Akimov, V. et al. Nat. Struct. Mol. Biol. 25, 631–640 (2018).

Current approaches to enrich ubiquitinated proteins for analysis and identification by mass spectrometry suffer from various limitations. For example, ubiquitin binding domains successfully capture modified proteins, but do not provide information about ubiquitination sites. A popular approach that uses an antibody to detect a diglycine remnant of ubiquitin left over after tryptic digestion shows some bias toward certain sequences and is unable to distinguish ubiquitination from other ubiquitin-like protein modifications. Akimov et al. describe a new approach that uses an antibody dubbed UbiSite, which recognizes a 13-amino-acid remnant, specific to ubiquitin, left on ubiquitinated proteins after digestion with the protease LysC. Using proteasomal inhibitors and UbiSite-based enrichment, the researchers identified more than 63,000 ubiquitination sites on more than 9,000 proteins in human Hep2 and Jurkat cell lines. They found that ubiquitination was widespread, affecting proteins involved in all cellular processes in all locations in the cell.