| 1. |
Taihei Torigoe, Masatomo Takahashi*, Omidreza Heravizadeh, Kazuki Ikeda, Kohta Nakatani, Takeshi Bamba, Yoshihiro Izumi*, Predicting retention time in unified-hydrophilic-interaction/anion-exchange liquid chromatography high-resolution tandem mass spectrometry (unified-HILIC/AEX/HRMS/MS) for comprehensive structural annotation of polar metabolome, Analytical Chemistry, 10.1021/acs.analchem.3c04618, 96, 3, 1275-1283, 2024.01, The accuracy of the structural annotation of unidentified peaks obtained in metabolomic analysis using liquid chromatography/tandem mass spectrometry (LC/MS/MS) can be enhanced using retention time (RT) information as well as precursor and product ions. Unified-hydrophilic-interaction/anion-exchange liquid chromatography high-resolution tandem mass spectrometry (unified-HILIC/AEX/HRMS/MS) has been recently developed as an innovative method ideal for nontargeted polar metabolomics. However, the RT prediction for unified-HILIC/AEX has not been developed because of the complex separation mechanism characterized by the continuous transition of the separation modes from HILIC to AEX. In this study, we propose an RT prediction model of unified-HILIC/AEX/HRMS/MS, which enables the comprehensive structural annotation of polar metabolites. With training data for 203 polar metabolites, we ranked the feature importance using a random forest among 12,420 molecular descriptors (MDs) and constructed an RT prediction model with 26 selected MDs. The accuracy of the RT model was evaluated using test data for 51 polar metabolites, and 86.3% of the ΔRTs (difference between measured and predicted RTs) were within ±1.50 min, with a mean absolute error of 0.80 min, indicating high RT prediction accuracy. Nontargeted metabolomic data from the NIST SRM 1950-Metabolites in frozen human plasma were analyzed using the developed RT model and in silico MS/MS prediction, resulting in a successful structural estimation of 216 polar metabolites, in addition to the 62 identified based on standards. The proposed model can help accelerate the structural annotation of unknown hydrophilic metabolites, which is a key issue in metabolomic research.. |
| 2. |
Kohta Nakatani, Yoshihiro Izumi*, Hironobu Umakoshi, Maki Yokomoto-Umakoshi, Tomoko Nakaji, Hiroki Kaneko, Hiroshi Nakao, Yoshihiro Ogawa, Kazutaka Ikeda, Takeshi Bamba*, Wide-scope targeted analysis of bioactive lipids in human plasma by LC/MS/MS, Journal of Lipid Research, 10.1016/j.jlr.2023.100492, 65, 1, Article number 100492, 2024.01, Quantitative information on blood metabolites can be used in developing advanced medical strategies such as early detection and prevention of disease. Monitoring bioactive lipids such as steroids, bile acids, and PUFA metabolites could be a valuable indicator of health status. However, a method for simultaneously measuring these bioactive lipids has not yet been developed. Here, we report a LC/MS/MS method that can simultaneously measure 144 bioactive lipids, including steroids, bile acids, and PUFA metabolites, from human plasma, and a sample preparation method for these targets. Protein removal by methanol precipitation and purification of bioactive lipids by solid-phase extraction improved the recovery of the targeted compounds in human plasma samples, demonstrating the importance of sample preparation methods for a wide range of bioactive lipid analyses. Using the developed method, we studied the plasma from healthy human volunteers and confirmed the presence of bioactive lipid molecules associated with sex differences and circadian rhythms. The developed method of bioactive lipid analysis can be applied to health monitoring and disease biomarker discovery in precision medicine.. |
| 3. |
Noriyuki Tomiyasu, Masatomo Takahashi, Kenji Toyonaga, Sho Yamasaki, Takeshi Bamba, Yoshihiro Izumi*, Efficient lipidomic approach for the discovery of lipid ligands for immune receptors by combining LC-HRMS/MS analysis with fractionation and reporter cell assay, Analytical and Bioanalytical Chemistry, 10.1007/s00216-023-05111-w, in press, 2023.12, C-type lectin receptors (CLRs), which are pattern recognition receptors responsible for triggering innate immune responses, recognize damaged self-components and immunostimulatory lipids from pathogenic bacteria; however, several of their ligands remain unknown. Here, we propose a new analytical platform combining liquid chromatography-high-resolution tandem mass spectrometry with microfractionation capability (LC-FRC-HRMS/MS) and a reporter cell assay for sensitive activity measurements to develop an efficient methodology for searching for lipid ligands of CLR from microbial trace samples (crude cell extracts of approximately 5 mg dry cell/mL). We