Research Topics

1. Development and application of analytical methods for structural details on biological molecules (Dohmae, Suzuki, Masuda, Nakayama,Watanabe)

Genetic code is transcripted into RNA followed by translation to proteins. Many proteins capture appropriate functions and suffer regulations by post-translational modifications (PTMs). Direct analysis of the modifications of amino acid residues and their positions within a protein sequence is still required in post genome sequence era. Furthermore, binding site of a low molecular reagent to a protein is the most important information for an approach using chemical biology with bio-probe.

To characterize protein structure containing PTMs, we recommend the combination of three procedures. 1) Observation of molecular weight of whole protein using electro-spray ionization mass spectrometry. 2) Liquid chromatography(LC)-mass spectrometric analysis of enzymatic digest. 3) Amino acid analysis of acid hydrolyzates.

We have applied these methods to several cases in collaborative researches. For example, we analyzed protein methylation sites by histone lysine methyltransferases (Collaboration with Dr. Hamamoto, The University of Tokyo). And we also determined N-glycosylation site of cathepsin L2 and have been analyzed C-mannnosylation of proteins (Collaboration with Dr. Shimizu, Keio University). Furthermore, we have analyzed acrorein binding site of proteins in patients (Collaboration with Dr. Igarashi, Amine Pharma Research Institute Co.,Ltd.). We and professor Lee of Pusan National University have determined directly novel lipidation structures including N-monoacyl-S-monoacylglyceryl structure in low-GC Gram positive bacterial lipoproteins. We have held the Chemical Biology Core Facility Educational Seminar Series. The 9th and 10th seminar about mass spectrometry for biologist were held for introduction of recent mass spectrometry.

Development of quantitative analysis of biomolecules (Masuda, Suzuki, Dohmae)

Quantification of post-translational modifications (PTMs) of proteins is necessary for epigenetics or proteomics research. To complement qualitative analyses by mass spectroscopy such as determination of positions or kinds of PTMs, we have developed ultra-sensitive amino acid analysis. Amino acid analysis, which is independent on the sequence of amino acids in proteins and is based on degradation of proteins of peptides to amino acids, is absolutely quantitative method and is applicable to any kind of protein. We carried out a highly sensitive amino acid analysis using a fluorescent derivatizing reagent, 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). We examined pre-treatment of hydrolyzed samples to isolate the modified amino acids from large amount of other normal non-modified amino acids.

Using this system, we quantified PTMs of mRNA-protein complex known as P-bodies which was associated with protein arginine methyltransferases (joint-research with Dr. Matsumoto of Cell. Biochem. Lab.) and of protein substrate of demethylating enzyme (joint-research with Dr. Unoki of Kyushu Univ.). We also proved that synthesized proteins contained amino acids introduced by artificial genetic codes, which suggests the existence of a limited set of genetic codes in ancient times (joint-research with Dr. Kiga of Tokyo institute of technology). Subunit ratio of protein-RNA complexes was determined by gel chromatography in combination with amino acid analysis (joint-research with Dr. Shinkai of Func. Biol. I. Res. Team).

Studies on structural biology and its related technologies(Miyatake, Dohmae)

SPring-8 is the most excellent synchrotron facility in the world, but researchers on the off-site often face difficulties to collect data at the maximum efficiency in the limited beam-times. The researchers must collect and validate quality of a lot of data set, especially in the challenges for difficult crystals. Thus our team and the division of synchrotron radiation instrumentation (SPring-8 center) together built a mail-in data collection system in Wako campus area for remote accumulation of the diffraction data at SPring-8. We have been carried out some structural biological collaboration with other laboratories in Wako campus. We are ready to support the users from data collection to data analysis, prep ration of presentations and more. On the other hand, crystallization of target proteins is one of the most difficult processes for the recent X-ray crystallography, so that automated device for macromolecular crystallization is strongly desired. In this point of view, we have developed a novel device for protein crystallization based on dynamic light scattering in corporation with a company under the financial support of the RIKEN Collaboration Center Program (in collaboration with Nikkiso Co., Ltd.).

Identification and characterization of RNA by mass spectrometry (Nakayama, Akiyama, Koike)

We are developing a method to correlate tandem mass spectra of sample RNA nucleolytic fragments with an RNA nucleotide sequence in a DNA/RNA sequence database, thereby allowing tandem mass spectrometry (MS/MS)-based identification of RNA in biological samples. We have developed a database search engine, Ariadne, which identifies RNA by two probability-based evaluation steps of MS/MS data. In the first step, the software evaluates the matches between the masses of product ions generated by MS/MS of an RNase digest of sample RNA and those calculated from a candidate nucleotide sequence in a DNA/RNA sequence database, which then predicts the nucleotide sequences of these RNase fragments. In the second step, the candidate sequences are mapped for all RNA entries in the database, and each entry is scored for a function of occurrences of the candidate sequences to identify a particular RNA. Ariadne can also predict post-transcriptional modifications of RNA, such as methylation of nucleotide bases and/or ribose, by estimating mass shifts from the theoretical mass values. To expand the identification capability of Ariadne to larger DB such as human genome, we have improved the algorithm in the second step. As a result of the improvement, the method allows to identify RNA components simultaneously from mixtures of RNAs by searching human genome.
→ Link to Ariadne service