Department of Molecular Sciences, Amgen, Seattle, Washington 98119.
Department of Inflammation, Amgen, Seattle, Washington 98119.
J Biol Chem. 2011 Dec 2;286(48):41530-41538. doi: 10.1074/jbc.M111.248856. Epub 2011 Oct 10.
Protein ubiquitination is a key regulatory process essential to life at a cellular level; significant efforts have been made to identify ubiquitinated proteins through proteomics studies, but the level of success has not reached that of heavily studied post-translational modifications, such as phosphorylation. HRD1, an E3 ubiquitin ligase, has been implicated in rheumatoid arthritis, but no disease-relevant substrates have been identified. To identify these substrates, we have taken both peptide and protein level approaches to enrich for ubiquitinated proteins in the presence and absence of HRD1. At the protein level, a two-step strategy was taken using cells expressing His(6)-tagged ubiquitin, enriching proteins first based on their ubiquitination and second based on the His tag with protein identification by LC-MS/MS. Application of this method resulted in identification and quantification of more than 400 ubiquitinated proteins, a fraction of which were found to be sensitive to HRD1 and were therefore deemed candidate substrates. In a second approach, ubiquitinated peptides were enriched after tryptic digestion by peptide immunoprecipitation using an antibody specific for the diglycine-labeled internal lysine residue indicative of protein ubiquitination, with peptides and ubiquitination sites identified by LC-MS/MS. Peptide immunoprecipitation resulted in identification of over 1800 ubiquitinated peptides on over 900 proteins in each study, with several proteins emerging as sensitive to HRD1 levels. Notably, significant overlap exists between the HRD1 substrates identified by the protein-based and the peptide-based strategies, with clear cross-validation apparent both qualitatively and quantitatively, demonstrating the effectiveness of both strategies and furthering our understanding of HRD1 biology.
蛋白质泛素化是细胞水平生命的关键调节过程；尽管已经做出了巨大努力通过蛋白质组学研究来鉴定泛素化蛋白质，但成功的程度尚未达到像磷酸化这样的高度研究的翻译后修饰。HRD1 是一种 E3 泛素连接酶，与类风湿关节炎有关，但尚未鉴定出与疾病相关的底物。为了鉴定这些底物，我们已经采取了肽和蛋白质水平的方法，在存在和不存在 HRD1 的情况下富集泛素化蛋白质。在蛋白质水平上，我们使用表达 His(6)-标记泛素的细胞采取了两步策略，首先根据它们的泛素化进行蛋白质富集，然后根据 His 标签进行富集，然后通过 LC-MS/MS 进行蛋白质鉴定。该方法的应用导致鉴定和定量了超过 400 种泛素化蛋白质，其中一部分对 HRD1 敏感，因此被视为候选底物。在第二种方法中，通过肽免疫沉淀用针对二甘氨酸标记的内部赖氨酸残基的抗体从胰蛋白酶消化后富集泛素化肽，通过 LC-MS/MS 鉴定肽和泛素化位点。肽免疫沉淀在每项研究中都鉴定出超过 1800 种泛素化肽，涉及超过 900 种蛋白质，其中几种蛋白质对 HRD1 水平敏感。值得注意的是，基于蛋白质和基于肽的策略鉴定的 HRD1 底物之间存在明显的重叠，在定性和定量上都有明显的交叉验证，这证明了两种策略的有效性，并进一步加深了我们对 HRD1 生物学的理解。
