Lecture 23: Metabolomics Technology
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1 MBioS 478/578 Bioinformatics Mark Lange Lecture 23: Metabolomics Technology Definitions and Background Technologies Nuclear Magnetic Resonance Mass Spectrometry General Introduction Fourier-Transform Mass Spectrometry Gas Chromatography Mass Spectrometry Liquid Chromatography Mass Spectrometry Capillary Electrophoresis Mass Spectrometry Applications
2 MBioS 478/578 Bioinformatics Mark Lange Definitions and Background The metabolome represents the collection of all metabolites in a biological organism, which are the end products of its gene expression. First use of the term "metabolome" in the literature: Oliver, S.G., Winson, M.K., Kell, D.B., Baganz, F. (1998). Systematic functional analysis of the yeast genome, Trends Biotechnol. 16(10): Metabolomics is the "systematic study of the unique chemical fingerprints that specific cellular processes leave behind" - specifically, the study of their small-molecule metabolite profiles The word metabonomics is also used, particularly in the context of drug toxicity assessment or when NMR-based techniques are used. There is some disagreement over the exact differences between 'metabolomics' and 'metabonomics'; in general, the term 'metabolomics' is more commonly used.
3 MBioS 478/578 Bioinformatics Mark Lange Definitions and Background For investigators of selected biochemical pathways, it is also often not necessary to view the effects of perturbation on all branches of metabolism. Instead, the analytical procedure can be focused on a smaller number of pre- defined metabolites. Sample preparation and data acquisition can be focused on the chemical properties of these compounds with the chance to reduce matrix effects. This process is called metabolite profiling (or sometimes metabolic profiling ). For functional genomic or plant breeding programmes, as well as for diagnostic usage in industrial or clinical routines, it might not be necessary to determine the levels of all metabolites individually. Instead, a rapid classification of samples according to their origin or their biological relevance might be more adequate in order to maintain a high throughput. This process can be called metabolic fingerprinting. General problems encountered when characterizing the metabolome are the highly complex nature and the enormous chemical diversity of physiologically relevant metabolites. At present, the metabolome cannot be computed from the genome. (Trethewey, R.N. (2004) Curr. Opin. Plant Biol. 7, ) It has been estimated that plants produce more than 200,000 metabolites. (Fiehn, O. (2002) Plant Mol. Biol. 48, )
4 Role of post-genomic technologies in systems biology (Nicholson, Connelly, Lindon, Holmes Nature Rev. Drug Discov. 1, ) Real World Omics World Inputs: Noxious agent Stressor Mutation Gene expression Indicator for end-point Outputs: Biological end-points Pathology Protein profile Metabolic profile Indicator/ end-point Real world end-point
5 Integrative analysis of genes, proteins and metabolites (Fiehn O, Weckwerth W Deciphering metabolic networks. Eur. J. Biochem. 270, )
6 Problems: No single extraction procedure works for all metabolites because conditions that stabilize one type of compound will destroy other types or interfere with their analysis. Examples: nucleotides (ATP, S-adenosylmethionine, etc) are extracted using perchloric acid, a strong acid and oxidizing agent that attacks other compounds; alkaloids are extracted in alkaline media that destroy aldehydic compounds; thiols and 2-oxo acids are so labile that they must be extracted using aggressive reagents that convert them to stable derivatives but which interfere with determination of other analytes. Therefore the extraction protocol has to be tailored to the metabolites to be profiled. In practice, these considerations mean that metabolic profiling has up to now largely been confined to fairly stable compounds that can be extracted together. These include major primary metabolites (sugars, sugar phosphates, amino acids, and organic acids) and certain secondary metabolites (e.g., phenylpropanoids, alkaloids). The most comprehensive profiling covers a few hundred such compounds, the majority of which remain unidentified. Many crucial metabolites are therefore currently being missed.
7 MBioS 478/578 Bioinformatics Mark Lange Technologies Nuclear Magnetic Resonance Advantages Can be coupled with liquid chromatography (increased resolution) Applicable with a wide range of metabolites Can be used to determine structure of metabolites Disadvantages Time-consuming for most nuclei Relatively insensitive not suited for low abundance compounds
8 (
9 MBioS 478/578 Bioinformatics Mark Lange Technologies Mass Spectrometry General Introduction (
10 Fragmentation Patterns
11 MBioS 478/578 Bioinformatics Mark Lange Technologies Fourier-Transform Mass Spectrometry Advantages Crude extracts can be injected directly (no chromatography separation) Very fast fingerprinting method Capable of ultra-high resolution (permits mass to be determined to four or five decimal places Empirical molecular formulae can be assigned to peaks Disadvantages $ $ $ High maintenance Inability to distinguish isomers of the same molecular mass
12 Fourier-Transform Mass Spectrometry (
13
14 MBioS 478/578 Bioinformatics Mark Lange Technologies Gas Chromatography Mass Spectrometry Advantages Very high chromatographic resolving power Good selection of stationary phases Readily interfaced to a mass spectrometer Disadvantages Compounds must be sufficiently volatile Compounds must be thermally stable Limited to nonpolar and slightly polar molecules
15 Gas Chromatography Mass Spectrometry
16 MBioS 478/578 Bioinformatics Mark Lange Technologies Liquid Chromatography Mass Spectrometry Advantages Capable of analyzing a wide range of metabolites (thermally labile, high polarity, high molecular mass) Good selection of stationary phases Readily interfaced to a mass spectrometer Disadvantages Fragmentation rules not well established MS libraries missing Limited resolution (new developments: UPLC, chip-lc)
17 ESI
18 APCI
19 MBioS 478/578 Bioinformatics Mark Lange Technologies Capillary Electrophoresis Mass Spectrometry Advantages Short analysis times High separation efficiency Enables chiral separations Disadvantages Methods development difficult MS libraries missing
20
21 Nature Biotechnology 18, (2000) Metabolite profiling for plant functional genomics Oliver Fiehn, Joachim Kopka, Peter Dörmann, Thomas Altmann, Richard N. Trethewey & Lothar Willmitzer
22 Nature Biotechnology 18, (2000) Metabolite profiling for plant functional genomics Oliver Fiehn, Joachim Kopka, Peter Dörmann, Thomas Altmann, Richard N. Trethewey & Lothar Willmitzer
23 J. Chromatography A 993, (2003) Comprehensive chemical derivatization for gas chromatography mass spectrometry-based multi-targeted profiling of the major phytohormones Claudia Birkemeyer, Ania Kolasa and Joachim Kopka ABA Cytokinin IAA SA MJA GA
24 Anal. Chem., 77 (10), , 2005 Automated 20 kpsi RPLC-MS and MS/MS with Chromatographic Peak Capacities of and Capabilities in Proteomics and Metabolomics Y. Shen, R. Zhang, R.J. Moore, J. Kim, T.O. Metz, K.K. Hixson, R. Zhao, E.A. Livesay, H.R. Udseth, R.D. Smith
25 MBioS 478/578 Bioinformatics Mark Lange Lecture 23: Metabolomics Technology Any questions?
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