A Mass Spectrometry-Based Strategy for Mapping Modification Sites for the Ubiquitin-Like Modifier NEDD8

The identification of modification sites for ubiquitin and ubiquitin-like modifiers is an essential step in the elucidation of controlled processes. The ubiquitin-like modifier NEDD8 is an important regulator of plethora of biological processes both under homeostatic and proteotoxic stress conditions. Here, we describe a detailed protocol for proteome-wide identification of NEDDylation sites. The approach is based on the use of cell lines stably expressing the NEDD8^R74K mutant. Digestion of samples with Lysyl endopeptidase generates peptides with a di-glycine remnant only from proteins modified with NEDD8^R74K but not with ubiquitin or ISG15. The isolation of these peptides with anti-di-glycine antibodies (K-ε-GG) allows the identification of NEDDylation sites by liquid chromatography tandem mass spectrometry (LC-MS/MS).