Departments of Protein Chemistry, Genentech, Inc, South San Francisco, California 94080, USA.
Mol Cell Proteomics. 2011 May;10(5):M110.003756. doi: 10.1074/mcp.M110.003756. Epub 2010 Nov 3.
Ubiquitinated substrates can be recruited to macromolecular complexes through interactions between their covalently bound ubiquitin (Ub) signals and Ub receptor proteins. To develop a functional understanding of the Ub system in vivo, methods are needed to determine the composition of Ub signals on individual substrates and in protein mixtures. Mass spectrometry has emerged as an important tool for characterizing the various forms of Ub. In the Ubiquitin-AQUA approach, synthetic isotopically labeled internal standard peptides are used to quantify unbranched peptides and the branched -GG signature peptides generated by trypsin digestion of Ub signals. Here we have built upon existing methods and established a comprehensive platform for the characterization of Ub signals. Digested peptides and isotopically labeled standards are analyzed either by selected reaction monitoring on a QTRAP mass spectrometer or by narrow window extracted ion chromatograms on a high resolution LTQ-Orbitrap. Additional peptides are now monitored to account for the N terminus of ubiquitin, linear polyUb chains, the peptides surrounding K33 and K48, and incomplete digestion products. Using this expanded battery of peptides, the total amount of Ub in a sample can be determined from multiple loci within the protein, minimizing possible confounding effects of complex Ub signals, digestion abnormalities, or use of mutant Ub in experiments. These methods have been useful for the characterization of in vitro, multistage ubiquitination and have now been extended to reactions catalyzed by multiple E2 enzymes. One question arising from in vitro studies is whether individual protein substrates in cells may be modified by multiple forms of polyUb. Here we have taken advantage of recently developed polyubiquitin linkage-specific antibodies recognizing K48- and K63-linked polyUb chains, coupled with these mass spectrometry methods, to further evaluate the abundance of mixed linkage Ub substrates in cultured mammalian cells. By combining these two powerful tools, we show that polyubiquitinated substrates purified from cells can be modified by mixtures of K48, K63, and K11 linkages.
泛素化底物可以通过其共价结合的泛素（Ub）信号和 Ub 受体蛋白之间的相互作用被招募到大分子复合物中。为了在体内对 Ub 系统有功能上的理解，需要开发确定单个底物和蛋白质混合物中 Ub 信号组成的方法。质谱分析已成为表征各种形式 Ub 的重要工具。在 Ub 水凝胶（Ubiquitin-AQUA）方法中，使用合成的同位素标记内部标准肽来定量未分支的肽和由 Ub 信号的胰蛋白酶消化产生的分支-GG 特征肽。在这里，我们在现有方法的基础上建立了一个全面的 Ub 信号表征平台。通过在 QTRAP 质谱仪上进行选择反应监测，或通过高分辨率 LTQ-Orbitrap 上的窄窗口提取离子色谱图来分析消化后的肽和同位素标记的标准品。现在还监测了更多的肽，以解释泛素的 N 末端、线性多 Ub 链、K33 和 K48 周围的肽以及不完全消化产物。使用这个扩展的肽库，可以从蛋白质内的多个位置确定样品中 Ub 的总量，最大限度地减少复杂 Ub 信号、消化异常或在实验中使用突变 Ub 可能产生的干扰影响。这些方法已用于体外多阶段泛素化的表征，并已扩展到由多种 E2 酶催化的反应。体外研究中出现的一个问题是，细胞中的单个蛋白质底物是否可能被多种形式的多 Ub 修饰。在这里，我们利用最近开发的识别 K48 和 K63 连接的多 Ub 链的多泛素连接特异性抗体，结合这些质谱方法，进一步评估培养的哺乳动物细胞中混合连接 Ub 底物的丰度。通过结合这两种强大的工具，我们表明从细胞中纯化的多泛素化底物可以被 K48、K63 和 K11 连接的混合物修饰。