also developed an in-house lipidomic library containing accurate mass and fragmentation patterns of more than 10,000 lipid molecules predicted in silico for 90 lipid subclasses and 35 acyl side chain fatty acids. Using the developed LC-FRC-HRMS/MS system, the lipid extracts of Helicobacter pylori were separated and fractionated, and HRMS and HRMS/MS spectra were obtained simultaneously. The fractionated lipid extract samples in 96-well plates were thereafter subjected to reporter cell assays using nuclear factor of activated T cells (NFAT)-green fluorescent protein (GFP) reporter cells expressing mouse or human macrophage-inducible C-type lectin (Mincle). A total of 102 lipid molecules from all fractions were annotated using an in-house lipidomic library. Furthermore, a fraction that exhibited significant activity in the NFAT-GFP reporter cell assay contained α-cholesteryl glucoside, a type of glycolipid, which was successfully identified as a lipid ligand molecule for Mincle. Our analytical platform has the potential to be a useful tool for efficient discovery of lipid ligands for immunoreceptors.. |
| 4. |
Naoya Nishimura, Noriyuki Tomiyasu, Shota Torigoe, Satoru Mizuno, Hanako Fukano, Eri Ishikawa, Harutaka Katano, Yoshihiko Hoshino, Kazuhiro Matsuo, Masatomo Takahashi, Yoshihiro Izumi*, Takeshi Bamba, Koichi Akashi, Sho Yamasaki*, Mycobacterial mycolic acids trigger inhibitory receptor Clec12A to suppress host immune responses., Tuberculosis, 10.1016/j.tube.2022.102294, 94, 138, Article number 102294, 2023.01, Mycobacteria often cause chronic infection. To establish persistence in the host, mycobacteria need to evade host immune responses. However, the molecular mechanisms underlying the evasion strategy are not fully understood. Here, we demonstrate that mycobacterial cell wall lipids trigger an inhibitory receptor to suppress host immune responses. Mycolic acids are major cell wall components and are essential for survival of mycobacteria. By screening inhibitory receptors that react with mycobacterial lipids, we found that mycolic acids from various mycobacterial species bind to mouse Clec12A, and more potently to human Clec12A. Clec12A is a conserved inhibitory C-type lectin receptor containing immunoreceptor tyrosine-based inhibitory motif (ITIM). Innate immune responses, such as MCP-1 production, and PPD-specific recall T cell responses were augmented in Clec12A-deficient mice after infection. In contrast, human Clec12A transgenic mice were susceptible to infection with M. tuberculosis. These results suggest that mycobacteria dampen host immune responses by hijacking an inhibitory host receptor through their specific and essential lipids, mycolic acids. The blockade of this interaction might provide a therapeutic option for the treatment or prevention of mycobacterial infection.. |
| 5. |
Kohta Nakatani, Yoshihiro Izumi*, Masatomo Takahashi, Takeshi Bamba*, Unified-hydrophilic-interaction/anion-exchange liquid chromatography mass spectrometry (unified-HILIC/AEX/MS): A single-run method for comprehensive and simultaneous analysis of polar metabolome., Analytical Chemistry, 10.1021/acs.analchem.2c03986, 94, 48, 16877-16866, 2022.12, One of the technical challenges in the field of metabolomics is the development of a single-run method to detect the full complement of polar metabolites in biological samples. However, an ideal method to meet this demand has not yet been developed. Herein, we proposed a simple methodology that enables the comprehensive and simultaneous analysis of polar metabolites using unified-hydrophilic-interaction/anion-exchange liquid chromatography mass spectrometry (unified-HILIC/AEX/MS) with a polymer-based mixed amines column composed of methacrylate-based polymer particles with primary, secondary, tertiary, and quaternary amines as functional groups. The optimized unified-HILIC/AEX/MS method is composed of two consecutive chromatographic separations, HILIC-dominant separation for cationic, uncharged, and zwitterionic polar metabolites [retention times (RTs) = 0-12.8 min] and AEX-dominant separation for polar anionic metabolites (RTs = 12.8-26.5 min), by varying the ratio of acetonitrile to 40 mM ammonium bicarbonate solution (pH 9.8). A total of 400 polar metabolites were analyzed simultaneously through a combination of highly efficient separation using unified-HILIC/AEX and remarkably sensitive detection using multiple reaction monitoring-based triple quadrupole mass spectrometry (unified-HILIC/AEX/MS/MS). A nontargeted metabolomic approach using unified-HILIC/AEX high-resolution mass spectrometry (unified-HILIC/AEX/HRMS) also provided more comprehensive information on polar metabolites (3242 metabolic features) in HeLa cell extracts than the conventional HILIC/HRMS method (2068 metabolic features). Our established unified-HILIC/AEX/MS/MS and unified-HILIC/AEX/HRMS methods have several advantages over conventional techniques, including polar metabolome coverage, throughput, and accurate quantitative performance, and represent potentially useful tools for in-depth studies on metabolism and biomarker discovery.. |
