An Efficient Identification Method of a Specific Binding Protein for a Bioactive Compound Using On-Bead Digestion and Mass Spectrometry

作者：Sakai Mitsuru^*; Furuya Minoru; Endo Hiroko; Yamaoka Kazuyoshi; Kondo Shiro; Koike Tohru

来源：Bulletin of the Chemical Society of Japan, 2008, 81(12): 1599-1604.

DOI：10.1246/bcsj.81.1599

Affinity chromatography is widely utilized as a tool in biomedical research and biotechnology. This purification method provides increased sensitivity for various kinds of analyses, such as mass spectrometry. This report describes a protocol to identify a specific binding protein for a bioactive compound (BAC) using BAC-affinity beads and nanoflow liquid chromatography tandem mass spectrometry (nano-LC/MS/MS). After the target protein was digested directly oil the affinity beads, the obtained peptide fragments were determined by MS analysis. The binding-specificity was shown by I competition experiment using beads lacking bound BAC. In this study, we describe two examples for benzenesul-fonamide- and methotrexate-binding proteins.

• 出版日期2008-12-15