, 2012). In this study mass spectrometry was applied to detect the S-adenosylhomocysteine product ( Lin et al., 2012). One of the largest classes of histone demethylases contain a Jumonji C domain (JMJD) and members of this family are Fe2+ and 2-oxoglutarate-dependent oxygenases which demethylate specific lysine residues of histone

tails. The JMJD2 human subfamily contains six members Ipilimumab (JMJD2A-F). Assays for JMJD demethylases have been developed to enable the discovery of chemical probes which could be useful for validating the role of these enzymes in disease. Like other demethlyases, the Jumonji-demethylases produce formaldehyde and this byproduct can be detected using formaldehyde dehydrogenase (FDH) (Lizcano et al., 2000). Miniaturized fluorescent assays (through detection of the NADH produced by FDH) have been developed and applied to HTS using this approach (King et al., 2010). However, Sotrastaurin nmr assays that are specific for the methyl mark are desirable and TR-FRET-based assays have been developed using antibodies that are specific for

the first demethylated product. An assay using Eu-antiH3K9me2 has been used to measuring demethylation of a H2K9me3 labeled peptide (Yu et al., 2012). The RapidFire system has also been applied to JMJD2C where the methylated products of a peptide were measured (Hutchinson et al., 2012). The inherent basicity of histone peptides can be an issue for MS due to the high number of charged states but the JMJD2C assay was developed using a truncated and mutated peptide with suitable performance on the MS which also maintained similar kinetic parameters to the native peptide. Signaling pathways in cells are often controlled by the stability and localization of proteins operating at critical nodes in the pathway. Protein stability and localization can be regulated through the why conjugation of ubiquitin or ubiquitin-like proteins to various protein targets.

Both ubiquitin ligases and ubiquitin specific proteases, known as deubiquitylases (DUBs) or ubiquitin C-terminal hydrolases (UCHs) are involved in this regulation. Dysregulation of the ubiquitin pathway has been associated with a number of diseases and therefore several assays have been developed for this class of enzymes. Coupled enzyme assays have been developed for measurement of isopeptidase activity of DUBs. These assays involve fusion of a ubiquitin chain to the N-terminus of enzymes such as phospholipase A2 or enterokinase which require a free N-terminus to be functional. Cleavage of the ubiquitin chain by a DUB results in an active coupling enzyme which is readily measured with a fluorescent substrate (Tian et al., 2011). Additionally, the use of different reporter enzymes can enable multiplex assays where more than one DUB activity is measured in the assay (Tian et al., 2011). Suitable counter-screens against the coupling enzyme are required before interpreting the results from these assays. Ubiquitin ligase (EC 6.3.2.