Tomáš Pluskal

MZmine 2: Modular framework for processing, visualizing, and analyzing mass spectrometry-based molecular profile data

BackgroundMass spectrometry (MS) coupled with online separation methods is commonly applied for differential and quantitative profiling of biological samples in metabolomic as well as proteomic research. Such approaches are used for systems biology, functional genomics, and biomarker discovery, among others. An ongoing challenge of these molecular profiling approaches, however, is the development of better data processing methods. Here we introduce a new generation of a popular open-source data processing toolbox, MZmine 2.ResultsA key concept of the MZmine 2 software design is the strict separation of core functionality and data processing modules, with emphasis on easy usability and support for high-resolution spectra processing. Data processing modules take advantage of embedded visualization tools, allowing for immediate previews of parameter settings. Newly introduced functionality includes the identification of peaks using online databases, MSn data support, improved isotope pattern support, scatter plot visualization, and a new method for peak list alignment based on the random sample consensus (RANSAC) algorithm. The performance of the RANSAC alignment was evaluated using synthetic datasets as well as actual experimental data, and the results were compared to those obtained using other alignment algorithms.ConclusionsMZmine 2 is freely available under a GNU GPL license and can be obtained from the project website at: http://mzmine.sourceforge.net/. The current version of MZmine 2 is suitable for processing large batches of data and has been applied to both targeted and non-targeted metabolomic analyses.

Highly Accurate Chemical Formula Prediction Tool Utilizing High-Resolution Mass Spectra, MS/MS Fragmentation, Heuristic Rules, and Isotope Pattern Matching

Mass spectrometry is commonly applied to qualitatively and quantitatively profile small molecules, such as peptides, metabolites, or lipids. Modern mass spectrometers provide accurate measurements of mass-to-charge ratios of ions, with errors as low as 1 ppm. Even such high mass accuracy, however, is not sufficient to determine the unique chemical formula of each ion, and additional algorithms are necessary. Here we present a universal software tool for predicting chemical formulas from high-resolution mass spectrometry data, developed within the MZmine 2 framework. The tool is based on the use of a combination of heuristic techniques, including MS/MS fragmentation analysis and isotope pattern matching. The performance of the tool was evaluated using a real metabolomic data set obtained with the Orbitrap MS detector. The true formula was correctly determined as the highest-ranking candidate for 79% of the tested compounds. The novel isotope pattern-scoring algorithm outperformed a previously published method in 64% of the tested Orbitrap spectra. The software described in this manuscript is freely available and its source code can be accessed within the MZmine 2 source code repository.

Synergistic roles of the proteasome and autophagy for mitochondrial maintenance and chronological lifespan in fission yeast

Regulations of proliferation and quiescence in response to nutritional cues are important for medicine and basic biology. The fission yeast Schizosaccharomyces pombe serves as a model, owing to the shift of proliferating cells to the metabolically active quiescence (designate G0 phase hereafter) by responding to low nitrogen source. S. pombe G0 phase cells keep alive for months without growth and division. Nitrogen replenishment reinstates vegetative proliferation phase (designate VEG). Some 40 genes required for G0 maintenance were identified, but many more remain to be identified. We here show, using mutants, that the proteasome is required for maintaining G0 quiescence. Functional outcomes of proteasome in G0 and VEG phases appear to be distinct. Upon proteasome dysfunction, a number of antioxidant proteins and compounds responsive to ROS (reactive oxygen species) are produced. In addition, autophagy-mediated destruction of mitochondria occurs, which suppresses the loss of viability by eliminating ROS-generating mitochondria. These defensive responses are found in G0 but not in VEG, suggesting that the main function of proteasome in G0 phase homeostasis is to minimize ROS. Proteasome and autophagy are thus collaborative to support the lifespan of S. pombe G0 phase.

Specific biomarkers for stochastic division patterns and starvation-induced quiescence under limited glucose levels in fission yeast

Glucose as a source of energy is centrally important to our understanding of life. We investigated the cell division–quiescence behavior of the fission yeast Schizosaccharomyces pombe under a wide range of glucose concentrations (0–111 mm). The mode of S. pombe cell division under a microfluidic perfusion system was surprisingly normal under highly diluted glucose concentrations (5.6 mm, 1/20 of the standard medium, within human blood sugar levels). Division became stochastic, accompanied by a curious division‐timing inheritance, in 2.2–4.4 mm glucose. A critical transition from division to quiescence occurred within a narrow range of concentrations (2.2–1.7 mm). Under starvation (1.1 mm) conditions, cells were mostly quiescent and only a small population of cells divided. Under fasting (0 mm) conditions, division was immediately arrested with a short chronological lifespan (16 h). When cells were first glucose starved prior to fasting, they possessed a substantially extended lifespan (∼14 days). We employed a quantitative metabolomic approach for S. pombe cell extracts, and identified specific metabolites (e.g. biotin, trehalose, ergothioneine, S‐adenosyl methionine and CDP‐choline), which increased or decreased at different glucose concentrations, whereas nucleotide triphosphates, such as ATP, maintained high concentrations even under starvation. Under starvation, the level of S‐adenosyl methionine increased sharply, accompanied by an increase in methylated amino acids and nucleotides. Under fasting, cells rapidly lost antioxidant and energy compounds, such as glutathione and ATP, but, in fasting cells after starvation, these and other metabolites ensuring longevity remained abundant. Glucose‐starved cells became resistant to 40 mm H2O2 as a result of the accumulation of antioxidant compounds.

