Identification of novel p53-binding proteins by biomolecular interaction analysis combined with tandem mass spectrometry.
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.
Kikuchi
Jiro
J
Gene Try Co, Ltd, Tokyo.
Furukawa
Yusuke
Y
Hayashi
Nakanobu
N
D000595:Amino Acid Sequence; D001921:Brain / Q000378:metabolism; D001923:Brain Chemistry; D005240:Feasibility Studies; D005260:Female; D006454:Hemoglobins / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000737:chemistry / Q000378:metabolism; D008832:Microchemistry / Q000379:methods*; D008969:Molecular Sequence Data; D036103:Nanotechnology / Q000379:methods; D010920:Placenta / Q000737:chemistry / Q000378:metabolism; D011247:Pregnancy; D011485:Protein Binding; D020539:Sequence Analysis, Protein / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D020349:Surface Plasmon Resonance / Q000379:methods*; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000302:isolation & purification
D006454:Hemoglobins; D016159:Tumor Suppressor Protein p53