MOLECULAR BIOTECHNOLOGY Volume 23, 2003 Protein Identification by BIA-MS/MS 203 RESEARCH 203 Molecular Biotechnology 2003 Humana Press Inc. All rights of any nature whatsoever reserved. 1073–6085/2003/23:3/203–212/$20.00 *Author to whom all correspondence and reprint requests should be addressed: * ,2 Division of Stem Cell Regulation, Center for Molecular Medicine, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi-machi Tochigi 329-0498, Japan; 1 Gene Try Co., Ltd, Tokyo 171-0022 Abstract Identification of Novel p53-Binding Proteins by Biomolecular Interaction Analysis Combined with Tandem Mass Spectrometry Jiro Kikuchi, 1,2 Yusuke Furukawa, 2,* and Nakanobu Hayashi 1 Electrospray tandem mass spectrometry (ESI-MS/MS) was combined with biomolecular interaction analy- sis (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 power- ful tool for analyzing complex biological materials in a simple but highly specific and sensitive manner. Index Entries: Surface plasmon resonance (SPR); BIA; ESI-MS/MS; proteome; p53. 1. Introduction There has been considerable recent interest in and success with genome techniques, such as the rapid screening of mutation/polymorphisms and the genome-wide monitoring of gene expression. However, more emphasis should be placed on protein-based methods, because the ultimate tar- gets of the majority of biochemical and medical research are proteins. To obtain an integrated view of disease mechanisms and cellular pro- cesses at the protein level, a large-scale analysis of the entire protein population (proteome) in tar- get cells, tissues, and body fluids is essential. This type of analysis is now called proteomics. Biomolecular interaction analysis (BIA) is an affinity-based biosensor technology optimized for monitoring the interaction of two biomolecules in real time. This method utilizes surface plasmon resonance (SPR) to detect the changes in a refrac- tive index on the surface of a sensor chip. These changes are in linear correlation with surface con- centrations of the molecules and are displayed as resonance units (RU) vs time in a sensorgram (1). The measurement is made quickly in real time with little if any chemical modification of samples. The SPR-BIA serves two main purposes in pro- teomics: (1) as a sensitive instrument to confirm and quantify the specific binding between two biomolecules and (2) as a support to purify small samples for further analysis. This method is supe- rior to other affinity-based methods such as immu- noprecipitation, especially when sample volumes are limited. Most proteomic approaches rely exclusively on mass spectrometry (MS) for protein identification because of its high sensitivity and the ability to search mass-spectral data in rapidly expanding protein and expressed-sequence-tag (EST) data- bases (2). Two techniques of MS were proven to be efficient for analyzing large biomolecules: matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) (3) and electrospray ionization mass spectrometry (ESI-MS) (4). An alternative sensitive method for protein identifica- tion is mass spectrometric sequencing using tan- dem mass spectrometry (MS/MS) (5,6).