Takanori Harada

Live single-cell video-mass spectrometry for cellular and subcellular molecular detection and cell classification

The molecular content from the cytoplasm of a live, single mammalian cell and its organelle were trapped with a nano-electrospray ionization (ESI) tip acting as a micropipette under a video microscope, and hundreds of small molecular peaks were detected by direct nano-ESI mass spectrometry (MS). Granule- or cytoplasm-specific peaks in a mast cell (RBL 2H3) model were extracted by paired t-test to demonstrate their specific localization. Some of the typical and specific molecules were successfully identified by MS/MS analysis. This method was also applied to the cell classification of seven types of cell lines at the single-cellular level by principal component analysis (PCA), revealing seven clusters in the multivariate score plot.

In situ molecular analysis of plant tissues by live single-cell mass spectrometry.

We report the development of a rapid, direct molecular analysis of live, single plant cells viewed under a video microscope in their natural environment. A nanoelectrospray tip was used to extract the contents of a single leaf, stem, or petal cell from Pelargonium zonale, and the samples were analyzed on an Orbitrap mass spectrometer by nanoelectrospray ionization. Around a thousand m/z peaks belonging to metabolites and other compounds in each sample were obtained and processed by using statistical tools to find the cell specific molecular peaks. Hybrid high-resolution mass spectrometry analysis was performed to confirm the structure of specific metabolites from the analyzed samples. This method is useful for identifying specific molecules in live single cells from plant tissue and will allow different cell types and stages from different sites in the plant to be compared with morphological observations.

Conformational stabilities of 1-methoxy-2-(methylthio)ethane and relevant intramolecular CH⋯O interaction studied by matrix-isolation infrared spectroscopy and density functional calculations

Abstract Conformational stabilities of 1-methoxy-2-(methylthio)ethane were studied by matrix-isolation infrared spectroscopy, and the relevant intramolecular 1,5-CH⋯O interaction was examined by density functional calculations. The conformer with trans–trans–gauche± around the CH3O–CH2–CH2–SCH3 bonds is the most stable in an argon matrix and the conformer with trans–gauche±–gauche∓ is the second most stable. The energy difference between the two conformers was determined to be 0.7±0.4 kJ mol −1 using thermal effusive sources. The 1,5-CH⋯O interaction is responsible for the high stability of the trans–gauche±–gauche∓ conformer, since the nonbonded (C)H⋯O distance associated with this interaction is shorter than the corresponding van der Waals separation by 0.15 A. The energy of the 1,5-CH⋯O interaction was evaluated to be 4.0 kJ mol −1 .

Conformational and vibrational analyses of 2-methoxyethanol and 2-(methylthio)ethanol by density functional theory

Abstract Conformational and vibrational analyses were performed on 2-methoxyethanol (ME) and 2-(methylthio)ethanol (MTE) by density functional theory (DFT). The energies, molecular geometries and vibrational wavenumbers were calculated for the TGg′, GGg′, TTt and TGt conformers of ME and the GGg′, G′Gg′, TGg′ and GGt conformers of MTE (T or t: trans; G or g: gauche) by the BLYP, B3LYP and B3PW91 methods using the 6-31G∗ basis set. The calculations by the HF and MP2 methods were also carried out on the same conformers. The calculated energies are consistent with the experimental findings that the TGg′ conformer of ME and the GGg′ and G′Gg′ conformers of MTE are present in low-temperature matrix. The DFT calculations give the stabilization energy by the OH⋯S hydrogen bonding substantially the same as the energy by the OH⋯O hydrogen bonding. The vibrational wavenumbers for the TGg′ conformer of ME and the GGg′ conformer of MTE are successfully predicted by the MP2, B3LYP and B3PW91 methods using the uniform scale factors for the respective methods. The experimental large wavenumber difference between ME and MTE of the intramolecular hydrogen bonded O-H stretching vibrations is reproduced much better with the DFT calculations than with the ab initio MO calculations. The present study has shown that Beckes three-parameter exchange functional methods overall give the most accurate results.

Novel Computational Methodologies for Structural Modeling of Spacious Ligand Binding Sites of G-Protein-Coupled Receptors: Development and Application to Human Leukotriene B4 Receptor

This paper describes a novel method to predict the activated structures of G-protein-coupled receptors (GPCRs) with high accuracy, while aiming for the use of the predicted 3D structures in in silico virtual screening in the future. We propose a new method for modeling GPCR thermal fluctuations, where conformation changes of the proteins are modeled by combining fluctuations on multiple time scales. The core idea of the method is that a molecular dynamics simulation is used to calculate average 3D coordinates of all atoms of a GPCR protein against heat fluctuation on the picosecond or nanosecond time scale, and then evolutionary computation including receptor-ligand docking simulations functions to determine the rotation angle of each helix of a GPCR protein as a movement on a longer time scale. The method was validated using human leukotriene B4 receptor BLT1 as a sample GPCR. Our study demonstrated that the proposed method was able to derive the appropriate 3D structure of the active-state GPCR which docks with its agonists.