A Proteomic Approach for Identification of Secreted Proteins during the Differentiation of 3T3-L1 Preadipocytes to Adipocytes

doi:10.1074/mcp.M200006-MCP200

A Proteomic Approach for Identification of Secreted Proteins during the Differentiation of 3T3-L1 Preadipocytes to Adipocytes*

^‡Center for Experimental Bioinformatics, University of Southern Denmark, Odense M, DK-5230 Denmark
^§Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461
^‖Departments of Medicine and Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110
^¶MDS-Proteomics, Staermosegaardsvej 6, Odense M, DK-5230 Denmark
^‡‡Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142
^¶¶Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts 02115

§§To whom correspondence may be addressed. E-mail: mann{at}bmb.sdu.dk.

Abstract

We have undertaken a systematic proteomic approach to purify and identify secreted factors that are differentially expressed in preadipocytes versus adipocytes. Using one-dimensional gel electrophoresis combined with nanoelectrospray tandem mass spectrometry, proteins that were specifically secreted by 3T3-L1 preadipocytes or adipocytes were identified. In addition to a number of previously reported molecules that are up- or down-regulated during this differentiation process (adipsin, adipocyte complement-related protein 30 kDa, complement C3, and fibronectin), we identified four secreted molecules that have not been shown previously to be expressed differentially during the process of adipogenesis. Pigment epithelium-derived factor, a soluble molecule with potent antiangiogenic properties, was found to be highly secreted by preadipocytes but not adipocytes. Conversely, we found hippocampal cholinergic neurostimulating peptide, neutrophil gelatinase-associated lipocalin, and haptoglobin to be expressed highly by mature adipocytes. We also used liquid chromatography-based separation followed by automated tandem mass spectrometry to identify proteins secreted by mature adipocytes. Several additional secreted proteins including resistin, secreted acidic cysteine-rich glycoprotein/osteonectin, stromal cell-derived factor-1, cystatin C, gelsolin, and matrix metalloprotease-2 were identified by this method. To our knowledge, this is the first study to identify several novel secreted proteins by adipocytes by a proteomic approach using mass spectrometry.

Footnotes

Published, MCP Papers in Press, February 14, 2002, DOI 10.1074/mcp.M200006-MCP200
↵¹ The abbreviations used are: PEDF, pigment epithelium-derived factor; Acrp30, adipocyte complement-related protein 30; HCNP, hippocampal cholinergic neurostimulating peptide; LC, liquid chromatography; MS/MS, tandem spectrometry; NGAL, neutrophil gelatinase-associated lipocalin precursor; SPARC, secreted acidic cysteine-rich glycoprotein; DMEM, Dulbecco’s modified Eagle’s medium; MOPS, 4-morpholinepropanesulfonic acid; SDF, stromal cell-derived factor.
↵* This work was supported in part by a generous grant from the Danish National Research Foundation (to the Center for Experimental Bioinformatics).
↵** Supported by grants from the American Diabetes Association and the Clinical Nutrition Research Unit of Washington University (5P30 DK56341).Supported by grants from the American Diabetes Association and the Clinical Nutrition Research Unit of Washington University (5P30 DK56341).
↵‖‖ Supported by Howard Temin Award KO1 CA75447 from NCI, National Institutes of Health and by a travel award from the Plasmid Foundation, Roskilde, Denmark. To whom correspondence may be addressed: Visiting Scientist, Center for Experimental Bioinformatics, University of Southern Denmark, Odense M, DK-5230 Denmark. pandey{at}cebi.sdu.dk.
- Received January 11, 2002.
- Revision received February 8, 2002.