Kaiser Peter, Wohlschlegel James
Department ofBiological Chemistry, University of California, Irvine, California, USA.
Methods Enzymol. 2005;399:266-77. doi: 10.1016/S0076-6879(05)99018-6.
The identification of protein modification sites is an important step toward understanding the biological role of covalent modifications. For example, the mapping of phosphorylation sites and analyses of phosphorylation site mutants have tremendously contributed to our knowledge of different cellular processes. Given the diverse functions of ubiquitination, similar studies with ubiquitin attachment site mutants are becoming increasingly important in understanding the molecular roles of ubiquitination. Relatively few studies to date have mapped ubiquitination sites, and in almost all cases the identification of the acceptor lysines were based on indirect evidence (Petroski and Deshaies, 2003; Scherer et al., 1995); that is, mutation of particular lysines to arginines blocked ubiquitination. Direct evidence for ubiquitin attachment sites has been obtained by mapping of hydroxylamine-derived peptides from ubiquitinated proteins (Chau et al., 1989); however, these experiments can be very challenging. Recent advances in protein mass spectrometry have enabled ubiquitinated lysine residues to be identified directly, thereby providing more convincing evidence for the exact location of the modification (Flick et al., 2004; Peng et al., 2003). In addition to mapping attachment sites, mass spectrometry can also be used to determine the type of ubiquitin chain linkage (Flick et al., 2004; Peng et al., 2003). In vivo evidence for the covalent attachment of the carboxyl terminus of one ubiquitin molecule to lysine residues in several locations in a different ubiquitin molecule demonstrates the complexity of ubiquitin biology (Peng et al., 2003). These different ubiquitin chain topologies can dramatically affect the molecular function of ubiquitin chains (Hoege et al., 2002; Spence et al., 1995), and, hence, the mass spectrometric determination of the ubiquitin chain architecture can provide important insight into the mechanisms of ubiquitin function. This chapter describes mass spectrometric approaches for identifying ubiquitin acceptor lysines on target proteins and analyzing the ubiquitin chain topology.
蛋白质修饰位点的鉴定是理解共价修饰生物学作用的重要一步。例如，磷酸化位点的定位以及磷酸化位点突变体的分析极大地促进了我们对不同细胞过程的了解。鉴于泛素化功能的多样性，对泛素连接位点突变体进行类似研究对于理解泛素化的分子作用变得越来越重要。迄今为止，相对较少的研究对泛素化位点进行了定位，几乎在所有情况下，受体赖氨酸的鉴定都是基于间接证据（彼得罗斯基和德谢斯，2003年；谢勒等人，1995年）；也就是说，将特定赖氨酸突变为精氨酸会阻止泛素化。通过对泛素化蛋白质中羟胺衍生肽的定位获得了泛素连接位点的直接证据（周等人，1989年）；然而，这些实验可能极具挑战性。蛋白质质谱技术的最新进展使得能够直接鉴定泛素化的赖氨酸残基，从而为修饰的确切位置提供更有说服力的证据（弗利克等人，2004年；彭等人，2003年）。除了定位连接位点，质谱还可用于确定泛素链连接的类型（弗利克等人，2004年；彭等人，2003年）。一个泛素分子的羧基末端与另一个不同泛素分子中多个位置的赖氨酸残基共价连接的体内证据证明了泛素生物学的复杂性（彭等人，2003年）。这些不同的泛素链拓扑结构可显著影响泛素链的分子功能（赫格等人，2002年；斯彭斯等人，1995年），因此，通过质谱测定泛素链结构可深入了解泛素功能机制。本章介绍了用于鉴定靶蛋白上泛素受体赖氨酸并分析泛素链拓扑结构的质谱方法。