| 6. |
Yuki Soma, Yoshihiro Izumi, Takehiko Shimohira, Masatomo Takahashi, Yuri Imado, Saki Tominaga, Kanako Tokito, Kosuke Hata, Shoji Shinadama, Mana Oshiro, Yoshihiro Hayakawa, Takeshi Bamba*, In-needle pre-column derivatization for amino acid quantification (iPDAQ) using HPLC., Metabolites, 10.3390/metabo12090807, 12, 9, 807, 2022.08, Pre-column fluorescent derivatization has been used for the fast quantification of amino acids using high-performance liquid chromatography (HPLC) systems. However, it generally requires an offline in-vial derivatization process with multiple derivatization reagents. The offline derivatization requires the same number of reaction vials as the number of sample vials for use as a reaction chamber for the derivatization reaction in an autosampler. Therefore, the number of samples analyzed per batch using the pre-column derivatization method is halved. To benefit from the pre-column derivatization method, we transformed the derivatization process from an offline chamber process to an online in-needle process (in-needle Pre-column Derivatization for Amino acids Quantification; iPDAQ). Fluorescent derivatization in the injection needle obviated the need for vacant vials as reaction chambers. Consequently, the throughput per batch improved up to two times, and the consumption of derivatization reagents was reduced to less than one-tenth of that in the conventional vial method. We demonstrated to separate and quantify the amino acids in various biological samples. Herein, we presented a novel HPLC-based amino acid quantification method that enables the continuous analysis of a large number of samples. The iPDAQ facilitates accurate amino acid quantification due to the automation of derivatization and achieves improvement in the throughput and reduction of analysis labor.. |
| 7. |
Shin Nishiumi*, Yoshihiro Izumi*, Akiyoshi Hirayama*, Masatomo Takahashi, Motonao Nakao, Kosuke Hata, Daisuke Saigusa, Eiji Hishinuma, Naomi Matsukawa, Suzumi M. Tokuoka, Yoshihiro Kita, Fumie Hamano, Nobuyuki Okahashi, Kazutaka Ikeda, Hiroki Nakanishi, Kosuke Saito, Masami Yokota Hirai, Masaru Yoshida, Yoshiya Oda, Fumio Matsuda, Takeshi Bamba, Comparative evaluation of plasma metabolomic data from multiple laboratories., Metabolites, 10.3390/metabo12020135, 12, 2, 135, 2022.01, In mass spectrometry-based metabolomics, the differences in the analytical results from different laboratories/machines are an issue to be considered because various types of machines are used in each laboratory. Moreover, the analytical methods are unique to each laboratory. It is important to understand the reality of inter-laboratory differences in metabolomics. Therefore, we have evaluated whether the differences in analytical methods, with the exception sample pretreatment and including metabolite extraction, are involved in the inter-laboratory differences or not. In this study, nine facilities are evaluated for inter-laboratory comparisons of metabolomic analysis. Identical dried samples prepared from human and mouse plasma are distributed to each laboratory, and the metabolites are measured without the pretreatment that is unique to each laboratory. In these measurements, hydrophilic and hydrophobic metabolites are analyzed using 11 and 7 analytical methods, respectively. The metabolomic data acquired at each laboratory are integrated, and the differences in the metabolomic data from the laboratories are evaluated. No substantial difference in the relative quantitative data (human/mouse) for a little less than 50% of the detected metabolites is observed, and the hydrophilic metabolites have fewer differences between the laboratories compared with hydrophobic metabolites. From evaluating selected quantitatively guaranteed metabolites, the proportion of metabolites without the inter-laboratory differences is observed to be slightly high. It is difficult to resolve the inter-laboratory differences in metabolomics because all laboratories cannot prepare the same analytical environments. However, the results from this study indicate that the inter-laboratory differences in metabolomic data are due to measurement and data analysis rather than sample preparation, which will facilitate the understanding of the problems in metabolomics studies involving multiple laboratories.. |
