Mass spectrometry (MS) has become the method of choice for the large-scale analysis of protein ubiquitylation. There exist a number of proposed methods for mapping ubiquitin sites, each with different pros and cons. We present here a protocol for the MS analysis of the ubiquitin-proteome captured by TUBEs and subsequent data analysis. Using dedicated software and algorithms, specific information on the presence of ubiquitylated peptides can be obtained from the MS search results. In addition, a quantitative and functional analysis of the ubiquitylated proteins and their interacting partners helps to unravel the biological and molecular processes they are involved in.
Keywords: Chloroacetamide (CAA); Collision induced dissociation (CID); Gene Ontology (GO); Iodoacetamide (IAA); Mass spectrometry (MS); Posttranslational modification (PTM); Tandem Ubiquitin Binding Entities (TUBEs); Ubiquitin.