title: Combination of MS protein identification and bioassay of chromatographic fractions to identify biologically active substances from complex protein sources.
abstract: 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.
authors.affiliation: Astellas Pharm Inc., 21 Miyukigaoka, Tsukuba-shi 305-8585, Japan.
authors.forename: Sadao
authors.initials: S
authors.lastname: Kuromitsu
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Yokota
authors.forename: Masashi
authors.initials: M
authors.lastname: Hiramoto
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Yuri
authors.forename: Masanori
authors.initials: M
authors.lastname: Naitou
authors.forename: Naoto
authors.initials: N
authors.lastname: Nakamura
authors.forename: Shigeki
authors.initials: S
authors.lastname: Kawabata
authors.forename: Masato
authors.initials: M
authors.lastname: Kobori
authors.forename: Masao
authors.initials: M
authors.lastname: Katoh
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Furuchi
authors.forename: Haruhisa
authors.initials: H
authors.lastname: Mita
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Yamada
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000595:Amino Acid Sequence; D001681:Biological Assay; D016022:Case-Control Studies; D002460:Cell Line; D002852:Chromatography, Ion Exchange / Q000379:methods*; D018856:Cystitis, Interstitial / Q000652:urine; D004815:Epidermal Growth Factor / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008875:Middle Aged; D008969:Molecular Sequence Data; D010449:Peptide Mapping
chemical_list: D004815:Epidermal Growth Factor