MCP Waters-The Science of What's Possible
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Originally published In Press as doi:10.1074/mcp.M400126-MCP200 on February 9, 2005.
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Molecular & Cellular Proteomics 4:618-625, 2005.
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Intact-protein-based High-resolution Three-dimensional Quantitative Analysis System for Proteome Profiling of Biological Fluids*

Hong Wang{ddagger}, Shawn G. Clouthier{ddagger},§, Vladimir Galchev{ddagger}, David E. Misek{ddagger}, Ulrich Duffner§, Chang-Ki Min§, Rong Zhao{ddagger}, John Tra{ddagger}, Gilbert S. Omenn§, James L. M. Ferrara{ddagger},§ and Samir M. Hanash{ddagger},¶

From the Departments of {ddagger} Pediatrics and § Internal Medicine, University of Michigan, Ann Arbor, Michigan 48109

The substantial complexity and vast dynamic range of protein abundance in biological fluids, notably serum and plasma, present a formidable challenge for comprehensive protein analysis. Integration of multiple technologies is required to achieve high-resolution and high-sensitivity proteomics analysis of biological fluids. We have implemented an orthogonal three-dimensional intact-protein analysis system (IPAS), coupled with protein tagging and immunodepletion of abundant proteins, to quantitatively profile the human plasma proteome. Following immunodepletion, plasma proteins in each of paired samples are concentrated and labeled with a different Cy dye, before mixing. Proteins are subsequently separated in three dimensions according to their charge, hydrophobicity, and molecular mass. Differences in the abundance of resolved proteins are determined based on Cy dye ratios. We have applied this strategy to profile the plasma proteome for changes that occur with acute graft-versus-host disease (GVHD), following allogeneic bone marrow transplantation (BMT). Using capillary HPLC ESI Q-TOF MS, we identified 75 proteins in the micromolar to femtomolar range that exhibited quantitative differences between the pre- and post-GVHD samples. These proteins included serum amyloid A, apolipoproteins A-I/A-IV, and complement C3 that are well-known acute-phase reactants likely reflecting the post-BMT inflammatory state. In addition, we identified some potentially interesting immunologically relevant molecules including vitamin D-binding protein, fetuin, vitronectin, proline-rich protein 3 and 4, integrin-{alpha}, and leukocyte antigen CD97. IPAS provides a combination of comprehensive profiling and quantitative analysis, with a substantial dynamic range, for disease-related applications.

To whom correspondence should be addressed: Dept. of Pediatrics, University of Michigan, Rm. A520 MSRB-1, 1150 West Medical Center Dr., Ann Arbor, MI 48109-0656. Tel.: 734-763-9311; E-mail: shanash{at}umich.edu


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