Ken Aoshima

MassBank: a public repository for sharing mass spectral data for life sciences.

MassBank is the first public repository of mass spectra of small chemical compounds for life sciences (<3000 Da). The database contains 605 electron-ionization mass spectrometry (EI-MS), 137 fast atom bombardment MS and 9276 electrospray ionization (ESI)-MS(n) data of 2337 authentic compounds of metabolites, 11 545 EI-MS and 834 other-MS data of 10,286 volatile natural and synthetic compounds, and 3045 ESI-MS(2) data of 679 synthetic drugs contributed by 16 research groups (January 2010). ESI-MS(2) data were analyzed under nonstandardized, independent experimental conditions. MassBank is a distributed database. Each research group provides data from its own MassBank data servers distributed on the Internet. MassBank users can access either all of the MassBank data or a subset of the data by specifying one or more experimental conditions. In a spectral search to retrieve mass spectra similar to a query mass spectrum, the similarity score is calculated by a weighted cosine correlation in which weighting exponents on peak intensity and the mass-to-charge ratio are optimized to the ESI-MS(2) data. MassBank also provides a merged spectrum for each compound prepared by merging the analyzed ESI-MS(2) data on an identical compound under different collision-induced dissociation conditions. Data merging has significantly improved the precision of the identification of a chemical compound by 21-23% at a similarity score of 0.6. Thus, MassBank is useful for the identification of chemical compounds and the publication of experimental data.

Circulating miRNA biomarkers for Alzheimer's disease.

A minimally invasive diagnostic assay for early detection of Alzheimers disease (AD) is required to select optimal patient groups in clinical trials, monitor disease progression and response to treatment, and to better plan patient clinical care. Blood is an attractive source for biomarkers due to minimal discomfort to the patient, encouraging greater compliance in clinical trials and frequent testing. MiRNAs belong to the class of non-coding regulatory RNA molecules of ∼22 nt length and are now recognized to regulate ∼60% of all known genes through post-transcriptional gene silencing (RNAi). They have potential as useful biomarkers for clinical use because of their stability and ease of detection in many tissues, especially blood. Circulating profiles of miRNAs have been shown to discriminate different tumor types, indicate staging and progression of the disease and to be useful as prognostic markers. Recently their role in neurodegenerative diseases, both as diagnostic biomarkers as well as explaining basic disease etiology has come into focus. Here we report the discovery and validation of a unique circulating 7-miRNA signature (hsa-let-7d-5p, hsa-let-7g-5p, hsa-miR-15b-5p, hsa-miR-142-3p, hsa-miR-191-5p, hsa-miR-301a-3p and hsa-miR-545-3p) in plasma, which could distinguish AD patients from normal controls (NC) with >95% accuracy (AUC of 0.953). There was a >2 fold difference for all signature miRNAs between the AD and NC samples, with p-values<0.05. Pathway analysis, taking into account enriched target mRNAs for these signature miRNAs was also carried out, suggesting that the disturbance of multiple enzymatic pathways including lipid metabolism could play a role in AD etiology.

Identification of a new plasma biomarker of Alzheimer's disease using metabolomics technology.

We performed unbiased analysis of steroid-related compounds to identify novel Alzheimers disease (AD) plasma biomarkers using liquid chromatography-atmospheric pressure chemical ionization-mass spectroscopy. The analysis revealed that desmosterol was found to be decreased in AD plasma versus controls. To precisely quantify variations in desmosterol, we established an analytical method to measure desmosterol and cholesterol. Using this LC-based method, we discovered that desmosterol and the desmosterol/cholesterol ratio are significantly decreased in AD. Finally, the validation of this assay using 109 clinical samples confirmed the decrease of desmosterol in AD as well as a change in the desmosterol/cholesterol ratio in AD. Interestingly, we could also observe a difference between mild cognitive impairment and control. In addition, the decrease of desmosterol was somewhat more significant in females. Receiver operating characteristic (ROC) analysis between controls and AD, using plasma desmosterol shows a score of 0.80, indicating a good discrimination power for this marker in the two reference populations and confirms the potential usefulness of measuring plasma desmosterol levels for diagnosing AD. Further analysis showed a significant correlation of plasma desmosterol with Mini-Mental State Examination scores. Although larger sample populations will be needed to confirm this diagnostic marker sensitivity, our studies demonstrate a sensitive and accurate method of detecting plasma desmosterol concentration and suggest that plasma desmosterol could become a powerful new specific biomarker for early and easy AD diagnosis.