| 8. |
Kosuke Hata, Yuki Soma, Toshiyuki Yamashita, Masatomo Takahashi, Kuniyo Sugitate, Takeshi Serino, Hiromi Miyagawa, Kenichi Suzuki, Kayoko Yamada, Takatomo Kawamukai, Teruhisa Shiota, Yoshihiro Izumi*, Takeshi Bamba*, Calibration-curve-locking database for semi-quantitative metabolomics by gas chromatography/mass spectrometry, Metabolites, 10.3390/metabo11040207, 11, 4, Article number 207, 2021.03, Calibration-Curve-Locking Databases (CCLDs) have been constructed for automatic compound search and semi-quantitative screening by gas chromatography/mass spectrometry (GC/MS) in several fields. CCLD felicitates the semi-quantification of target compounds without calibration curve preparation because it contains the retention time (RT), calibration curves, and electron ionization (EI) mass spectra, which are obtained under stable apparatus conditions. Despite its usefulness, there is no CCLD for metabolomics. Herein, we developed a novel CCLD and semi-quantification framework for GC/MS-based metabolomics. All analytes were subjected to GC/MS after derivatization under stable apparatus conditions using (1) target tuning, (2) RT locking technique, and (3) automatic derivatization and injection by a robotic platform. The RTs and EI mass spectra were obtained from an existing authorized database. A quantifier ion and one or two qualifier ions were selected for each target metabolite. The calibration curves were obtained as plots of the peak area ratio of the target compounds to an internal standard versus the target compound concentration. These data were registered in a database as a novel CCLD. We examined the applicability of CCLD for analyzing human plasma, resulting in time-saving and labor-saving semi-qualitative screening without the need for standard substances.. |
| 9. |
Tatsuya Fushimi, Yoshihiro Izumi*, Masatomo Takahashi, Kosuke Hata, Yoshihiro Murano, and Takeshi Bamba, Dynamic metabolome analysis reveals the metabolic fate of medium-chain fatty acid in AML12 cells, Journal of Agricultural and Food Chemistry, org/10.1021/acs.jafc.0c04723, 68, 43, 11997-12010, 2020.10, Several studies in hepatocyte cell lines reported that medium-chain fatty acids (MCFAs) with 6–12 carbons showed different metabolic properties from long-chain fatty acids (LCFAs). However, these studies reported unclear effects of different fatty acid molecules on hepatocyte metabolism. This study is aimed to capture the metabolic kinetics of MCFA assimilation in AML12 cells treated with octanoic acid (FA 8:0), decanoic acid (FA 10:0), or lauric acid (FA12:0) [LCFA; oleic acid (FA 18:1)] via metabolic profiling and dynamic metabolome analysis with 13C-labeling. The concentrations of total ketone bodies in the media of cells treated with FA 8:0 or FA 10:0 were 3.22- or 3.69-fold higher than those obtained with FA 18:1 treatment, respectively. FA 12:0 treatment did not significantly increase ketone body levels compared to DMSO treatment (control), whereas FA 12:0 treatment increased intracellular triacylglycerol (TG) levels 15.4 times compared to the control. Metabolic profiles of FA 12:0-treated samples differed from those of the FA 8:0-treated and FA 10:0-treated samples, suggesting that metabolic assimilation of MCFAs differed significantly depending on the MCFA type. Furthermore, the dynamic metabolome analysis clearly revealed that FA 8:0 was rapidly and quantitatively oxidized to acetyl-CoA and assimilated into ketone bodies, citrate cycle intermediates, and glucogenic amino acids but not readily into TGs.. |
| 10. |
Toshiaki Yoshioka, Yoshihiro Izumi*, Masatomo Takahashi, Koji Suzuki, Yasuhisa Miyamoto, Yasushi Nagatomi, Takeshi Bamba*, Identification of Acrylamide Adducts Generated during Storage of Canned Milk Coffee, Journal of Agricultural and Food Chemistry, 10.1021/acs.jafc.9b08139, 68, 12, 3859-3867, 2020.03, Since coffee is a significant contributor to the consumption of acrylamide, its reduction is required. Acrylamide is produced during the roasting of coffee beans, but the roasting process is an essential step in determining the taste of coffee. Acrylamide content in coffee has been suggested to decrease by reacting with proteins and/or other substances during storage, but details are unknown. Investigation of acrylamide adducts may contribute to a strategy for acrylamide reduction in coffee. In this study, a stable isotope labeling technique, combined with high-resolution mass spectrometry, allows the identification of acrylamide adducts (3-hydroxypyridine-acrylamide and pyridine-acrylamide) in canned milk coffee. Other acrylamide adducts derived from milk coffee proteins, Lys-acrylic acid and CysSO2-acrylic acid, were identified. During a 4-month storage period, the formation of these four adducts was found to reduce the total content of acrylamide by 75.3% in canned milk coffee. Therefore, endogenous proteins can be used in acrylamide reduction.. |
