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Originally published In Press as doi:10.1074/mcp.M500143-MCP200 on June 28, 2005.
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Molecular & Cellular Proteomics 4:1503-1521, 2005.
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Proteomic Analysis of Human Neutrophil Granules*,S

George Lominadze{ddagger}, David W. Powell§, Greg C. Luerman{ddagger}, Andrew J. Link§, Richard A. Ward and Kenneth R. McLeish{ddagger},¶,||,**

From the Departments of Medicine and {ddagger} Biochemistry and Molecular Biology, University of Louisville and the || Veterans Affairs Medical Center, Louisville, Kentucky 40202 and the § Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

Stimulated exocytosis of intracellular granules plays a critical role in conversion of inactive, circulating neutrophils to fully activated cells capable of chemotaxis, phagocytosis, and bacterial killing. The functional changes induced by exocytosis of each of the granule subsets, gelatinase (tertiary) granules, specific (secondary) granules, and azurophil (primary) granules, are poorly defined. To improve the understanding of the role of exocytosis of these granule subsets, a proteomic analysis of the azurophil, specific, and gelatinase granules from human neutrophils was performed. Two different methods for granule protein identification were applied. First, two-dimensional (2D) gel electrophoresis followed by MALDI-TOF MS analysis of peptides obtained by in-gel trypsin digestion of proteins was performed. Second, peptides from tryptic digests of granule membrane proteins were separated by two-dimensional microcapillary chromatography using strong cation exchange and reverse phase microcapillary high pressure liquid chromatography and analyzed with electrospray ionization tandem mass spectrometry (2D HLPC ESI-MS/MS). Our analysis identified 286 proteins on the three granule subsets, 87 of which were identified by MALDI MS and 247 were identified by 2D HPLC ESI-MS/MS. The increased sensitivity of 2D HPLC ESI-MS/MS, however, resulted in identification of over 500 proteins from subcellular organelles contaminating isolated granules. Defining the proteome of neutrophil granule subsets provides a basis for understanding the role of exocytosis in neutrophil biology. Additionally, the described methods may be applied to mobilizable compartments of other secretory cells.

** To whom correspondence should be addressed: Molecular Signaling Group, Kidney Disease Program, Donald E. Baxter Research Bldg., 570 S. Preston St., University of Louisville, Louisville, KY 40202. Tel.: 502-852-0014; Fax: 502-852-4384; E-mail: k.mcleish{at}louisville.edu


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