Department of Biochemistry, Institute of Biochemistry and Technical Biochemistry, University of Stuttgart, Stuttgart, Germany.
Methods Mol Biol. 2022;2529:313-325. doi: 10.1007/978-1-0716-2481-4_15.
Posttranslational methylation of amino acid side chains in proteins mainly occurs on lysine, arginine, glutamine, and histidine residues. It is introduced by different protein methyltransferases (PMTs) and regulates many aspects of protein function including stability, activity, localization, and protein/protein interactions. Although the biological effects of PMTs are mediated by their methylation substrates, the full substrate spectrum of most PMTs is not known. For many PMTs, their activity on a particular potential substrate depends, among other factors, on the peptide sequence containing the target residue for methylation. In this protocol, we describe the application of SPOT peptide arrays to investigate the substrate specificity of PMTs and identify novel substrates. Methylation of SPOT peptide arrays makes it possible to study the methylation of many different peptides in one experiment at reasonable costs and thereby provides detailed information about the specificity of the PMT under investigation. In these experiments, a known substrate sequence is used as template to design a SPOT peptide array containing peptides with single amino acid exchanges at all positions of the sequence. Methylation of the array with the PMT provides detailed preferences for each amino acid at each position in the substrate sequence, yielding a substrate sequence specificity profile. This information can then be used to identify novel potential PMT substrates by in silico data base searches. Methylation of novel substrate candidates can be validated in SPOT arrays at peptide level, followed by validation at protein level in vitro and in cells.
蛋白质中氨基酸侧链的翻译后甲基化主要发生在赖氨酸、精氨酸、谷氨酰胺和组氨酸残基上。它是由不同的蛋白质甲基转移酶（PMTs）引入的，调节蛋白质功能的许多方面，包括稳定性、活性、定位和蛋白质/蛋白质相互作用。尽管 PMTs 的生物学效应是通过其甲基化底物介导的，但大多数 PMTs 的完整底物谱尚不清楚。对于许多 PMTs 来说，其对特定潜在底物的活性取决于包含目标甲基化残基的肽序列等因素。在本方案中，我们描述了应用 SPOT 肽阵列来研究 PMTs 的底物特异性并鉴定新的底物。SPOT 肽阵列的甲基化使得可以在一个实验中以合理的成本研究许多不同肽的甲基化，从而提供有关所研究的 PMT 的特异性的详细信息。在这些实验中，使用已知的底物序列作为模板来设计包含在该序列的所有位置都有单个氨基酸交换的肽的 SPOT 肽阵列。用 PMT 对该阵列进行甲基化，可以提供底物序列中每个位置的每个氨基酸的详细偏好，从而得到一个底物序列特异性图谱。然后，可以通过在数据库中搜索来识别新的潜在 PMT 底物。可以在肽水平上在 SPOT 阵列中验证新的候选底物，然后在体外和细胞中在蛋白质水平上进行验证。