| 11. |
Hiroaki Takeda, Yoshihiro Izumi*, Shohei Tamura, Tomonari Koike, Yui Koike, Masashi Shiomi, Takeshi Bamba, Lipid Profiling of Serum and Lipoprotein Fractions in Response to Pitavastatin Using an Animal Model of Familial Hypercholesterolemia, Journal of Proteome Research, 10.1021/acs.jproteome.9b00602, 19, 3, 1100-1108, 2020.03, Statins are widely used for the treatment of atherosclerotic cardiovascular diseases. They inhibit cholesterol biosynthesis in the liver and cause pleiotropic effects, including anti-inflammatory and antioxidant effects. To develop novel therapeutic drugs, the effect of blood-borne lipid molecules on the pleiotropic effects of statins must be elucidated. Myocardial infarction-prone Watanabe heritable hyperlipidemic (WHHLMI) rabbits, an animal model for hypercholesterolemia, are suitable for the determination of lipid molecules in the blood in response to statins because their lipoprotein metabolism is similar to that of humans. Herein, lipid molecules were investigated by lipidome analysis in response to pitavastatin using WHHLMI rabbits. Various lipid molecules in the blood were measured using a supercritical fluid chromatography triple quadrupole mass spectrometry. Cholesterol and cholesterol ester blood concentrations decreased by reducing the secretion of very low density lipoproteins from the liver. Independent of the inhibition effects of cholesterol biosynthesis, the concentrations of some lipids with anti-inflammation and antioxidant effects (phospholipid molecules with n-6 fatty acid side chains, lysophosphatidylcholines, phosphatidylethanolamine plasmalogens, and ceramide molecules) were significantly altered. These findings may lead to further investigation of the mechanism of statin action.. |
| 12. |
Kosuke Hata, Yoshihiro Izumi*, Takeshi Hara, Masaki Matsumoto*, Takeshi Bamba, In-Line Sample Processing System with an Immobilized Trypsin-Packed Fused-Silica Capillary Tube for the Proteomic Analysis of a Small Number of Mammalian Cells, Analytical chemistry, 10.1021/acs.analchem.9b03993, 92, 4, 2997-3005, 2020.02, Omics analysis at single-cell resolution has helped to demonstrate the shaping of cellular heterogeneity on the basis of the expression of various molecules. However, in-depth proteomic analysis of low-quantity samples has remained challenging because of difficulties associated with the measurement of large numbers of proteins by shotgun proteomics using nanoflow liquid chromatography tandem mass spectrometry (nano-LC/MS/MS). To meet such a demand, we developed a method called in-line sample preparation for efficient cellular proteomics (ISPEC) in which cells were captured, directly lysed, and digested with immobilized trypsin within fused-silica capillaries. ISPEC minimized sample loss during the sample preparation processes with a relatively small number of mammalian cells ( |
| 13. |
Nao Nishida-Aoki, Yoshihiro Izumi*, Hiroaki Takeda, Masatomo Takahashi, Takahiro Ochiya*, Takeshi Bamba, Lipidomic analysis of cells and extracellular vesicles from high-and low-metastatic triple-negative breast cancer, Metabolites, 10.3390/metabo10020067, 10, 2, Article number: 67, 2020.02, Extracellular vesicles (EVs) are lipid bilayer nanovesicles secreted from almost all cells including cancer. Cancer-derived EVs contribute to cancer progression and malignancy via educating the surrounding normal cells. In breast cancer, epidemiological and experimental observations indicated that lipids are associated with cancer malignancy. However, lipid compositions of breast cancer EVs and their contributions to cancer progression are unexplored. In this study, we performed a widely targeted quantitative lipidomic analysis in cells and EVs derived from high-and low-metastatic triple-negative breast cancer cell lines, using supercritical fluid chromatography fast-scanning triple-quadrupole mass spectrometry. We demonstrated the differential lipid compositions between EVs and cells of their origin, and between high-and low-metastatic cell lines. Further, we demonstrated EVs from highly metastatic breast cancer accumulated unsaturated diacylglycerols (DGs) compared with EVs from lower-metastatic cells, without increasing the amount in cells. The EVs enriched with DGs could activate the protein kinase D signaling pathway in endothelial cells, which can lead to stimulated angiogenesis. Our results indicate that lipids are selectively loaded into breast cancer EVs to support tumor progression.. |
| 14. |
