title: 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.
authors.affiliation: Institute of Biochemistry, Graz University of Technology, Austria.
authors.forename: Maria
authors.initials: M
authors.lastname: Morak
authors.forename: Hannes
authors.initials: H
authors.lastname: Schmidinger
authors.forename: Peter
authors.initials: P
authors.lastname: Krempl
authors.forename: Gerald
authors.initials: G
authors.lastname: Rechberger
authors.forename: Manfred
authors.initials: M
authors.lastname: Kollroser
authors.forename: Ruth
authors.initials: R
authors.lastname: Birner-Gruenberger
authors.forename: Albin
authors.initials: A
authors.lastname: Hermetter
mesh_terms: D000818:Animals; D002232:Carbocyanines / Q000737:chemistry / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D004950:Esterases / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D008049:Lipase / Q000378:metabolism*; D008066:Lipolysis; D051379:Mice; D008969:Molecular Sequence Data; D015394:Molecular Structure; D020543:Proteome; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D002232:Carbocyanines; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D020543:Proteome; D004950:Esterases; D008049:Lipase