Mollah Sahana, Wertz Ingrid E, Phung Qui, Arnott David, Dixit Vishva M, Lill Jennie R
Applied Biosystems, Foster City, CA 94404, USA.
Rapid Commun Mass Spectrom. 2007;21(20):3357-64. doi: 10.1002/rcm.3227.
Post-translational modifications of proteins including phosphorylation, glycosylation, acetylation and ubiquitination facilitate the regulation of many cellular processes and intracellular signaling events. Ubiquitination plays a key role in the functional regulation and degradation of many classes of proteins, and the study of ubiquitination and poly-ubiquitination has emerged as one of the most active areas in proteomic research. A variety of mass spectrometric methods have been described for the identification of ubiquitination sites, the study of poly-ubiquitin topology and the identification of ubiquitin substrates. The most popular workflow for both ubiquitination site mapping and poly-ubiquitination chain topology characterization is to take advantage of the Gly-Gly signature on the substrate's lysine residue observed after tryptic digestion. Although a number of protocols have been described for the mapping of ubiquitination sites, one major challenge is that ubiquitination is typically heterogeneous, and several lysine residues may be ubiquitinated within a protein. When multiple ubiquitination sites are present, multiple analyses are often required to cover all of the potential modification sites which in turn can necessitate the usage of larger quantities of material. In addition, the level of ubiquitination on endogenous and recombinant proteins may be of low intensity, adding further analytical challenges in the identification of this modification. The use of the multiple reaction monitoring (MRM)-initiated detection and sequencing workflow (MIDAS) for the identification of phosphorylation sites has previously been described. Here, we explore the use of an MRM workflow for ubiquitination site mapping on the substrate protein, receptor interacting protein (RIP).
蛋白质的翻译后修饰，包括磷酸化、糖基化、乙酰化和泛素化，有助于调节许多细胞过程和细胞内信号事件。泛素化在许多类蛋白质的功能调节和降解中起关键作用，对泛素化和多聚泛素化的研究已成为蛋白质组学研究中最活跃的领域之一。已经描述了多种质谱方法用于鉴定泛素化位点、研究多聚泛素拓扑结构以及鉴定泛素底物。用于泛素化位点定位和多聚泛素链拓扑结构表征的最常用工作流程是利用胰蛋白酶消化后在底物赖氨酸残基上观察到的甘氨酰 - 甘氨酸特征。尽管已经描述了许多用于泛素化位点定位的方案，但一个主要挑战是泛素化通常是异质性的，并且蛋白质内的几个赖氨酸残基可能被泛素化。当存在多个泛素化位点时，通常需要进行多次分析以覆盖所有潜在的修饰位点，这反过来可能需要使用大量材料。此外，内源性和重组蛋白质上的泛素化水平可能较低，这在鉴定这种修饰时增加了进一步的分析挑战。先前已经描述了使用多反应监测（MRM）启动的检测和测序工作流程（MIDAS）来鉴定磷酸化位点。在这里，我们探索使用MRM工作流程来对底物蛋白受体相互作用蛋白（RIP）进行泛素化位点定位。
