Anagho Holda A, Elsborg Jonas D, Hendriks Ivo A, Buch-Larsen Sara C, Nielsen Michael L
Proteomics program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
Methods Mol Biol. 2023;2609:251-270. doi: 10.1007/978-1-0716-2891-1_15.
ADP-ribosylation is a posttranslational modification (PTM) that has crucial functions in a wide range of cellular processes. Although mass spectrometry (MS) in recent years has emerged as a valuable tool for profiling ADP-ribosylation on a system level, the use of conventional MS methods to profile ADP-ribosylation sites in an unbiased way remains a challenge. Here, we describe a protocol for identification of ADP-ribosylated proteins in vivo on a proteome-wide level, and localization of the amino acid side chains modified with this PTM. The method relies on the enrichment of ADP-ribosylated peptides using the Af1521 macrodomain (Karras GI, Kustatscher G, Buhecha HR, Allen MD, Pugieux C, Sait F, Bycroft M, Ladurner AG, EMBO J 24:1911-1920, 2005), followed by liquid chromatography-high-resolution tandem MS (LC-MS/MS) with electron transfer dissociation-based peptide fragmentation methods, resulting in accurate localization of ADP-ribosylation sites. This protocol explains the step-by-step enrichment and identification of ADP-ribosylated peptides from cell culture to data processing using the MaxQuant software suite.
ADP核糖基化是一种翻译后修饰（PTM），在广泛的细胞过程中具有关键作用。尽管近年来质谱（MS）已成为在系统水平上分析ADP核糖基化的一种有价值的工具，但使用传统MS方法以无偏差方式分析ADP核糖基化位点仍然是一项挑战。在这里，我们描述了一种在全蛋白质组水平上体内鉴定ADP核糖基化蛋白质以及定位经此PTM修饰的氨基酸侧链的方案。该方法依赖于使用Af1521宏结构域富集ADP核糖基化肽段（Karras GI, Kustatscher G, Buhecha HR, Allen MD, Pugieux C, Sait F, Bycroft M, Ladurner AG, EMBO J 24:1911 - 1920, 2005），随后采用基于电子转移解离的肽段碎裂方法进行液相色谱 - 高分辨率串联质谱（LC - MS/MS）分析，从而实现ADP核糖基化位点的精确定位。本方案详细说明了从细胞培养物中逐步富集和鉴定ADP核糖基化肽段直至使用MaxQuant软件套件进行数据处理的过程。
