Also, protein ubiquitination in JNK inhibitor in vitro synapses of rat brains was also studied using this approach [ 28]. Advantages and challenges are also discussed in recent reviews [ 24 and 29]. There are some limitations to this approach in that there is some ambiguity in assigning gly-gly modifications on lysine residues to ubiquitination, as for instance NEDD8 modification also leads to the same tag present on lysine side chains after proteolytic trypsin digestion. To overcome this, other tags on the basis of the detection of LRGG-lysine have been used in MS experiments (Figure 2). However, this approach is not feasible for the detection of protein

modifications with other ubiquitin-like proteins, such as SUMOylation. Recent attempts to overcome this without the need to introduce SUMO C-terminal mutations were reported in which the application of aspartic acid cleavage, caspase, elastase and trypsin digestion protocols were used to generate SUMO tags on lysine residues that can facilitate Selleck SB431542 detection of modifications by SUMO1 and SUMO2/3 [30 and 31]. Such approaches permit the survey of a wider range of ubiquitin and ubiquitin-like modification profiles on proteomes under normal physiological and pathological conditions in the future. Advances in the sensitivity and throughput

of mass spectrometry (MS) based discovery capabilities have continued to spur experiments that are focused on characterising the expression of conjugating (E1/E2/E3s) and deconjugating enzymes (DUBs), but also their interactors and/or substrates. For instance, whole Selleckchem Etoposide cell proteome studies can now provide insight into the turnover and levels of several thousands of cellular proteins in one single experiment [32••, 33 and 34••]. Of particular note is a study reporting on a reference proteomes of 11 cell lines illustrating differences

in the steady state level of a number of proteins [32••]. This is the first time that comprehensive information on the abundance of components of the ubiquitin system is available in different cell types. Interestingly, the abundance of ubiquitin-specific enzymes appears to vary to a great extent as demonstrated for a selection of E3 ligases and DUBs (Figure 3). This information can help to better understand their biological function when combined with functional assays, cell type specificity and regulation. Also, direct co-immunoprecipitation of either E3 ligase components or DUBs directly has given better clues about the enzyme’s function through the discovery of interactors and/or substrates [35, 36 and 37]. However, these approaches have their limitations in terms of the identification of cognate substrates as often direct enzyme-substrate affinities are low.