Kohta Nakatani, Yoshihiro Izumi*, Kosuke Hata, Takeshi Bamba, An analytical system for single-cell metabolomics of typical mammalian cells based on highly sensitive nano-liquid chromatography tandem mass spectrometry, Mass Spectrometry, 10.5702/massspectrometry.A0080, 9, 1, 2020.01, The rapid development of next-generation sequencing techniques has enabled single-cell genomic and transcriptomic analyses, which have revealed the importance of heterogeneity in biological systems. However, analytical methods to accurately identify and quantify comprehensive metabolites from single mammalian cells with a typical diameter of 10–20 µm are still in the process of development. The aim of this study was to develop a single-cell metabolomic analytical system based on highly sensitive nanoliquid chromatography tandem mass spectrometry (nano-LC-MS/MS) with multiple reaction monitoring. A packed nano-LC column (3-µm particle-size pentafluorophenylpropyl Discovery HSF5 of dimensions 100 µm i.d.×180 mm) was prepared using a slurry technique. The optimized nano-LC-MS/MS method showed 3–132-fold (average value, 26-fold) greater sensitivity than semimicro-LC-MS/MS, and the detection limits for several hydrophilic metabolites, including amino acids and nucleic acid related metabolites were in the sub-fmol range. By combining live single-cell sampling and nano-LC-MS/MS, we successfully detected 18 relatively abundant hydrophilic metabolites (16 amino acids and 2 nucleic acid related metabo-lites) from single HeLa cells (n=22). Based on single-cell metabolic profiles, the 22 HeLa cells were classified into three distinct subclasses, suggesting differences in metabolic function in cultured HeLa cell populations. Our single-cell metabolomic analytical system represents a potentially useful tool for in-depth studies focused on cell metabolism and heterogeneity.. |
| 15. |
Yoshihiro Izumi, Fumio Matsuda*, Akiyoshi Hirayama, Kazutaka Ikeda, Yoshihiro Kita, Kanta Horie, Daisuke Saigusa, Kosuke Saito, Yuji Sawada, Hiroki Nakanishi, Nobuyuki Okahashi, Masatomo Takahashi, Motonao Nakao, Kosuke Hata, Yutaro Hoshi, Motohiko Morihara, Kazuhiro Tanabe, Takeshi Bamba*, Yoshiya Oda, Inter-laboratory comparison of metabolite measurements for metabolomics data integration, Metabolites, 10.3390/metabo9110257, 9, 11, Article number: 257, 2019.11, Background: One of the current problems in the field of metabolomics is the difficulty in integrating data collected using different equipment at different facilities, because many metabolomic methods have been developed independently and are unique to each laboratory. Methods: In this study, we examined whether different analytical methods among 12 different laboratories provided comparable relative quantification data for certain metabolites. Identical samples extracted from two cell lines (HT-29 and AsPc-1) were distributed to each facility, and hydrophilic and hydrophobic metabolite analyses were performed using the daily routine protocols of each laboratory. Results: The results indicate that there was no difference in the relative quantitative data (HT-29/AsPc-1) for about half of the measured metabolites among the laboratories and assay methods. Data review also revealed that errors in relative quantification were derived from issues such as erroneous peak identification, insufficient peak separation, a difference in detection sensitivity, derivatization reactions, and extraction solvent interference. Conclusion: The results indicated that relative quantification data obtained at different facilities and at different times would be integrated and compared by using a reference materials shared for data normalization.. |
| 16. |
Masatomo Takahashi, Yoshihiro Izumi*, Fukumatsu Iwahashi, Yasumune Nakayama, Mitsuhiko Iwakoshi, Motonao Nakao, Seiji Yamato, Eiichiro Fukusaki, Takeshi Bamba*, Highly Accurate Detection and Identification Methodology of Xenobiotic Metabolites Using Stable Isotope Labeling, Data Mining Techniques, and Time-Dependent Profiling Based on LC/HRMS/MS, Analytical Chemistry, 10.1021/acs.analchem.8b01388, 90, 15, 9068-9076, 2018.08, A generally applicable method to discover xenobiotic metabolites is important to safely and effectively develop xenobiotics. We propose an advanced method to detect and identify comprehensive xenobiotic metabolites using stable isotope labeling, liquid chromatography coupled with benchtop quadrupole Orbitrap high-resolution tandem mass spectrometry (LC/HRMS/MS), data mining techniques (alignment, peak picking, and paired-peaks filtering), in silico metabolism prediction, and time-dependent profiling. The LC/HRMS analysis was carried out using Arabidopsis T87 cultured cells treated with unlabeled, or 13C- or 2H-labeled 2,4-dichlorophenoxyacetic acid (2,4-D). Paired-peak filtering enabled accurate detection of 83 candidates for 2,4-D metabolites without any false positive peaks derived from solvents or the biological matrix. We confirmed 10 previously reported 2,4-D metabolites and identified 16 novel 2,4-D metabolites. Our method provides accurate detection and identification of comprehensive xenobiotic metabolites and represents a potentially useful tool to elucidate xenobiotic metabolism.. |
