Quantitative metabolomics based on gas chromatography mass spectrometry: status and perspectives

Metabolomics involves the unbiased quantitative and qualitative analysis of the complete set of metabolites present in cells, body fluids and tissues (the metabolome). By analyzing differences between metabolomes using biostatistics (multivariate data analysis; pattern recognition), metabolites relevant to a specific phenotypic characteristic can be identified. However, the reliability of the analytical data is a prerequisite for correct biological interpretation in metabolomics analysis. In this review the challenges in quantitative metabolomics analysis with regards to analytical as well as data preprocessing steps are discussed. Recommendations are given on how to optimize and validate comprehensive silylation-based methods from sample extraction and derivatization up to data preprocessing and how to perform quality control during metabolomics studies. The current state of method validation and data preprocessing methods used in published literature are discussed and a perspective on the future research necessary to obtain accurate quantitative data from comprehensive GC-MS data is provided

Phylogeny in Aid of the Present and Novel Microbial Lineages: Diversity in Bacillus

Bacillus represents microbes of high economic, medical and biodefense importance. Bacillus strain identification based on 16S rRNA sequence analyses is invariably limited to species level. Secondly, certain discrepancies exist in the segregation of Bacillus subtilis strains. In the RDP/NCBI databases, out of a total of 2611 individual 16S rDNA sequences belonging to the 175 different species of the genus Bacillus, only 1586 have been identified up to species level. 16S rRNA sequences of Bacillus anthracis (153 strains), B. cereus (211 strains), B. thuringiensis (108 strains), B. subtilis (271 strains), B. licheniformis (131 strains), B. pumilus (83 strains), B. megaterium (47 strains), B. sphaericus (42 strains), B. clausii (39 strains) and B. halodurans (36 strains) were considered for generating species-specific framework and probes as tools for their rapid identification. Phylogenetic segregation of 1121, 16S rDNA sequences of 10 different Bacillus species in to 89 clusters enabled us to develop a phylogenetic frame work of 34 representative sequences. Using this phylogenetic framework, 305 out of 1025, 16S rDNA sequences presently classified as Bacillus sp. could be identified up to species level. This identification was supported by 20 to 30 nucleotides long signature sequences and in silico restriction enzyme analysis specific to the 10 Bacillus species. This integrated approach resulted in identifying around 30% of Bacillus sp. up to species level and revealed that B. subtilis strains can be segregated into two phylogenetically distinct groups, such that one of them may be renamed

Advances in structure elucidation of small molecules using mass spectrometry

The structural elucidation of small molecules using mass spectrometry plays an important role in modern life sciences and bioanalytical approaches. This review covers different soft and hard ionization techniques and figures of merit for modern mass spectrometers, such as mass resolving power, mass accuracy, isotopic abundance accuracy, accurate mass multiple-stage MS(n) capability, as well as hybrid mass spectrometric and orthogonal chromatographic approaches. The latter part discusses mass spectral data handling strategies, which includes background and noise subtraction, adduct formation and detection, charge state determination, accurate mass measurements, elemental composition determinations, and complex data-dependent setups with ion maps and ion trees. The importance of mass spectral library search algorithms for tandem mass spectra and multiple-stage MS(n) mass spectra as well as mass spectral tree libraries that combine multiple-stage mass spectra are outlined. The successive chapter discusses mass spectral fragmentation pathways, biotransformation reactions and drug metabolism studies, the mass spectral simulation and generation of in silico mass spectra, expert systems for mass spectral interpretation, and the use of computational chemistry to explain gas-phase phenomena. A single chapter discusses data handling for hyphenated approaches including mass spectral deconvolution for clean mass spectra, cheminformatics approaches and structure retention relationships, and retention index predictions for gas and liquid chromatography. The last section reviews the current state of electronic data sharing of mass spectra and discusses the importance of software development for the advancement of structure elucidation of small molecules

Metabolomics in Animal Cell Culture

Metabolomics is defined as a global quantitative assessment of metabolites within a biological system. Metabolic profiling of cell cultures has many potential applications as well as advantages to currently utilized methods for cell-line testing. Metabolite concentrations represent sensitive markers of genomic changes and responses of cells to external stimuli. Effects of drugs or toxins on cell cultures can be observed through the changes in metabolite concentrations. When cell cultures used for production of various biomolecules metabolomics can aid in optimization of cell growth. Metabolomics can also be used as a method for routine monitoring of extracellular metabolic changes in real time measurements. Nuclear magnetic resonance spectroscopy and mass spectrometry are major analytical platforms used for metabolomics measurements. These methods provide detailed, non-biased and highly complementary chemical analyses of metabolic changes within cells (fingerprint) and in excreted metabolites (footprint). This chapter provides review of current applications of metabolomics in cell cultures with an overview of experimental and data analysis methodologies.Peer reviewed: YesNRC publication: Ye