Research Topics in 2012

Development and application of analytical methods for structural details on biological molecules

Development of quantitative analysis of biomolecules

Studies on structural biology and its related technologies

Identification and characterization of RNA by mass spectrometry

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

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 gone in a research project, the research on the innovative development and the practical application of new drugs for hepatitis B, and started global analysis of PTMs. In this fiscal year, the latest mass spectrometer (Thermo Q-Exactive) with the highest sensitivity and resolution was introduced to global analysis of PTMs by shotgun mass spectrometry. By the shotgun analysis of a human cell (THP-1, human monocyte), 10000-15000 peptides derived from 1500-2000 proteins per one-time analysis were identified. Finally, 23000 peptides from 3500 proteins were identified by accumulated analyses. Among these peptides, about 1% of acetylated or methylated peptides were found, the results which suggested the possibility of the global identification of PTMs by the shotgun analysis.

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 and discovered alteration in lipidation status of S. aureus lipoproteins under a combination of environmental and growth conditions. This is the first report showing that the environment mediates lipid-modified structural alterations of bacterial lipoproteins.

We have held the Chemical Biology Core Facility Educational Seminar Series(http://www.riken.jp/BiomolChar/log.html).

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

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. However, one separation mode of chromatography is not enough to quantify a small amount of PTM-amino acids in the presence of a large amount of other normal amino acids. Then we developed 2D chromatography system in which amino acids of a hydrolyzed sample are separated by a graphite column before AAA using ion-exchange chromatography. By using this system we successfully quantified novel histone modification, hydroxylysine (joint-research with Dr. Unoki of Kyushu Univ.). It was confirmed that histone demethylase, JMJD6, mediated histone lysyl 5-hydroxylation.

Studies on structural biology and its related technologies(Miyatake)

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, preparation of presentations, papers 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 (H.M.: in collaboration with YMC Co., Ltd.).

Identification and characterization of RNA by mass spectrometry (Nakayama)

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. We further developed a visualization tool for the database search results.→ Link to Ariadne service

Researc Topics 2002-