Functional Proteomic Analysis of Lipases and Esterases in Cultured Human Adipocytes Maximilian Schicher, † Maria Morak, † Ruth Birner-Gruenberger, §,⊥ Heidemarie Kayer, † Bojana Stojcic, † Gerald Rechberger, ‡ Manfred Kollroser, § and Albin Hermetter* ,† Institute of Biochemistry, Graz University of Technology, Graz, Austria, Institute of Molecular Biosciences, University of Graz, Graz, Austria, and Institute of Forensic Medicine, Medical University of Graz, Graz, Austria Received June 9, 2010 This study reports on the analysis of the lipolytic proteome of cultured human fat cells. We used specific affinity tags to detect and identify the lipolytic and esterolytic enzymes in human subcutaneous (Sc) and visceral (Visc) adipocytes. For this purpose, differentiated fat cells were incubated with a fluorescent suicide inhibitor followed by protein separation using one- or two-dimensional gel electrophoresis. After detection by fluorescence laser scanning, the labeled proteins were tryptically digested and peptides were identified by mass spectrometry. In addition, a biotinylated probe was used for specific enzyme labeling with subsequent avidin affinity isolation of the tagged proteins. Finally, we determined the quantitative differences in protein expression levels between subcutaneous and visceral adipocytes using differential activity-based gel electrophoresis (DABGE). We found that the lipase/esterase patterns of both cell types are very similar, except for some proteins that were only found in Sc cells. Two novel enzyme candidates identified in this study were overexpressed and characterized using biologically relevant glycerolipid substrates in vitro. Both of them showed pronounced hydrolytic activities on hydrophobic acylglycerols and therefore may be considered lipases. The physiological functions of the novel lipolytic proteins in vivo are currently subject to investigation. Keywords: subcutaneous fat • visceral fat • fluorescent lipids • abhydrolases • patatin-like phospholipases • lipid-associated disorders • proteomics Introduction The bulk lipid depots of higher organisms are essentially represented by two different types of adipose tissue, namely, the subcutaneous (Sc) and the visceral (Visc) fat. Mobilization of free fatty acids through degradation of the constituent triacylglycerols (TAG) and cholesterol esters is a key process in energy homeostasis. 1-4 Impairment of their specific meta- bolic functions may be associated with lipid metabolic disor- ders such as insulin resistance, obesity, and the metabolic syndrome. 5-7 Thus, knowledge of lipid metabolism in these lipid “organs” and the enzymes catalyzing lipid synthesis and catabolism is important for an understanding of the role of adipose tissue in pathophysiology. Enzymatic hydrolysis of intracellular triacylglycerols into glycerol and free fatty acids is mainly catalyzed by three lipases, namely, adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and monoglyceride lipase (MGL). ATGL prefer- entially catalyzes the first step of TAG hydrolysis, leading to the formation of diacylglycerols (DAG) and free fatty acids. HSL mainly hydrolyzes DAG, but also accepts triacylglycerols, monoacylglycerols (MAG), and cholesterol esters (CE) as substrates. MGL is responsible for monoacylglycerol hydrolysis, finally leading to the formation of glycerol and fatty acids. 8,9 In mouse adipose tissue, many other lipase and esterase candidates were identified by our laboratory using an activity- based proteomics approach. 10 In contrast, information on lipases in human adipocytes 11,12 and adipose tissue 2 is scarce. Pe ´rez-Pe ´rez et al. analyzed the protein patterns of subcutane- ous and visceral adipose tissue using conventional proteomic analysis based on unselective protein staining and found, next to a very large number of other proteins, several hydrolytic enzymes (carboxylesterase 1, proteasome subunit alpha, glu- tathione S-transferase and monoglyceride lipase). Here we present the results from a specific functional proteomic analysis aimed at the selective detection of the lipolytic and esterolytic proteins of cultured human adipocytes originating from subcutaneous and visceral adipose tissues. For this purpose, we used specific enzyme probes containing fluorescent or biotinylated p-nitrophenyl phosphonate esters that specifically, covalently, and stoichiometrically bind to the active sites of esterolytic and lipolytic enzymes. 13 After specific tagging of the lipases and esterases with the functional fluorescent probes, they were separated by gel electrophoresis, detected with a laser scanner, and identified by LC-MS/MS after * To whom correspondence should be addressed. Dr. Albin Hermetter, Institute of Biochemistry, Graz University of Technology, Petersgasse 12/II, A-8010 Graz, Austria. Tel: +43-316-873 6457. Fax: +43-316-873 6952. E-mail: albin.hermetter@tugraz.at. † Institute of Biochemistry, Graz University of Technology. ‡ Institute of Molecular Biosciences, University of Graz. § Institute of Forensic Medicine, Medical University of Graz. ⊥ Present address: Center for Medical Research, Medical University of Graz, Graz, Austria. 6334 Journal of Proteome Research 2010, 9, 6334–6344 10.1021/pr1005795  2010 American Chemical Society Published on Web 10/13/2010