Details of a Researcher - Soga, Tomoyoshi

Papers - Soga, Tomoyoshi

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Degradation of ppGpp by Nudix Pyrophosphatase Modulates the Transition of Growth Phase in the Bacterium Thermus thermophilus

Ooga, T., Ohashi, Y., Kuramitsu, S., Koyama, Y., Tomita, M., Soga, T., Masui, R.

J. Biol. Chem. 284 ( 23 ) 15549 - 15556 2009.06

Research paper (scientific journal), Joint Work, Accepted

A major bacterial alarmone, guanosine 3′,5′-bispyrophosphate (ppGpp), controls cellular growth under conditions of nutritional starvation. For most bacteria, intracellular ppGpp levels are tightly controlled by the synthesis/degradation cycle of RelA and SpoT activities. This study shows a novel ppGpp regulatory protein governing the cellular growth of Thermus thermophilus, Ndx8, a member of the Nudix pyrophosphatase family that degrades ppGpp to yield guanosine 3′,5′-bisphosphate. The ndx8-null mutant strain exhibited early stage growth arrest accompanied by the stationary phase-specific morphologies and global transcriptional modulation under nutritionally defined conditions. Several possible substrate compounds of Ndx8, which specifically accumulated in the ndx8 mutant cells, were identified by employing a capillary electrophoresis time-of-flight mass spectrometry-based metabolomics approach. Among them, the hydrolytic activity of Ndx8 for ppGpp was significant not only in vitro but also in vivo. Finally, the elimination of ppGpp synthetic activity suppressed the observed phenotype of the ndx8 mutation, suggesting that the function of Ndx8 as a growth regulator is involved in ppGpp accumulation, which is thought to act as a trigger of the growth phase transition. These results suggest a novel mechanism of ppGpp-mediated growth control by the functional relay between Ndx8 and SpoT activity as ppGpp scavengers.

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Quantitative Metabolome Profiling of Colon and Stomach Cancer Microenvironment by Capillary Electrophoresis Time-of-flight Mass Spectrometry

Hirayama, A., Kami, K., Sugimoto, M., Sugawara, M., Toki, N., Onozuka, H., Kinoshita, T., Saito, N., Ochiai, A., Tomita, M., Esumi, H., Soga, T.

Cancer Res. 69 ( 1 ) 4918 - 4925 2009.06

Research paper (scientific journal), Joint Work, Accepted

Most cancer cells predominantly produce energy by glycolysis rather than oxidative phosphorylation via the tricarboxylic acid (TCA) cycle, even in the presence of an adequate oxygen supply (Warburg effect). However, little has been reported regarding the direct measurements of global metabolites in clinical tumor tissues. Here, we applied capillary electrophoresis time-of-flight mass spectrometry, which enables comprehensive and quantitative analysis of charged metabolites, to simultaneously measure their levels in tumor and grossly normal tissues obtained from 16 colon and 12 stomach cancer patients. Quantification of 94 metabolites in colon and 95 metabolites in stomach involved in glycolysis, the pentose phosphate pathway, the TCA and urea cycles, and amino acid and nucleotide metabolisms resulted in the identification of several cancer-specific metabolic traits. Extremely low glucose and high lactate and glycolytic intermediate concentrations were found in both colon and stomach tumor tissues, which indicated enhanced glycolysis and thus confirmed the Warburg effect. Significant accumulation of all amino acids except glutamine in the tumors implied autophagic degradation of proteins and active glutamine breakdown for energy production, i.e., glutaminolysis. In addition, significant organ-specific differences were found in the levels of TCA cycle intermediates, which reflected the dependency of each tissue on aerobic respiration according to oxygen availability. The results uncovered unexpectedly poor nutritional conditions in the actual tumor microenvironment and showed that capillary electrophoresis coupled to mass spectrometry-based metabolomics, which is capable of quantifying the levels of energy metabolites in tissues, could be a powerful tool for the development of novel anticancer agents that target cancer-specific metabolism.

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Mono-(2-ethylhexyl) phthalate Targets Glycogen Debranching Enzyme and Affects Glycogen Metabolism in Rat Testis

Kuramori, C., Hase, Y., Hoshikawa, K., Watanabe, K., Nishi, T., Hishiki, T., Soga, T., Nashimoto, A., Kabe, Y., Yamaguchi, Y., Watanabe, H., Kataoka, K., Suematsu, M., Handa, H.

