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This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: M800491-MCP200v1 8/6/1318    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Glossary Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Kuromitsu, S. Articles by Yamada, T. Search for Related Content PubMed PubMed Citation Articles by Kuromitsu, S. Articles by Yamada, T. Social Bookmarking                      What's this?

Molecular & Cellular Proteomics 8:1318-1323, 2009.
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Combination of MS Protein Identification and Bioassay of Chromatographic Fractions to Identify Biologically Active Substances from Complex Protein Sources

Sadao Kuromitsu, Hiroyuki Yokota^,, Masashi Hiramoto, Masatoshi Yuri, Masanori Naitou, Naoto Nakamura, Shigeki Kawabata, Masato Kobori, Masao Katoh, Kiyoshi Furuchi, Haruhisa Mita^¶ and Tetsuo Yamada^¶

From the Astellas Pharm Inc., 21 Miyukigaoka, Tsukuba-shi 305-8585, Japan and
¶National Hospital Organization Sagamihara National Hospital, Sagamihara 228-8522, Japan

Purification of biologically active proteins from complex biological sources is a difficult task, usually requiring large amounts of sample and many separation steps. We found an active substance in a serum response element-dependent luciferase reporter gene bioassay in interstitial cystitis urine that we attempted to purify with column chromatography and the bioassay. With anion-exchange Mono Q and C₄ reversed-phase columns, apparently sharp active peaks were obtained. However, more than 20 kinds of proteins were identified from the active fractions with MS, indicating that the purification was not complete. As further purification was difficult, we chose a candidate molecule by means of studying the correlation between MS protein identification scores and bioassay responses of chromatographic fractions near the active peaks. As a result, epidermal growth factor (EGF) was nominated as a candidate molecule among the identified proteins because the elution profile of EGF was consistent with that of the bioassay, and the correlation coefficient of EGF between MS protein identification scores and bioassay responses was the highest among all the identified proteins. With recombinant EGF and anti-EGF and anti-EGF receptor antibodies, EGF was confirmed to be the desired substance in interstitial cystitis urine. This approach required only 20 ml of urine sample and two column chromatographic steps. The combination of MS protein identification and bioassay of chromatographic fractions may be useful for identifying biologically active substances from complex protein sources.

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