Differential activity-based gel electrophoresis for comparative analysis of lipolytic and esterolytic activities

Abstract

We established a novel technique for differential activity-based gel electrophoresis (DABGE) of lipolytic enzymes from two different biological samples. For this purpose, a set of three fluorescent suicide inhibitors was developed. These probes possess the same substrate analogous structures but carry different cyanine dyes (Cy2b, Cy3, and Cy5) as reporter fluorophores. For comparison of enzyme profiles, two samples are individually labeled with a different probe followed by mixing, gel electrophoresis, fluorescence imaging, and identification of the tagged proteins by MS/MS. Protocols for quantitative determination of active enzymes were developed on the basis of lipolytic proteomes that had been admixed with defined amounts of known lipases and esterases. A detailed analysis of the fluorescence intensities showed that the found enzyme ratios very closely reflected the relative amounts of the labeled enzymes that were used for spiking. The DABGE method was used to compare the lipolytic proteomes of brown and white adipose tissue showing specific enzyme patterns of both samples. This study represents the first application of this technology for comparative analysis of lipases and esterases. Further applications of this technique can be expected to provide entirely new information on lipid enzymology in health and disease with high precision.-Morak, M., H. Schmidinger, P. Krempl, G. Rechberger, M. Kollroser, R. Birner-Gruenberger, and A. Hermetter. Differential activity-based gel electrophoresis for comparative analysis of lipolytic and esterolytic activities. In the postgenomic era, researchers are now challenging the proteome with methods like two-dimensional gel electrophoresis or multidimensional chromatography followed by mass spectrometry and various other methods for abundance-based proteome profiling. However, the amount of proteins present at a certain state of the cell might not correlate with the enzyme activities responsible for the metabolic fluxes, cell management, and signal transduction. Therefore, elucidation of changes in protein activity is the ultimate goal of functional proteomics.Methods have been developed for specific detection of enzymes on the basis of their catalytic activities. For this purpose, activity recognition probes (ARPs) (1) or activitybased probes are used. Basically, an ARP is a molecule consisting of i) a recognition site targeting a certain enzyme species, ii) a properly positioned reactive site that forms a covalent bond with the target, and iii) a tag for visualization and/or isolation of the covalently bound target (2-4). Many reactive groups have been developed so far for identifying different types of enzyme activity, i.e., fluorophosphonates for serine hydrolases (5), p-nitrophenyl organophosphonates for lipases and esterases (3, 6), epoxides for cysteine proteases (7), and sulfonate esters for various enzyme classes (8, 9). Two-dimensional (2D) gel electrophoresis is a well-established technique for simultaneous separation and display of hundreds to thousan...

Adipose Triglyceride Lipase (ATGL) and Hormone-Sensitive Lipase (HSL) Deficiencies Affect Expression of Lipolytic Activities in Mouse Adipose Tissues

“…Effects on ATGL-and HSL-Deficiency-The lipolytic proteomes of adipose tissue homogenates isolated from ATGLϪ/Ϫ and HSLϪ/Ϫ mice were analyzed and compared with the enzyme patterns of the wt tissues using the DABGE method (20). This technique was developed for the comparison of the enzyme components of two different samples in one electrophoresis gel.…”

“…Active enzymes more abundant in the wt tissue appear green (red/dye-swap), those more abundant in the ko sample appear red (green/dye-swap), and enzymes which are equally abundant in both samples are yellow. The green and red fluorescence intensities of the spots were determined and the enzymes ratios of the ko versus wt mice were calculated as described before (20). Results for 2D and 1D gels are shown in Tables I and II, respectively.…”

“…2. The relative ratios were calculated using the formula described by Morak et al (20). Active enzymes are considered less abundant if relative ratios are lower that Ϫ1.9 and more abundant if ratios are higher than 1.9.…”

“…This study aimed at identifying the effects of ATGL and HSL-deficiency on the expression of other lipolytic enzymes in adipose tissue. For this purpose, we compared the lipolytic proteomes of BAT and WAT from ATGL (Ϫ/Ϫ) and HSL (Ϫ/Ϫ) mice with the enzyme patterns of wt tissues using differential activity-based gel electrophoresis (DABGE) (20). This method is based on activity-recognition probes containing same substrate analogous structures but carrying different fluorophores for specific detection of the individual enzyme patterns of two different tissues.…”

Activity-based Proteomics of Enzyme Superfamilies: Serine Hydrolases as a Case Study

“…These findings and others (33) suggest that multiple enzymes can contribute to 2-AG hydrolysis in mammalian tissues. Other biological applications of ABPP include characterization of SH activities in adipose tissue (34), immune cell function (35), cancer metastasis (36), bacterial (37,38) and fungal (39) pathogenesis, and natural product biosynthesis (40), as well as the analysis of other enzyme classes in cancer (41,42) and infectious diseases (43).…”