| 17. |
Hiroaki Takeda, Yoshihiro Izumi, Masatomo Takahashi, Thanai Paxton, Shohei Tamura, Tomonari Koike, Ying Yu, Noriko Kato, Katsutoshi Nagase, Masashi Shiomi, Takeshi Bamba*, Widely-targeted quantitative lipidomics method by supercritical fluid chromatography triple quadrupole mass spectrometry, Journal of Lipid Research, 10.1194/jlr.D083014, 59, 7, 1283-1293, 2018.01, Lipidomics, the mass spectrometry-based comprehensive analysis of lipids, has attracted attention as an analytical approach to provide novel insight into lipid metabolism and to search for biomarkers. However, an ideal method for both comprehensive and quantitative analysis of lipids has not been fully developed. Here, we have proposed a practical methodology for widely targeted quantitative lipidome analysis using supercritical fluid chromatography fast-scanning triple-quadrupole mass spectrometry (SFC/ QqQMS) and theoretically calculated a comprehensive lipid multiple reaction monitoring (MRM) library. Lipid classes can be separated by SFC with a normal-phase diethylaminebonded silica column with high resolution, high throughput, and good repeatability. Structural isomers of phospholipids can be monitored by mass spectrometric separation with fatty acyl-based MRM transitions. SFC/QqQMS analysis with an internal standard-dilution method offers quantitative information for both lipid class and individual lipid molecular species in the same lipid class. Additionally, data acquired using this method has advantages, including reduction of misidentification and acceleration of data analysis. Using the SFC/QqQMS system, alteration of plasma lipid levels in myocardial infarction-prone rabbits to the supplementation of EPA was first observed. Our developed SFC/QqQMS method represents a potentially useful tool for in-depth studies focused on complex lipid metabolism and biomarker discovery.. |
| 18. |
Yoshihiro Izumi, Shimpei Aikawa, Fumio Matsuda, Tomohisa Hasunuma, Akihiko Kondo*, Aqueous size-exclusion chromatographic method for the quantification of cyanobacterial native glycogen, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 10.1016/j.jchromb.2013.04.037, 930, 90-97, 2013.07, Cyanobacterial glycogen has gained interest as a valuable biomass feedstock for biofuel production. However, an ideal method for native glycogen quantification has not been developed. Here, we have proposed a simple methodology that enables the quantitative determination of cyanobacterial glycogen concentration with high repeatability using aqueous size-exclusion chromatography with a differential refractive index detector (SEC/RID). Our SEC/RID system also allows size distributions for native glycogen based on hydrodynamic volumes (Vh), which is proportional to the product of the molecular mass (M) and intrinsic viscosity [η], obtained by universal calibration using linear homopolymers of known M with Mark-Houwink-Sakurada parameters. The universal calibration curve achieved a broad linear range (Vh parameter [η]M=2×102-8×108mLg-1) with a high correlation coefficient (R2=0.9942), because the developed system is equipped with an OHpak SB-806M HQ aqueous column containing four types of polyhydroxy methacrylate-based particles with different particle and pore sizes. Based on the SEC/RID system, response of molecular size distribution of glycogen in microalgae to the cultivation condition was first observed. Our established SEC/RID method has several advantages over conventional techniques, including the simultaneous quantitative and size distribution analyses of glycogen, and represents a potentially useful tool to elucidate the relationship between structural properties and the roles of glycogen in metabolism.. |
| 19. |
Yoshihiro Izumi, Shin Takimura, Shinichi Yamaguchi, Junko Iida, Takeshi Bamba, Eiichiro Fukusaki*, Application of electrospray ionization ion trap/time-of-flight mass spectrometry for chemically-synthesized small RNAs, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2011.11.007, 113, 3, 412-419, 2012.03, In this study, we have demonstrated an accurate and rapid small RNA analytical method with both sequence determination and detailed modification analysis by electrospray ionization-ion trap/time-of-flight mass spectrometry (ESI-IT/TOFMS). To develop this ideal method, we have examined the performance of ESI-IT/TOFMS using various chemically-synthesized model sequences of modified or unmodified microRNAs (miRNAs). The deconvoluted mass of a 22-nucleotide (nt) miRNA was obtained from a multiply charged precursor ion (MS
