Ubiquitin and Ubiquitin-Like modifiers (UBLs) have emerged as major players in cell biology. However, the branched nature of these modifications creates additional difficulties for studying them by MS.
The Ubiquitin family of small protein-modifiers includes, in addition to Ubiquitin itself, other proteins with relatively poor primary sequence conservation but that all adopt the so-called “Ubiquitin fold”. The most famous are SUMO, NEDD8, ATG-8 and -12 and ISG15, but others include URM1, FAT10, FUB1, UBL5, UFM1 and MUB.
Just like other PTMs, study of protein modification by Ubiquitin and other UBLs relies on modified protein (pictured) or peptide enrichment. Trypsin or GluC – but not Lys-C – digestion of Ubiquitin-, NEDD8 and ISG15-modified proteins generates a signature di-glycine modified lysine (K-ε-GG) which can be purified with specific antibodies. While tryptic digestion of SUMO generates a much longer, impractical tag, Tammsalu et al. have also created the T90K SUMO2 mutant which, upon LysC digestion, creates mutant-specific K-ε-GG modified lysines (1).
(1) Proteome-wide identification of SUMO modification sites by mass spectrometry (2014), Triin Tammsalu, Ivan Matic, Ellis G Jaffray, Adel F M Ibrahim, Michael H Tatham & Ronald T Hay. Sci Signal. 7 (323)