Toxicol. Sci. 109 ( 1 ) 143 - 151 2009.05

Research paper (scientific journal), Joint Work, Accepted

Phthalate esters are commonly used plasticizers; however, some are suspected to cause reproductive toxicity. Administration of high doses of di-(2-ethylhexyl) phthalate (DEHP) induces germ cell death in male rodents. Mono-(2-ethylhexyl) phthalate (MEHP), a hydrolyzed metabolite of DEHP, appears to be responsible for this testicular toxicity; however, the underlying mechanism of this chemical's action remains unknown. Here, using a one-step affinity purification procedure, we identified glycogen debranching enzyme (GDE) as a phthalate-binding protein. GDE has oligo-1,4-1,4-glucanotransferase and amylo-1,6-glucosidase activities, which are responsible for the complete degradation of glycogen to glucose. Our findings demonstrate that MEHP inhibits the activity of oligo-1,4-1,4-glucanotransferase, but not of amylo-1,6-glucosidase. Among various phthalate esters tested, MEHP specifically binds to and inhibits GDE. We also show that DEHP administration affects glycogen metabolism in rat testis. Thus, inhibition of GDE by MEHP may play a role in germ cell apoptosis in the testis.

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Cystathionine β-synthase as a Carbon Monoxide-sensitive Regulator of Bile Excretion

Shintani, T., Iwabuchi, T., Soga, T., Kato,Y., Yamamoto, T., Takano, N., Hishiki, T., Ueno, Y., Ikeda, S., Sakurakawa, T., Ishikawa, K., Goda, N., Yitagawa, Y., Kajiyama, M., Matsumoto, K., *Suematsu, M.

Hepatology 49 ( 1 ) 141 - 150 2009.01

Research paper (scientific journal), Joint Work, Accepted

Carbon monoxide (CO) is a stress-inducible gas generated by heme oxygenase (HO) eliciting adaptive responses against toxicants; however, mechanisms for its reception remain unknown. Serendipitous observation in metabolome analysis in CO-overproducing livers suggested roles of cystathionine beta-synthase (CBS) that rate-limits transsulfuration pathway and H(2)S generation, for the gas-responsive receptor. Studies using recombinant CBS indicated that CO binds to the prosthetic heme, stabilizing 6-coordinated CO-Fe(II)-histidine complex to block the activity, whereas nitric oxide (NO) forms 5-coordinated structure without inhibiting it. The CO-overproducing livers down-regulated H(2)S to stimulate HCO(3) (-)-dependent choleresis: these responses were attenuated by blocking HO or by donating H(2)S. Livers of heterozygous CBS knockout mice neither down-regulated H(2)S nor exhibited the choleresis while overproducing CO. In the mouse model of estradiol-induced cholestasis, CO overproduction by inducing HO-1 significantly improved the bile output through stimulating HCO(3) (-) excretion; such a choleretic response did not occur in the knockout mice.
Conclusion: Results collected from metabolome analyses suggested that CBS serves as a CO-sensitive modulator of H(2)S to support biliary excretion, shedding light on a putative role of the enzyme for stress-elicited adaptive response against bile-dependent detoxification processes.

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Time-resolved Metabolomics Reveals Metabolic Modulation in Rice Foliage

Sato, S., Arita, M., Soga, T., Nishioka, T., Tomita, M.