1
). The ion exhibited high mass accuracy ( 2
method using ion trap collision-induced dissociation, as well as automatic annotation analysis of product ions based on the accurate mass information, enabled the precise sequencing determination of intact miRNAs. Further, the detailed structural analysis of 3'-terminal modified nucleic acid in intact methylated miRNA was carried out using the MS
3
capability of the hybrid IT/TOFMS. The direct infusion method also provided a high throughput and good sensitivity because the analytical time and sample concentration needed in a series of experiments with reliable data were only 3min and 100nM, respectively. This study provides a novel approach for characterizing the intact chemically-synthesized small RNA without chemical and enzymatic digestions and would be widely applicable for the structural analysis of complicated modified small RNAs.. |
| 20. |
Yoshihiro Izumi, Atsushi Okazawa, Takeshi Bamba, Akio Kobayashi, Eiichiro Fukusaki*, Development of a method for comprehensive and quantitative analysis of plant hormones by highly sensitive nanoflow liquid chromatography-electrospray ionization-ion trap mass spectrometry, Analytica Chimica Acta, 10.1016/j.aca.2009.07.001, 648, 2, 215-225, 2009.08, In recent plant hormone research, there is an increased demand for a highly sensitive and comprehensive analytical approach to elucidate the hormonal signaling networks, functions, and dynamics. We have demonstrated the high sensitivity of a comprehensive and quantitative analytical method developed with nanoflow liquid chromatography-electrospray ionization-ion trap mass spectrometry (LC-ESI-IT-MS/MS) under multiple-reaction monitoring (MRM) in plant hormone profiling. Unlabeled and deuterium-labeled isotopomers of four classes of plant hormones and their derivatives, auxins, cytokinins (CK), abscisic acid (ABA), and gibberellins (GA), were analyzed by this method. The optimized nanoflow-LC-ESI-IT-MS/MS method showed ca. 5-10-fold greater sensitivity than capillary-LC-ESI-IT-MS/MS, and the detection limits (S/N = 3) of several plant hormones were in the sub-fmol range. The results showed excellent linearity (R2 values of 0.9937-1.0000) and reproducibility of elution times (relative standard deviations, RSDs, |
| 21. |
Yoshihiro Izumi, Shin'Ichiro Kajiyama*, Ryosuke Nakamura, Atsushi Ishihara, Atsushi Okazawa, Eiichiro Fukusaki, Yasuo Kanematsu, Akio Kobayashi, High-resolution spatial and temporal analysis of phytoalexin production in oats, Planta, 10.1007/s00425-008-0887-x, 229, 4, 931-943, 2009.03, The production of oat (Avena sativa L.) phytoalexins, avenanthramides, occurs in response to elicitor treatment with oligo-N- acetylchitooligosaccharides. In this study, avenanthramides production was investigated by techniques that provide high spatial and temporal resolution in order to clarify the process of phytoalexin production at the cellular level. The amount of avenanthramides accumulation in a single mesophyll cell was quantified by a combination of laser micro-sampling and low-diffuse nanoflow liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) techniques. Avenanthramides, NAD(P)H and chlorophyll were also visualized in elicitor-treated mesophyll cells using line-scanning fluorescence microscopy. We found that elicitor-treated mesophyll cells could be categorized into three characteristic cell phases, which occurred serially over time. Phase 0 indicated the normal cell state before metabolic or morphological change in response to elicitor, in which the cells contained abundant NAD(P)H. In phase 1, rapid NAD(P)H oxidation and marked movement of chloroplasts occurred, and this phase was the early stage of avenanthramides biosynthesis. In phase 2, avenanthramides accumulation was maximized, and chloroplasts were degraded. Avenanthramides appear to be synthesized in the chloroplast, because a fluorescence signal originating from avenanthramides was localized to the chloroplasts. Moreover, our results indicated that avenanthramides biosynthesis and the hypersensitive response (HR) occurred in identical cells. Thus, the avenanthramides production may be one of sequential events programmed in HR leading to cell death. Furthermore, the phase of the defense response was different among mesophyll cells simultaneously treated with elicitor. These results suggest that individual cells may have different susceptibility to the elicitor.. |