Quantitative and Wide-Ranging Profiling of Phospholipids in Human Plasma by Two-dimensional Liquid Chromatography/Mass Spectrometry

Normal-phase or reverse-phase liquid chromatography has been used in phospholipidomics for lipid separation prior to mass spectrometry analysis. However, separation using a single separation mode is often inadequate, as high-abundance phospholipids can mask large numbers of low-abundance lipids of interest. In order to detect and quantify low-abundance phospholipids, we present a novel two-dimensional (2D) approach for sensitive and quantitative global analysis of phospholipids. The methodology monitors individual glycerolipids and phospholipids through the use of a new quantitative normal-phase, solid-phase extraction procedure, followed by molecular characterization and relative quantification using an ion-trap Orbitrap equipped with a reverse-phase liquid chromatograph, with data processing by MS++ software. The CV (%) of the peak area of each lipid standard was less than 15% with this extraction method. When the method was applied to a liver sample, we could detect more phosphatidylserine (PS) compared to the previous method. Finally, our developed method was applied to Alzheimers disease (AD) plasma samples. Several hundred peaks were detected from a 60 μL plasma sample. A partial-least-squares discriminant analysis (PLS-DA) plot using peak area ratio gave a unique group of PLS scores which could distinguish plasma samples of Alzheimers disease (AD) patients from those of age-matched healthy controls.

Eribulin mesylate reduces tumor microenvironment abnormality by vascular remodeling in preclinical human breast cancer models

Eribulin mesylate is a synthetic macrocyclic ketone analog of the marine sponge natural product halichondrin B and an inhibitor of microtubule dynamics. Some tubulin‐binding drugs are known to have antivascular (antiangiogenesis or vascular‐disrupting) activities that can target abnormal tumor vessels. Using dynamic contrast‐enhanced MRI analyses, here we show that eribulin induces remodeling of tumor vasculature through a novel antivascular activity in MX‐1 and MDA‐MB‐231 human breast cancer xenograft models. Vascular remodeling associated with improved perfusion was shown by Hoechst 33342 staining and by increased microvessel density together with decreased mean vascular areas and fewer branched vessels in tumor tissues, as determined by immunohistochemical staining for endothelial marker CD31. Quantitative RT‐PCR analysis of normal host cells in the stroma of xenograft tumors showed that eribulin altered the expression of mouse (host) genes in angiogenesis signaling pathways controlling endothelial cell–pericyte interactions, and in the epithelial–mesenchymal transition pathway in the context of the tumor microenvironment. Eribulin also decreased hypoxia‐associated protein expression of mouse (host) vascular endothelial growth factor by ELISA and human CA9 by immunohistochemical analysis. Prior treatment with eribulin enhanced the anti‐tumor activity of capecitabine in the MDA‐MB‐231 xenograft model. These findings suggest that eribulin‐induced remodeling of abnormal tumor vasculature leads to a more functional microenvironment that may reduce the aggressiveness of tumors due to elimination of inner tumor hypoxia. Because abnormal tumor microenvironments enhance both drug resistance and metastasis, the apparent ability of eribulin to reverse these aggressive characteristics may contribute to its clinical benefits.

Quantitative Profiling of Polar Cationic Metabolites in Human Cerebrospinal Fluid by Reversed-Phase Nanoliquid Chromatography/Mass Spectrometry

Reversed-phase (RP) nanoliquid chromatography (LC)/mass spectrometry (MS) is widely used for proteome analysis, but hydrophilic metabolites are poorly retained on RP columns. We describe here the development and application of an efficient, robust, and quantitative nano-LC/MS method for cationic metabolome analysis in the positive ionization mode without any derivatization of analytes. Various stationary phases for nano-LC, coating of the internal wall of the capillary column, and various mobile phases were evaluated in terms of separation and peak shapes for 33 hydrophilic metabolites, including nonderivatized amino acids. Polar cationic compounds were strongly bound to mixed-functional RP with cation exchange mode resin, and the best separation was obtained with hydrophilic internal wall coating and a two-step trifluoroacetic acid (TFA) gradient in methanol as the mobile phase. Simple, but optimized, sample processing and the use of a high content of methanol allowed robust nano-LC/MS analysis. Our developed method was applied for biomarker discovery in Alzheimers disease (AD). Several hundred peaks were detected from 10 microL of cerebrospinal fluid (CSF). In a principal component analysis (PCA) plot using peak intensities without normalization, peak separation depended on the experimental date, not disease state. Therefore, constant amounts of two stable isotope-labeled amino acids, Val and Lys, were added as internal standards (ISs) to each sample before processing. These ISs were eluted in different gradient slopes in the two-step gradient, and the normalized peak ratios using the corresponding ISs gave a unique group of PCA scores which could distinguish AD CSF samples from age-matched control CSF samples.

