Proteomics Analysis of Human Coronary Atherosclerotic Plaque

doi:10.1074/mcp.M600259-MCP200

Proteomics Analysis of Human Coronary Atherosclerotic Plaque

A Feasibility Study of Direct Tissue Proteomics by Liquid Chromatography and Tandem Mass Spectrometry*S

From the ^‡Department of Cell Biology, Center for Vascular Biology, University of Connecticut School of Medicine, Farmington, Connecticut 06030, ^¶Proteomics Resource Center, Rockefeller University, New York, New York 10021, ^‖Holland Laboratories, American Red Cross, Rockville, Maryland 20855, and ^**Fred Hutchinson Cancer Research Center, Seattle, Washington 98109

‡‡To whom correspondence should be addressed: Dept. of Cell Biology, Center for Vascular Biology, University of Connecticut School of Medicine, 263 Farmington Ave., Farmington, CT 06030. Tel.: 860-679-2444; Fax: 860-679-1201; E-mail: han{at}nso.uchc.edu

Abstract

Cardiovascular disease presents significant variations in human populations with respect to the atherosclerotic plaque progression, inflammation, thrombosis, and rupture. To gain a more comprehensive picture of the pathogenic mechanism of atherosclerosis and the variations seen in patients, efficient methods to identify proteins from the normal and diseased arteries need to be developed. To accomplish this goal, we tested the feasibility and efficiency of protein identification by a recently developed method, termed direct tissue proteomics (DTP). We analyzed frozen and paraformaldehyde-fixed archival coronary arteries with the DTP method. We also validated the distinct expression of four proteins by immunohistochemistry. In addition, we demonstrated the compatibility of the DTP method with laser capture microdissection and the possibility of monitoring specific cytokines and growth factors by the absolute quantification of abundance method. Major findings from this feasibility study are that 1) DTP can be used to efficiently identify proteins from paraformaldehyde-fixed, paraffin-embedded, and frozen coronary arteries; 2) approximately twice the number of proteins were identified from the frozen sections when compared with the paraformaldehyde-fixed sections; 3) laser capture microdissection is compatible with DTP; and 4) detection of low abundance cytokines and growth factors in the coronary arteries required selective reaction monitoring experiments coupled to absolute quantification of abundance. The analysis of 35 human coronary atherosclerotic samples allowed identification of a total of 806 proteins. The present study provides the first large scale proteomics map of human coronary atherosclerotic plaques.

Footnotes

Published, MCP Papers in Press, March 5, 2007, DOI 10.1074/mcp.M600259-MCP200
↵¹ The abbreviations used are: SMC, smooth muscle cell; AQUA, absolute quantification of abundance; DAB, diaminobenzidine; DTP, direct tissue proteomics; H&E, hematoxylin and eosin; HFBA, heptafluorobutyric acid; IHC, immunohistochemistry; LCM, laser capture microdissection; MFG-E8, milk fat globule-EGF-factor 8; EGF, epidermal growth factor; MRM, multiple reaction monitoring; PDGF-β, platelet-derived growth factor β; PEDF, pigment epithelium-derived factor; SDF1-α, stromal cell-derived factor 1 α; TGF-β, transforming growth factor β; TUNEL, terminal deoxynucleotidyltransferase dUTP nick-end labeling; VEGF, vascular endothelial growth factor; 1D, one-dimensional.
↵² L. Wu, S. I. Hwang, K. Rezaul, L. J. Lu, V. Mayya, M. Girstein, J. K. Eng, D. H. Lundgren, and D. K. Han, in press.
↵* This work was supported by National Institutes of Health Grants HL67569 and HL70694. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.
↵§ Both authors contributed equally to this work.
- Received July 14, 2006.
- Revision received February 27, 2007.