Genetic and metabolomic dissection of the ergothioneine and selenoneine biosynthetic pathway in the fission yeast, S. pombe, and construction of an overproduction system.

Ergothioneine is a small, sulfur-containing metabolite (229 Da) synthesized by various species of bacteria and fungi, which can accumulate to millimolar levels in tissues or cells (e.g. erythrocytes) of higher eukaryotes. It is commonly marketed as a dietary supplement due to its proposed protective and antioxidative functions. In this study we report the genes forming the two-step ergothioneine biosynthetic pathway in the fission yeast, Schizosaccharomyces pombe. We identified the first gene, egt1+ (SPBC1604.01), by sequence homology to previously published genes from Neurospora crassa and Mycobacterium smegmatis. We showed, using metabolomic analysis, that the Δegt1 deletion mutant completely lacked ergothioneine and its precursors (trimethyl histidine/hercynine and hercynylcysteine sulfoxide). Since the second step of ergothioneine biosynthesis has not been characterized in eukaryotes, we examined four putative homologs (Nfs1/SPBC21D10.11c, SPAC11D3.10, SPCC777.03c, and SPBC660.12c) of the corresponding mycobacterial enzyme EgtE. Among deletion mutants of these genes, only one (ΔSPBC660.12c, designated Δegt2) showed a substantial decrease in ergothioneine, accompanied by accumulation of its immediate precursor, hercynylcysteine sulfoxide. Ergothioneine-deficient strains exhibited no phenotypic defects during vegetative growth or quiescence. To effectively study the role of ergothioneine, we constructed an egt1+ overexpression system by replacing its native promoter with the nmt1+ promoter, which is inducible in the absence of thiamine. We employed three versions of the nmt1 promoter with increasing strength of expression and confirmed corresponding accumulations of ergothioneine. We quantified the intracellular concentration of ergothioneine in S. pombe (0.3, 157.4, 41.6, and up to 1606.3 µM in vegetative, nitrogen-starved, glucose-starved, and egt1+-overexpressing cells, respectively) and described its gradual accumulation under long-term quiescence. Finally, we demonstrated that the ergothioneine pathway can also synthesize selenoneine, a selenium-containing derivative of ergothioneine, when the culture medium is supplemented with selenium. We further found that selenoneine biosynthesis involves a novel intermediate compound, hercynylselenocysteine.

Metabolomic Analysis of Fission Yeast at the Onset of Nitrogen Starvation

Microorganisms naturally respond to changes in nutritional conditions by adjusting their morphology and physiology. The cellular response of the fission yeast S. pombe to nitrogen starvation has been extensively studied. Here, we report time course metabolomic analysis during one hour immediately after nitrogen starvation, prior to any visible changes in cell morphology except for a tiny increase of cell length per division cycle. We semi-quantitatively measured 75 distinct metabolites, 60% of which changed their level over 2-fold. The most significant changes occurred during the first 15 min, when trehalose, 2-oxoglutarate, and succinate increased, while purine biosynthesis intermediates rapidly diminished. At 30–60 min, free amino acids decreased, although several modified amino acids—including hercynylcysteine sulfoxide, a precursor to ergothioneine—accumulated. Most high-energy metabolites such as ATP, S-adenosyl-methionine or NAD+ remained stable during the whole time course. Very rapid metabolic changes such as the shut-off of purine biosynthesis and the rise of 2-oxoglutarate and succinate can be explained by the depletion of NH4Cl. The changes in the levels of key metabolites, particularly 2-oxoglutarate, might represent an important mechanistic step to trigger subsequent cellular regulations.

Impaired coenzyme A synthesis in fission yeast causes defective mitosis, quiescence-exit failure, histone hypoacetylation and fragile DNA.

