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Originally published In Press as doi:10.1074/mcp.M500150-MCP200 on July 5, 2005.
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Molecular & Cellular Proteomics 4:1550-1557, 2005.
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Peptidyl-Lys Metalloendopeptidase-catalyzed 18O Labeling for Comparative Proteomics

Application to Cytokine/Lipolysaccharide-treated Human Retinal Pigment Epithelium Cell Line*,S

K. C. Sekhar Rao{ddagger}, Vikram Palamalai{ddagger}, Jane R. Dunlevy§ and Masaru Miyagi{ddagger},¶

From the Departments of {ddagger} Biochemistry and Molecular Biology and § Anatomy and Cell Biology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58203

We recently proposed a comparative proteomic method utilizing proteolytic 18O labeling of peptides catalyzed by peptidyl-Lys metalloendopeptidase (Lys-N) (Rao, K. C. S., Carruth, R. T., and Miyagi, M. (2005) Proteolytic 18O labeling by peptidyl-Lys metalloendopeptidase for comparative proteomics. J. Proteome Res. 4, 507–514). Unlike trypsin, which generates a mixture of isotopic isoforms resulting from the incorporation of one or two 18O atoms into each peptide species, Lys-N incorporates only a single 18O atom into the carboxyl terminus of each proteolytically generated peptide in H218O solvent. This study reports the first biological application of the Lys-N-based proteolytic 18O labeling method, characterizing the proteome changes of cytokine/lipopolysaccharide-treated verses untreated human retinal pigment epithelium (ARPE-19) cells. The study resulted not only in the identification of 584 proteins but also the determination of the relative abundances of 562 proteins in the two proteomes. The results demonstrate the usefulness of the Lys-N-based proteolytic 18O labeling method in comparative proteomic studies. The results also provide the most comprehensive description of the retinal pigment epithelium proteome to date.

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, 501 North Columbia Rd., Grand Forks, ND 58203. Tel.: 701-777-2760; Fax: 701-777-2382; E-mail: mmiyagi{at}medicine.nodak.edu


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