Identification of novel p53-binding proteins by biomolecular interaction analysis combined with tandem mass spectrometry |
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Authors: | Kikuchi Jiro Furukawa Yusuke Hayashi Nakanobu |
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Affiliation: | (1) Division of Stem Cell Regulation, Center for Molecular Medicine, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi-machi, 329-0498 Tochigi, Japan;(2) Gene Try Co., Ltd, 171-0022 Tokyo |
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Abstract: | Electrospray tandem mass spectrometry (ESI-MS/MS) was combined with biomolecular interaction analysis (BIA) to develop a method of direct protein identification after real-time analysis of protein-protein interactions. Using this method, called BIA-MS/MS, we detected multiple p53-interacting proteins in whole tissue extracts from human placenta and liver. Peptide sequencing revealed three proteins whose interaction with p53 had not been previously reported: a cyclin-dependent kinase inhibitor p57/Kip2, a serine/threonine protein phosphatase PP1C, and hemoglobin. Using our system, unambiguous sequence information can be obtained at the femto- to picomole level after repeating the recovery procedure five times. Furthermore, the association and dissociation constants are easily determined by kinetic analysis. This system provides a powerful tool for analyzing complex biological materials in a simple but highly specific and sensitive manner. |
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Keywords: | Surface plasmon resonance (SPR) BIA ESI-MS/MS proteome p53 |
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