Biosynthesis of coenzyme A (CoA) requires a five-step process using pantothenate and cysteine in the fission yeast Schizosaccharomyces pombe. CoA contains a thiol (SH) group, which reacts with carboxylic acid to form thioesters, giving rise to acyl-activated CoAs such as acetyl-CoA. Acetyl-CoA is essential for energy metabolism and protein acetylation, and, in higher eukaryotes, for the production of neurotransmitters. We isolated a novel S. pombe temperature-sensitive strain ppc1-537 mutated in the catalytic region of phosphopantothenoylcysteine synthetase (designated Ppc1), which is essential for CoA synthesis. The mutant becomes auxotrophic to pantothenate at permissive temperature, displaying greatly decreased levels of CoA, acetyl-CoA and histone acetylation. Moreover, ppc1-537 mutant cells failed to restore proliferation from quiescence. Ppc1 is thus the product of a super-housekeeping gene. The ppc1-537 mutant showed combined synthetic lethal defects with five of six histone deacetylase mutants, whereas sir2 deletion exceptionally rescued the ppc1-537 phenotype. In synchronous cultures, ppc1-537 cells can proceed to the S phase, but lose viability during mitosis failing in sister centromere/kinetochore segregation and nuclear division. Additionally, double-strand break repair is defective in the ppc1-537 mutant, producing fragile broken DNA, probably owing to diminished histone acetylation. The CoA-supported metabolism thus controls the state of chromosome DNA.

A transcript-specific eIF3 complex mediates global translational control of energy metabolism

The multi-subunit eukaryotic translation initiation factor eIF3 is thought to assist in the recruitment of ribosomes to mRNA. The expression of eIF3 subunits is frequently disrupted in human cancers, but the specific roles of individual subunits in mRNA translation and cancer remain elusive. Using global transcriptomic, proteomic, and metabolomic profiling, we found a striking failure of Schizosaccharomyces pombe cells lacking eIF3e and eIF3d to synthesize components of the mitochondrial electron transport chain, leading to a defect in respiration, endogenous oxidative stress, and premature aging. Energy balance was maintained, however, by a switch to glycolysis with increased glucose uptake, upregulation of glycolytic enzymes, and strict dependence on a fermentable carbon source. This metabolic regulatory function appears to be conserved in human cells where eIF3e binds metabolic mRNAs and promotes their translation. Thus, via its eIF3d-eIF3e module, eIF3 orchestrates an mRNA-specific translational mechanism controlling energy metabolism that may be disrupted in cancer.

Metabolism of skin-absorbed resveratrol into its glucuronized form in mouse skin.

Resveratrol (RESV) is a plant polyphenol, which is thought to have beneficial metabolic effects in laboratory animals as well as in humans. Following oral administration, RESV is immediately catabolized, resulting in low bioavailability. This study compared RESV metabolites and their tissue distribution after oral uptake and skin absorption. Metabolomic analysis of various mouse tissues revealed that RESV can be absorbed and metabolized through skin. We detected sulfated and glucuronidated RESV metabolites, as well as dihydroresveratrol. These metabolites are thought to have lower pharmacological activity than RESV. Similar quantities of most RESV metabolites were observed 4 h after oral or skin administration, except that glucuronidated RESV metabolites were more abundant in skin after topical RESV application than after oral administration. This result is consistent with our finding of glucuronidated RESV metabolites in cultured skin cells. RESV applied to mouse ears significantly suppressed inflammation in the TPA inflammation model. The skin absorption route could be a complementary, potent way to achieve therapeutic effects with RESV.

Klf1, a C2H2 Zinc Finger-Transcription Factor, Is Required for Cell Wall Maintenance during Long-Term Quiescence in Differentiated G0 Phase

Fission yeast, Schizoaccharomyces pombe, is a model for studying cellular quiescence. Shifting to a medium that lacks a nitrogen-source induces proliferative cells to enter long-term G0 quiescence. Klf1 is a Krüppel-like transcription factor with a 7-amino acid Cys2His2-type zinc finger motif. The deletion mutant, ∆klf1, normally divides in vegetative medium, but proliferation is not restored after long-term G0 quiescence. Cell biologic, transcriptomic, and metabolomic analyses revealed a unique phenotype of the ∆klf1 mutant in quiescence. Mutant cells had diminished transcripts related to signaling molecules for switching to differentiation; however, proliferative metabolites for cell-wall assembly and antioxidants had significantly increased. Further, the size of ∆klf1 cells increased markedly during quiescence due to the aberrant accumulation of Calcofluor-positive, chitin-like materials beneath the cell wall. After 4 weeks of quiescence, reversible proliferation ability was lost, but metabolism was maintained. Klf1 thus plays a role in G0 phase longevity by enhancing the differentiation signal and suppressing metabolism for growth. If Klf1 is lost, S. pombe fails to maintain a constant cell size and normal cell morphology during quiescence.