BMC Syst Biol. 2 51 2008.06

Research paper (scientific journal), Joint Work, Accepted

Background: To elucidate the interaction of dynamics among modules that constitute biological systems, comprehensive datasets obtained from "omics" technologies have been used. In recent plant metabolomics approaches, the reconstruction of metabolic correlation networks has been attempted using statistical techniques. However, the results were unsatisfactory and effective data-mining techniques that apply appropriate comprehensive datasets are needed.
Results: Using capillary electrophoresis mass spectrometry (CE-MS) and capillary electrophoresis diode-array detection (CE-DAD), we analyzed the dynamic changes in the level of 56 basic metabolites in plant foliage (Oryza sativa L. ssp. japonica) at hourly intervals over a 24-hr period. Unsupervised clustering of comprehensive metabolic profiles using Kohonen's self-organizing map (SOM) allowed classification of the biochemical pathways activated by the light and dark cycle. The carbon and nitrogen (C/N) metabolism in both periods was also visualized as a phenotypic linkage map that connects network modules on the basis of traditional metabolic pathways rather than pairwise correlations among metabolites. The regulatory networks of C/N assimilation/dissimilation at each time point were consistent with previous works on plant metabolism. In response to environmental stress, glutathione and spermidine fluctuated synchronously with their regulatory targets. Adenine nucleosides and nicotinamide coenzymes were regulated by phosphorylation and dephosphorylation. We also demonstrated that SOM analysis was applicable to the estimation of unidentifiable metabolites in metabolome analysis. Hierarchical clustering of a correlation coefficient matrix could help identify the bottleneck enzymes that regulate metabolic networks.
Conclusion: Our results showed that our SOM analysis with appropriate metabolic time-courses effectively revealed the synchronous dynamics among metabolic modules and elucidated the underlying biochemical functions. The application of discrimination of unidentified metabolites and the identification of bottleneck enzymatic steps even to non-targeted comprehensive analysis promise to facilitate an understanding of large-scale interactions among components in biological systems.

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Development of bottom-fermenting Saccharomyces strains that produce high SO2 levels, using integrated metabolome and transcriptome analysis

Yoshida, S., Imoto, J., Minato, T., Oouchi, R., Sugihara, M., Imai, T., Ishiguro, T., Mizutani, S., Tomita, M., Soga, T., Yoshimoto, H.

Appl. Environ. Microbiol. 74 ( 9 ) 2787 - 2796 2008.05

Research paper (scientific journal), Joint Work, Accepted

Sulfite plays an important role in beer flavor stability. Although breeding of bottom-fermenting Saccharomyces strains that produce high levels of SO2 is desirable, it is complicated by the fact that undesirable H2S is produced as an intermediate in the same pathway. Here, we report the development of a high-level SO2-producing bottom-fermenting yeast strain by integrated metabolome and transcriptome analysis. This analysis revealed that O-acetylhomoserine (OAH) is the rate-limiting factor for the production of SO2 and H2S. Appropriate genetic modifications were then introduced into a prototype strain to increase metabolic fluxes from aspartate to OAH and from sulfate to SO2, resulting in high SO2 and low H2S production. Spontaneous mutants of an industrial strain that were resistant to both methionine and threonine analogs were then analyzed for similar metabolic fluxes. One promising mutant produced much higher levels of SO2 than the parent but produced parental levels of H2S.

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Depiction of metabolome changes in histidine-starved Escherichia coli by CE-TOFMS

Ohashi, Y., Hirayama, A., Ishikawa, T., Nakamura, S., Shimizu, K., Ueno, Y., Tomita, M., *Soga, T.

Mol. BioSyst 4 ( 2 ) 135 - 147 2008.02

Research paper (scientific journal), Joint Work, Accepted

Metabolic changes in response to histidine starvation were observed in histidine-auxotrophic Escherichia coli using a capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS)-based metabolomics technique. Prior to the analysis, we prepared an E. coli metabolome list of 727 metabolites reported in the literature. An improved method for metabolite extraction was developed, which resulted in higher extraction efficiency in phosphate-rich metabolites, e.g., ATP and GTP. Based on the results, 375 charged, hydrophilic intermediates in primary metabolisms were analysed simultaneously, providing quantitative data of 198 metabolites. We confirmed that the intracellular levels of intermediates in histidine biosynthesis are rapidly accumulated in response to a drop in histidine level under histidine-starved conditions. Simultaneously, disciplined responses were observed in the glycolysis, tricarboxylic acid cycle, and amino acid and nucleotide biosynthesis pathways as regulated by amino acid starvation.

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Microelectrospray interface with coaxial sheath flow for high-resolution capillary electrophoresis/mass spectrometry separation.

Hashimoto, M., *Ishihama, Y., Tomita, M., Soga, T.

