Ubiquitylation is an elaborate post-translational modification involved in all biological processes. Its pleotropic effect is driven by the ability to form complex polyubiquitin chain architectures that can influence biological functions. In this study, we optimised sample preparation and chromatographic separation of Ubiquitin peptides for Absolute Quantification by Parallel Reaction Monitoring (Ub-AQUA-PRM). Using this refined Ub-AQUA-PRM assay, we were able to quantify all ubiquitin chain types in 10-min LC-MS/MS runs. We used this method to determine the ubiquitin chain-linkage composition in murine bone marrow-derived macrophages and different mouse tissues. We could show tissue-specific differences in ubiquitin levels in murine tissues, with polyubiquitin chain types contributing a small proportion to the total pool of ubiquitin. Interestingly, we observed enrichment of atypical (K33) ubiquitin chains in heart and muscle. Our approach enabled high-throughput screening of ubiquitin chain-linkage composition in different murine tissues and highlighted a possible role for atypical ubiquitylation in contractile tissues.

Significance

Large knowledge gaps exist in our understanding of ubiquitin chain-linkage composition in mammalian tissues. Defining this in vivo ubiquitin chain-linkage landscape could reveal the functional importance of different ubiquitin chain types in tissues. In this study, we refined the previously described Ub-AQUA-PRM assay to enable quantification of all ubiquitin chain types in a high-throughput manner. Using this assay, we provided new data on the ubiquitin chain-linkage composition in primary murine macrophages and tissues, and revealed an enrichment of atypical ubiquitin chains in contractile tissues. Our approach should thus enable rapid, high-throughput screening of ubiquitin chain-linkage composition in different sample types, as demonstrated in murine primary cells and tissues.

中文翻译：

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技术报告：靶向蛋白质组学分析揭示了收缩性鼠类组织中非典型泛素链的富集。

泛素化是涉及所有生物学过程的精心翻译后修饰。它的多效作用是由能够形成可能影响生物学功能的复杂多聚泛素链结构的能力驱动的。在这项研究中，我们优化了样品制备和泛素肽的色谱分离，以通过并行反应监测（Ub-AQUA-PRM）进行绝对定量。使用这种改进的Ub-AQUA-PRM分析，我们能够在10分钟的LC-MS / MS运行中定量所有泛素链类型。我们使用这种方法来确定鼠骨髓来源的巨噬细胞和不同小鼠组织中的泛素链连接组成。我们可以在鼠组织中显示泛素水平的组织特异性差异，其中多聚泛素链类型占泛素总库的比例很小。有趣的是，我们观察到心脏和肌肉中非典型（K33）泛素链的富集。我们的方法能够高通量筛选不同鼠类组织中的泛素链连接组成，并强调了在收缩组织中非典型泛素化的可能作用。

意义

在我们对哺乳动物组织中泛素链连接组成的理解上存在大量知识空白。定义这种体内泛素链连接态势可以揭示组织中不同泛素链类型的功能重要性。在这项研究中，我们完善了先前描述的Ub-AQUA-PRM测定法，以高通量的方式定量所有泛素链类型。使用该测定法，我们提供了关于原代鼠巨噬细胞和组织中泛素链连接组成的新数据，并揭示了收缩组织中非典型泛素链的富集。因此，我们的方法应该能够快速，高通量筛选不同样品类型中的遍在蛋白链连接组成，如在鼠原代细胞和组织中所证明的。