Degronomics: Mapping the Interacting Peptidome of a Ubiquitin Ligase Using an Integrative Mass Spectrometry Strategy
Degronomics: Mapping the Interacting Peptidome of a Ubiquitin Ligase Using an Integrative Mass Spectrometry Strategy
复制标题
DOI：
10.1021/acs.analchem.9b02331
复制
发表时间：
2019-10-15
影响因子：
7.4
通讯作者：
Bush, Matthew F.
中科院分区：
文献类型：
--
作者：
Canzani, Daniele;Rusnac, Domnita-Valeria;Bush, Matthew F.
人类细胞利用数百种独特的泛素E3连接酶来确保蛋白质组的保真度，并通过促进蛋白质降解来控制细胞功能。这些过程需要精确的选择性，但大多数E3的个体作用仍然很难表征，部分原因是与识别，定量和验证每个E3底物相关的挑战。我们报告了一个综合质谱（MS）的策略，用于表征与KLHDC 2相互作用的蛋白质片段，KLHDC 2是一种识别底物极端C-末端的人E3。使用天然MS、天然自上而下MS、去稳定样品的MS和液相色谱MS的组合，我们鉴定并定量了大肠杆菌中KLHDC 2结合肽组的几乎完整部分。coli细胞。该脱蛋白质组包括源自多种蛋白质的肽。虽然所有鉴定的蛋白质片段均以双甘氨酸或甘氨酰丙氨酸终止，但前面的氨基酸是不同的。这些结果大大扩展了我们对KLHDC 2可以识别的序列的理解，这为E3在人类中的潜在底物提供了深入了解。我们预计，这种综合MS策略可以更广泛地利用，以表征其他E3连接酶底物受体的degronomes，包括那些坚持更常见的N-末端规则的底物识别。因此，这项工作推进了“degronomics”，即，鉴定、定量和验证功能性E3：肽相互作用，以确定每个E3的单独作用。
Human cells make use of hundreds of unique ubiquitin E3 ligases to ensure proteome fidelity and control cellular functions by promoting protein degradation. These processes require exquisite selectivity, but the individual roles of most E3s remain poorly characterized in part due to the challenges associated with identifying, quantifying, and validating substrates for each E3. We report an integrative mass spectrometry (MS) strategy for characterizing protein fragments that interact with KLHDC2, a human E3 that recognizes the extreme C-terminus of substrates. Using a combination of native MS, native top-down MS, MS of destabilized samples, and liquid chromatography MS, we identified and quantified a near complete fraction of the KLHDC2-binding peptidome in E. coli cells. This degronome includes peptides that originate from a variety of proteins. Although all identified protein fragments are terminated by diglycine or glycylalanine, the preceding amino acids are diverse. These results significantly expand our understanding of the sequences that can be recognized by KLHDC2, which provides insight into the potential substrates of this E3 in humans. We anticipate that this integrative MS strategy could be leveraged more broadly to characterize the degronomes of other E3 ligase substrate receptors, including those that adhere to the more common N-end rule for substrate recognition. Therefore, this work advances "degronomics," i.e., identifying, quantifying, and validating functional E3:peptide interactions in order to determine the individual roles of each E3.