Rapid Commun. Mass Spectrom. 21 ( 22 ) 3579 - 3584 2007.11

Research paper (scientific journal), Joint Work, Accepted

We have fabricated a coaxial sheath liquid flow microelectrospray ionization (microESI) interface for capillary electrophoresis coupled with mass spectrometry (CE/MS). The ESI interface, which features a reduced probe diameter (130 microm i.d. x 174 microm o.d.) with a nebulizer-free format, can relatively easily electrospray a large amount of make-up sheath liquid (5-10 microL/min) over the long term (more than 80 runs) with a high degree of stability. The interface also provides higher separation qualities and improved detection sensitivities compared with a conventional ion spray (IS) interface.

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Metabolome Analysis by Capillary Electrophoresis – Mass Spectrometry

Monton M.R.N., Soga, T.

J. Chromatogr. A 1168 ( 1-2 ) 237 - 246 2007.10

Research paper (scientific journal), Joint Work, Accepted

Capillary electrophoresis (CE)-mass spectrometry (MS), as an analytical platform, has made significant contributions in advancing metabolomics research, if still limited up to this time. This review, covering reports published between 1998 and 2006, describes how CE-MS has been used thus far in this field, with the majority of the works dealing with targeted metabolite analyses and only a small fraction using it in the comprehensive context. It also discusses how some of the key features of CE-MS were exploited in selected metabolomic applications.

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Polymer Entrapment in Polymerized Silicate for Preparing Highly Stable Capillary Coatings in CE and CE-MS

Monton, R.M.N., Tomita, M., *Soga, T., *Ishihama, Y.

Anal. Chem. 79 ( 20 ) 7838 - 7844 2007.10

Research paper (scientific journal), Joint Work, Accepted

An easy-to-implement capillary coating strategy based on polymer entrapment in the network of polymerized silicate is described. In this manner, cationic polymers are tightly fixed onto the inner wall of the capillary for electroosmotic flow control without necessitating complex surface modification chemistries. The resulting coated capillary exhibited good stability over a wide range of pH, good reproducibility, strong endurance in more than 300 electrophoretic runs, and tolerance of commonly employed organic solvent additives in CE. Applications in CE-MS analysis of biologically important anions as well as sample enrichment are shown. Additionally, it was used as a durable base for attachment of multiple layers of charged polymers on the wall, via electrostatic interaction with the preceding layer. Thus, two novel types of highly stable coated capillaries, one with anodic EOF and the other cathodic, were developed.

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Direct measurement of isotopomer of intracellular metabolites using capillary electrophoresis time-of-flight mass spectrometry for efficient metabolic flux analysis

Toya, Y., Ishii, N., Hirasawa, T., Naba, M., Hirai, K., Sugawara, K., Igarashi, S., Shimizu, K., Tomita, M., *Soga, T.

J. Chromatogr. A 1159 ( 1-2 ) 134 - 141 2007.08

Research paper (scientific journal), Joint Work, Accepted

We have developed a metabolic flux analysis method that is based on 13C-labeling patterns of the intracellular metabolites directly measured by capillary electrophoresis time-of-flight mass spectrometry (CE–TOFMS). The flux distribution of the central carbon metabolism in Escherichia coli was determined by this new approach and the results were compared with findings obtained by conventional GC–MS analysis based on isotopomer of the proteinogenic amino acids. There were some differences in estimation results between new approach using CE–TOFMS and conventional approach using GC–MS. These were thought to be attributable to variations in measured mass distributions between amino acids and the corresponding precursors and to differences in the sensitivity of the exchange coefficients to mass distributions. However, our CE–TOFMS method facilitates high-throughput flux analysis without requiring complicated sample preparation such as hydrolysis of proteins and derivatization of amino acids.

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P-BOSS: A New Filtering Method for Treasure Hunting in Metabolomics

Morohashi, M., Shimizu, K., Ohashi, Y., Abe, J., Mori, H., Tomita, M., Soga, T.

J. Chromatogr. A 1159 ( 1-2 ) 142 - 148 2007.08

Research paper (scientific journal), Joint Work, Accepted

Metabolomics is expected to boost data driven research. In biomarker discovery, powerful filtering methods to remove noise and outliers are essential for screening significant candidates from the huge volume of omic data. Here we propose a post-measurement peak filtering method (named P-BOSS) for CE electrospray ionization–time-of-flight MS (CE–TOFMS) data. Combining outlier detection method functions in parallel, we applied P-BOSS to the data using Escherichia coli knockout mutants of the tryptophan and purine biosynthesis pathways. As the result, P-BOSS showed remarkably superior performance, reducing 65% of all peaks, while leaving significant peaks.

