Ubiquitin and ubiquitin-like modifiers (UBLs) have emerged as central components in cell biology. The branched nature of these modifications, however, creates additional challenges for analysis by mass spectrometry.
In addition to ubiquitin, the ubiquitin family of small protein-modifiers includes proteins with a relatively-low conservation of the amino acid primary sequence. Despite this, however, all can adopt the “ubiquitin fold”. The most well-known members of this family are SUMO, NEDD8, ATG-8, ATG-12 and ISG15.
Akin to other post-translational modifications (PTMs), the study of protein modification by ubiquitin and other UBLs relies on enrichment of either PTM-modified proteins (see image) or peptides. Tryptic digestion of ubiquitin-, NEDD8 and ISG15-modified proteins generates a signature di-glycine-modified lysine residue (K-ε-GG). This motif is recognised by specific antibodies and the modified proteins/peptides can be enriched from the background mixture of non-modified components.