Ubiquitinomics: History, methods, and applications in basic research and drug discovery

The ubiquitin‐proteasome system (UPS) was discovered about 40 years ago and is known to regulate a multitude of cellular processes including protein homeostasis. Ubiquitylated proteins are recognized by downstream effectors, resulting in alterations of protein abundance, activity, or localization. Not surprisingly, the ubiquitylation machinery is dysregulated in numerous diseases, including cancers and neurodegeneration. Mass spectrometry (MS)‐based proteomics has emerged as a transformative technology for characterizing protein ubiquitylation in an unbiased fashion. Here, we provide an overview of the different MS‐based approaches for studying protein ubiquitylation. We review various methods for enriching and quantifying ubiquitin modifications at the peptide or protein level, outline MS acquisition, and data processing approaches and discuss key challenges. Finally, we examine how MS‐based ubiquitinomics can aid both basic biology and drug discovery research.