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Analysis of Nucleotides by Pressure-Assisted Capillary Electrophoresis Mass Spectrometry Using Silanol Mask Technique

Soga, T.*, Ishikawa, T., Igarashi, S., Sugawara, K., Kakazu Y., Tomita, M.

J. Chromatogr. A 1159 ( 1-2 ) 125 - 133 2007.08

Research paper (scientific journal), Joint Work, Accepted

A method for the determination of nucleotides based on pressure-assisted capillary electrophoresis–electrospray ionization mass spectrometry (PACE–MS) is described. To prevent multi-phosphorylated species from adsorbing onto the fused-silica capillary, silanol groups were masked with phosphate ions by preconditioning the capillary with the background electrolyte containing phosphate. During preconditioning, nebulizer gas was turned off to avoid contamination of MS detector with phosphate ions. To detect nucleotides using the CE positive mode at a pH 7.5, it was necessary to apply air pressure to the inlet capillary during electrophoresis to supplement the electroosmotic flow (EOF) toward the cathode. Moreover, we exchanged the running electrolyte every analysis using the buffer replenishment system to obtain the required reproducibility. Under the optimized conditions, 14 phosphorylated species such as nucleotides, nicotinamide–adenine dinucleotides and coenzyme A (CoA) compounds were well determined in less than 20 min. The relative standard deviations (n = 6) of the method were better than 0.9% for migration times and between 1.7% and 8.1% for peak areas. The detection limits for these species were between 0.5 and 1.7 μmol/L with pressure injection of 50 mbar for 30 s (30 nL) at a signal-to-noise ratio of 3. This approach is robust and quantitative compared to the previous method, and its utility is demonstrated by the analysis of intracellular nucleotides and CoA compounds extracted from Escherichia coli wild type, pfkA and pfkB knockout mutants. The methodology was used to suggest that pfkA is the main functional enzyme.

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Model based definition of population heterogeneity and its effects on metabolism in sporulating Bacillus subtilis

Morohashi, M., *Ohashi, Y., Tani S., Ishii, K., Itaya, M., Nanamiya, H., Kawamura, F., Tomita, M., Soga, T.

J. Biochem. 142 ( 2 ) 183 - 191 2007.08

Research paper (scientific journal), Joint Work, Accepted

The soil bacterium Bacillus subtilis forms dormant, robust spores as a tactic to ensure survival under conditions of starvation. However, the sporulating culture includes sporulating and non-sporulating cells, because a portion of the cell population initiates sporulation in wild-type strain. We anticipated that the population effect must be considered carefully to analyse samples yielding population heterogeneity. We first built a mathematical model and simulated for signal transduction of the sporulation cue to see what mechanisms are responsible for generating the heterogeneity. The simulated results were confirmed experimentally, where heterogeneity is primarily modulated by negative feedback circuits, resulting in generation of a bistable response within the sporulating culture. We also confirmed that mutants relevant to negative feedback yield either sporulating or non-sporulating subpopulations. To see the effect of molecular mechanism between sporulating and non-sporulating cells in distinct manner, metabolome analysis was conducted using the above mutants. The metabolic profiles exhibited distinct characteristics with time regardless of whether sporulation was initiated or not. In addition, several distinct characteristics of metabolites were observed between strains, which was inconsistent with previously reported data. The results imply that careful consideration must be made in the interpretation of data obtained from cells yielding population heterogeneity.

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Multiple High-throughput Analyses Monitor the Response of E. coli to Perturbations

Ishii, N., Nakahigashi, K., Baba, T., Robert, M., Soga, T., Kanai, A., Hirasawa, T., Naba, M., Hirai, K., Hoque, A., Ho, P.Y., Kakazu, Y., Sugawara, K., Igarashi, S.,Harada, S., Masuda, T., Sugiyama, N., Togashi, T., Hasegawa, M., Takai, Y., Yugi, K., Arakawa, K., Iwata, N., Toya, Y., Nakayama, Y., Nishioka, T., Shimizu, K., Mori, H., *Tomita, M

Science 316 ( 5824 ) 593 - 597 2007.04

Research paper (scientific journal), Joint Work, Accepted

Analysis of cellular components at multiple levels of biological information can provide valuable functional insights. We performed multiple high-throughput measurements to study the response of Escherichia coli cells to genetic and environmental perturbations. Analysis of metabolic enzyme gene disruptants revealed unexpectedly small changes in messenger RNA and proteins for most disruptants. Overall, metabolite levels were also stable, reflecting the rerouting of fluxes in the metabolic network. In contrast, E. coli actively regulated enzyme levels to maintain a stable metabolic state in response to changes in growth rate. E. coli thus seems to use complementary strategies that result in a metabolic network robust against perturbations.