Quantitative phosphorus metabolomics using nanoflow liquid chromatography-tandem mass spectrometry and culture-derived comprehensive global internal standards.

Highly sensitive and quantitative analytical methods are essential for metabolomics. In this report, we introduce an analytical method focused on endogenous phosphorus metabolites, using nanoflow liquid chromatography-electrospray ionization tandem mass spectrometry (nanoLC-ESI-MS/MS) and culture-derived isotope-tagged metabolites as global internal standards for quantitative metabolomics. The nanoLC-ESI-MS/MS method employing a stone-arch microcolumn with amino propyl silica gel achieved good separation of phosphorus metabolites with forty- to hundred-fold increase of sensitivity compared with semimicro flow LC-ESI-MS. The quantitative reproducibility of the nanoLC-ESI-MS has been improved to the point where it is useful for studies of cellular metabolism. Focused metabolomics using culture-derived internal standards was employed to monitor 184 phosphorus-related metabolic changes in cancer cells treated with metabolic enzyme inhibitors, methotrexate, fluorouracil, and gemcitabine. We found marked perturbations of cellular metabolism, of which many, though not all, were in line with the known biological activities of these drugs.

Prediction of relaxin-3-induced downstream pathway resulting in anxiolytic-like behaviors in rats based on a microarray and peptidome analysis

Abstract The effect of the intracerebroventricular (i.c.v.) injection of relaxin-3 (RLX3) was evaluated using anxiety-related behavioral tests in rats. RLX3-injected animals showed normal locomotion activity in a habituated environment and declined anxiety cognition in the elevated plus maze test and the shock probe-burying test. The measurement of spontaneous locomotor activity in a novel environment also suggested that RLX3 reduced the stress response. To elucidate the regulatory mechanisms of the downstream signaling pathways underlying RLX3 activity and its relation to anxiolytic and hyperphagic behavior phenotypes, RLX3-i.c.v.-injected rat hypothalamic responses were examined using a microarray analysis. Ingenuity Pathway Analysis software listed the phenotype-relating genes and they showed characteristic expression patterns in the rat hypothalamus. When peptidome data sets for the same listed genes was analyzed using a semi-quantitative approach, the expressions of two neuropeptides were found to have increased. One of these neuropeptides, oxytocin (Oxt), exhibited increased expression in both the microarray and the peptidomic analysis, and a Western blot analysis validated the mass spectrometry results. A cross-omics data analysis is useful for predicting downstream signaling pathways, and the anxiolytic-like behavior of RLX3 may be mediated by an oxytocin signaling pathway in rats. These results suggest that RLX3 acts as an anxiolytic peptide and that the downstream pathways mediated by its receptors may be potential candidates for the treatment of anxieties in the future.

GlycanAnalysis Plug-in: a database search tool for N-glycan structures using mass spectrometry.

UNLABELLED Tandem mass spectrometry (MS/MS or MS(n)) is a potent technique for characterizing N-glycan structures. GlycanAnalysis searches a glycan database to support the identification of glycan structures from MS/MS spectra. It also calculates diagnostic ions of glycan structures registered in a glycan database (GlycomeDB or KEGG GLYCAN) and searches for MS/MS spectra of N-glycans that match diagnostic ions to determine the structures. This program functions as a plug-in for Mass++, a freeware mass spectrum visualization and analysis program. AVAILABILITY AND IMPLEMENTATION The executable files of Mass++ are available for free at http://www.first-ms3d.jp/english/. The GlycanAnalysis plug-in is included in the standard package of Mass++ for Windows. CONTACT k-morimt@shimadzu.co.jp or nishikaz@shimadzu.co.jp or acyshzw@shimadzu.co.jp SUPPLEMENTARY INFORMATION Supplementary material are available at Bioinformatics online.

Reduced plasma desmosterol-to-cholesterol ratio and longitudinal cognitive decline in Alzheimer's disease

We here examined whether plasma desmosterol‐to‐cholesterol ratio (DES/CHO) is decreased in patients with Alzheimers disease (AD) and investigated the association between plasma DES/CHO and longitudinal cognitive decline.