Fig. 3

Comparative and competitive gel-based ABPP reveals distinct SH activity profiles of glioma and healthy brain. Rat cerebellar membranes were included as an additional control to facilitate SH band comparison and identification. Proteomes (1 mg/ml) were treated for 1 h with DMSO or the indicated concentrations of the SH inhibitors, after which TAMRA-FP labelling was conducted for 1 h as detailed in Materials and Methods. The reaction was quenched and 10 μg protein was loaded per lane and separated by SDS-PAGE. TAMRA-FP labeled bands appear as black after in-gel imaging. Position of molecular weight markers (in kDa) is indicated on the gel. Based on previous studies from this and other laboratories [14, 19,20,21,22,23,24,25,26,27] many healthy brain-resident SHs were identified, as indicated at left. Note comparable activities of KIAA1363 and LYPLA1/2 doublets (black asterisk) in control brain and glioma. Note also prominent activity of MAGL doublet (double black asterisk) in control brain as opposed to hardly detectable activity in glioma. Note that the glioma contains two prominent SH bands, migrating at ~ 25 and ~ 30 kDa (white asterisks). It is noteworthy that the respective SH bands show low-to-non-detectable activity in control brain. Inhibitor profiling reveals that the glioma 30 kDa band is sensitive to all tested inhibitor whereas the ~ 25 kDa band is fully sensitive to PMSF and less so to deshiobiotin-FP (DeBi), but resistant to other tested inhibitors, including MAFP. The gel is representative of two independent ABPP runs with similar outcome. ABHD1, ABHD6, ABHD11, ABHD12, ABHD16A, α/β-hydrolase domain-containing 1, 6, 11, 12 and 16A; FAAH, fatty acid amide hydrolase; FASN, fatty acid synthase; KIAA1363, also known as AADACL1 or NCEH1 (neutral cholesteryl ester hydrolase 1); LYPLA1/2, lysophospholipase A1/A2 (also known as acyl-protein thioesterases 1/2 or APT1/APT2); MAGL, monoacylglycerol lipase; PREP, prolyl oligopeptidase, also known as POP. Image was adjusted for brightness and contrast