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Roles of Hemoglobin Allostery in Hypoxia-induced Metabolic Alterations in Erythrocytes: Simulation and Its Demonstration by Metabolome Analysis

Kinoshita, A., Tsukada, K., Soga, T., Hishiki, T., Ueno, Y., Nakayama, Y., Tomita, M., Suematsu, M

J. Biol. Chem. 282 ( 14 ) 10731 - 10741 2007.04

Research paper (scientific journal), Joint Work, Accepted

When erythrocytes are exposed to hypoxia, hemoglobin (Hb) stabilizes in the T-state by capturing 2,3-bisphosphoglycerate. This process could reduce the intracellular pool of glycolytic substrates, jeopardizing cellular energetics. Recent observations suggest that hypoxia-induced activation of glycolytic enzymes is correlated with their release from Band III (BIII) on the cell membrane. Based on these data, we developed a mathematical model of erythrocyte metabolism and compared hypoxia-induced differences in predicted activities of the enzymes, their products, and cellular energetics between models with and without the interaction of Hb with BIII. The models predicted that the allostery-dependent Hb interaction with BIII accelerates consumption of upstream glycolytic substrates such as glucose 6-phosphate and increases downstream products such as phosphoenolpyruvate. This prediction was consistent with metabolomic data from capillary electrophoresis mass spectrometry. The hypoxia-induced alterations in the metabolites resulted from acceleration of glycolysis, as judged by increased conversion of [13C]glucose to [13C]lactate. The allostery-dependent interaction of Hb with BIII appeared to contribute not only to maintenance of energy charge but also to further synthesis of 2,3-bisphosphoglycerate, which could help sustain stabilization of T-state Hb during hypoxia. Furthermore, such an activation of glycolysis was not observed when Hb was stabilized in R-state by treating the cells with CO. These results suggest that Hb allostery in erythrocytes serves as an O2-sensing trigger that drives glycolytic acceleration to stabilize intracellular energetics and promote the ability to release O2 from the cells.

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Visualization of Three-way Comparisons of Omics Data

Baran, R., Robert, M.*, Suematsu, M., Soga, T., Tomita, M.

BMC Bioinformatics 8 72 2007.03

Research paper (scientific journal), Joint Work, Accepted

Background: Density plot visualizations (also referred to as heat maps or color maps) are widely used in different fields including large-scale omics studies in biological sciences. However, the current color-codings limit the visualizations to single datasets or pairwise comparisons.
Results: We propose a color-coding approach for the representation of three-way comparisons. The approach is based on the HSB (hue, saturation, brightness) color model. The three compared values are assigned specific hue values from the circular hue range (e.g. red, green, and blue). The hue value representing the three-way comparison is calculated according to the distribution of three compared values. If two of the values are identical and one is different, the resulting hue is set to the characteristic hue of the differing value. If all three compared values are different, the resulting hue is selected from a color gradient running between the hues of the two most distant values (as measured by the absolute value of their difference) according to the relative position of the third value between the two. The saturation of the color representing the three-way comparison reflects the amplitude (or extent) of the numerical difference between the two most distant values according to a scale of interest. The brightness is set to a maximum value by default but can be used to encode additional information about the three-way comparison.
Conclusion: We propose a novel color-coding approach for intuitive visualization of three-way comparisons of omics data.

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MathDAMP: a package for differential analysis of metabolite profiles

Baran, R., Kochi, H., Saito, N., Suematsu, M., Soga, T., Nishioka, T., Robert, M., Tomita, M.

