Ubiquitin and ubiquitin-like modifiers (UBLs) such as small ubiquitin-like modifier (SUMO) can act as antagonists to one another by competing to occupy similar residues in the proteome. In addition, SUMO and ubiquitin can be coupled to each other at key lysine residues to form highly branched protein networks. The interplay between these modifications governs important biological processes such as double-strand break repair and meiotic recombination. We recently developed an approach that permits the identification of proteins that are modified by both SUMOylation and ubiquitylation. This protocol requires cells that express a mutant 6×His-SUMO3m protein that has had its C terminus modified from QQQTGG to RNQTGG, enabling the purification of SUMOylated peptides and their identification by tandem mass spectrometry (MS/MS). Cells are lysed under denaturing conditions, and the SUMOylated proteins are purified on nickel-nitrilotriacetic acid (Ni-NTA) resin via the 6×His on the SUMO3m construct. After on-bead digestion using trypsin, ubiquitylated peptides are enriched by immunoprecipitation, and the flow-through from this step is subjected to anti-SUMO immunoprecipitation. The SUMOylated peptides are fractionated on strong cation exchange (SCX) StageTips to enhance the coverage of the SUMO proteome. The ubiquitylated and SUMOylated peptides are analyzed separately by liquid chromatography (LC)-MS/MS and identified with MaxQuant. We demonstrate how this approach can be used to identify temporal changes in SUMOylated and ubiquitylated proteins in response to, for instance, heat shock and proteasome inhibition. The procedure requires 3 d when starting from cell pellets and yields >8,000 SUMO sites and >3,500 ubiquitin sites from 16 mg of cell extract.

中文翻译：

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使用顺序肽免疫纯化方法鉴定SUMOylation和泛素化之间的串扰。

泛素和类似泛素的修饰剂（UBL），例如小的泛素样修饰剂（SUMO），可以通过竞争在蛋白质组中占据相似的残基而彼此充当拮抗剂。此外，SUMO和泛素可以在赖氨酸的关键残基处相互偶联，形成高度分支的蛋白质网络。这些修饰之间的相互作用决定了重要的生物学过程，例如双链断裂修复和减数分裂重组。我们最近开发了一种方法，该方法可以识别同时被SUMOylation和泛素化修饰的蛋白质。此协议要求细胞表达突变的6×His-SUMO3m蛋白，其C端已从QQQTGG修饰为RNQTGG，从而能够纯化SUMO化肽并通过串联质谱（MS / MS）进行鉴定。在变性条件下裂解细胞，然后通过SUMO3m构建体上的6xHis在镍-三三乙酸镍（Ni-NTA）树脂上纯化SUMO化的蛋白。使用胰蛋白酶进行磁珠消化后，泛素化的肽通过免疫沉淀富集，并将来自此步骤的流出物进行抗SUMO免疫沉淀。将SUMOylated肽在强阳离子交换（SCX）StageTips上分级分离，以增强SUMO蛋白质组的覆盖范围。泛素化和SUMO酰化的肽分别通过液相色谱（LC）-MS / MS进行分析，并通过MaxQuant进行鉴定。我们演示了如何使用此方法来识别SUMOylated和泛素化蛋白的时间变化，以响应例如热休克和蛋白酶体抑制作用。