno & CEITEC - Central European Institute of Technology, Mendel University in Brno, Zemědělská 1, CZ-613 00 Brno, Czech Republic.
J Proteomics. 2013 Oct 30;92:2-27. doi: 10.1016/j.jprot.2013.05.040. Epub 2013 Jun 15.
Posttranslational modifications (PTMs) of proteins represent fascinating extensions of the dynamic complexity of living cells' proteomes. The results of enzymatically catalyzed or spontaneous chemical reactions, PTMs form a fourth tier in the gene - transcript - protein cascade, and contribute not only to proteins' biological functions, but also to challenges in their analysis. There have been tremendous advances in proteomics during the last decade. Identification and mapping of PTMs in proteins have improved dramatically, mainly due to constant increases in the sensitivity, speed, accuracy and resolution of mass spectrometry (MS). However, it is also becoming increasingly evident that simple gel-free shotgun MS profiling is unlikely to suffice for comprehensive detection and characterization of proteins and/or protein modifications present in low amounts. Here, we review current approaches for enriching and separating posttranslationally modified proteins, and their MS-independent detection. First, we discuss general approaches for proteome separation, fractionation and enrichment. We then consider the commonest forms of PTMs (phosphorylation, glycosylation and glycation, lipidation, methylation, acetylation, deamidation, ubiquitination and various redox modifications), and the best available methods for detecting and purifying proteins carrying these PTMs. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine.
蛋白质的翻译后修饰（PTMs）代表了活细胞蛋白质组动态复杂性的迷人扩展。这些修饰是酶催化或自发化学反应的结果，形成了基因-转录物-蛋白质级联的第四个层次，不仅对蛋白质的生物学功能有贡献，而且对其分析也提出了挑战。在过去的十年中，蛋白质组学取得了巨大的进展。蛋白质中 PTM 的鉴定和作图有了显著的改善，主要是由于质谱（MS）的灵敏度、速度、准确性和分辨率不断提高。然而，越来越明显的是，简单的无凝胶shotgun MS 分析可能不足以全面检测和表征低丰度存在的蛋白质和/或蛋白质修饰。在这里，我们回顾了目前用于富集和分离翻译后修饰蛋白质及其非 MS 检测的方法。首先，我们讨论了蛋白质组分离、分级和富集的一般方法。然后我们考虑了最常见的 PTM 形式（磷酸化、糖基化和糖化、脂化、甲基化、乙酰化、脱酰胺化、泛素化和各种氧化还原修饰），以及检测和纯化携带这些 PTM 的蛋白质的最佳可用方法。本文是题为“生物学和医学中的翻译后蛋白质修饰”的特刊的一部分。