BMC Bioinformatics 7 530 2006.12

Research paper (scientific journal), Joint Work, Accepted

Background: With the advent of metabolomics as a powerful tool for both functional and biomarker discovery, the identification of specific differences between complex metabolite profiles is becoming a major challenge in the data analysis pipeline. The task remains difficult, given the datasets' size, complexity, and common shifts in migration (elution/retention) times between samples analyzed by hyphenated mass spectrometry methods.
Results: We present a Mathematica (Wolfram Research, Inc.) package MathDAMP (Mathematica package for Differential Analysis of Metabolite Profiles), which highlights differences between raw datasets acquired by hyphenated mass spectrometry methods by applying arithmetic operations to all corresponding signal intensities on a datapoint-by-datapoint basis. Peak identification and integration is thus bypassed and the results are displayed graphically. To facilitate direct comparisons, the raw datasets are automatically preprocessed and normalized in terms of both migration times and signal intensities. A combination of dynamic programming and global optimization is used for the alignment of the datasets along the migration time dimension. The processed datasets and the results of direct comparisons between them are visualized using density plots (axes represent migration time and m/z values while peaks appear as color-coded spots) providing an intuitive overall view. Various forms of comparisons and statistical tests can be applied to highlight subtle differences. Overlaid electropherograms (chromatograms) corresponding to the vicinities of the candidate differences from any result may be generated in a descending order of significance for visual confirmation. Additionally, a standard library table (a list of m/z values and migration times for known compounds) may be aligned and overlaid on the plots to allow easier identification of metabolites.
Conclusion: Our tool facilitates the visualization and identification of differences between complex metabolite profiles according to various criteria in an automated fashion and is useful for data-driven discovery of biomarkers and functional genomics.

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Prediction of liquid chromatographic retention times of peptides generated by protease digestion of the Escherichia coli proteome using artificial neural networks

Shinoda, K., Sugimoto, M., Yachie, N., Sugiyama, N., Masuda, T., Robert, M., Soga, T., Tomita, M.,

J. Proteome Res 5 ( 12 ) 3312 - 3317 2006.12

Research paper (scientific journal), Joint Work, Accepted

We developed a computational method to predict the retention times of peptides in HPLC using artificial neural networks (ANN). We performed stepwise multiple linear regressions and selected for ANN input amino acids that significantly affected the LC retention time. Unlike conventional linear models, the trained ANN accurately predicted the retention time of peptides containing up to 50 amino acid residues. In 834 peptides, there was a strong correlation (R2 = 0.928) between measured and predicted retention times. We demonstrated the utility of our method by the prediction of the retention time of 121,273 peptides resulting from LysC-digestion of the Escherichia coli proteome. Our approach is useful for the proteome-wide characterization of peptides and the identification of unknown peptide peaks obtained in proteome analysis.

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HybGFS: a hybrid method for genome-fingerprint scanning

Shinoda, K., Yachie, N., Masuda, T., Sugiyama, N., Sugimoto, M., Soga, T., Tomita, M.,

BMC Bioinformatics 7 479 2006.10

Research paper (scientific journal), Joint Work, Accepted

Background: Protein identification based on mass spectrometry (MS) has previously been performed using peptide mass fingerprinting (PMF) or tandem MS (MS/MS) database searching. However, these methods cannot identify proteins that are not already listed in existing databases. Moreover, the alternative approach of de novo sequencing requires costly equipment and the interpretation of complex MS/MS spectra. Thus, there is a need for novel high-throughput protein-identification methods that are independent of existing predefined protein databases.
Results: Here, we present a hybrid method for genome-fingerprint scanning, known as HybGFS. This technique combines genome sequence-based peptide MS/MS ion searching with liquid-chromatography elution-time (LC-ET) prediction, to improve the reliability of identification. The hybrid method allows the simultaneous identification and mapping of proteins without a priori information about their coding sequences. The current study used standard LC-MS/MS data to query an in silico-generated six-reading-frame translation and the enzymatic digest of an entire genome. Used in conjunction with precursor/product ion-mass searching, the LC-ETs increased confidence in the peptide-identification process and reduced the number of false-positive matches. The power of this method was demonstrated using recombinant proteins from the Escherichia coli K12 strain.
Conclusion: The novel hybrid method described in this study will be useful for the large-scale experimental confirmation of genome coding sequences, without the need for transcriptome-level expression analysis or costly MS database searching.

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