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

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. http://en.wikipedia.org/wiki/Metabolome

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): 373–378.

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 http://en.wikipedia.org/wiki/Metabolomics

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.http://en.wikipedia.org/wiki/Metabolomics

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”). http://www.genomicglossaries.com/content/printpage.asp?REF=/content/metabolic_engineering.asp

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”.http://www.genomicglossaries.com/content/printpage.asp?REF=/content/metabolic_engineering.asp

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, 196-201)

It has been estimated that plants produce more than 200,000 metabolites.(Fiehn, O. (2002) Plant Mol. Biol. 48, 155-171)

Role of post-genomic technologies in systems biology(Nicholson, Connelly, Lindon, Holmes Nature Rev. Drug Discov. 1, 153 – 161)

Real World

Inputs:Noxious agent

StressorMutation

Outputs:Biological end-points

Pathology

‘Omics World’

Gene expression

Protein profile

Metabolic profile

Real world end-point

Indicator for end-point

Indicator/ end-point

Integrative analysis of genes, proteins and metabolites(Fiehn O, Weckwerth W. 2003. Deciphering metabolic networks. Eur. J. Biochem. 270, 579–588)

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.

http://www.hos.ufl.edu/meteng/HOS6231-2002/METABOLICPROFILINGANDMETABOLOMICS2005.htm

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

(http://www.chemguide.co.uk/analysis/nmr/background.html#top)

MBioS 478/578 Bioinformatics Mark Lange

Technologies

Mass Spectrometry – General Introduction

(http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/MassSpec/masspec1.htm)

Fragmentation Patterns

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

Fourier-Transform Mass Spectrometry(http://www.bumc.bu.edu/Dept/Content.aspx?DepartmentID=385&PageID=7205)

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

Gas Chromatography – Mass Spectrometry

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)

ESI

http://www.bris.ac.uk/nerclsmsf/techniques/hplcms.html

APCI http://www.bris.ac.uk/nerclsmsf/techniques/hplcms.html

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

Nature Biotechnology 18, 1157 - 1161 (2000)

Metabolite profiling for plant functional genomics

Oliver Fiehn, Joachim Kopka, Peter Dörmann, Thomas Altmann, Richard N. Trethewey & Lothar Willmitzer

Nature Biotechnology 18, 1157 - 1161 (2000)

Metabolite profiling for plant functional genomics

Oliver Fiehn, Joachim Kopka, Peter Dörmann, Thomas Altmann, Richard N. Trethewey & Lothar Willmitzer

Comprehensive chemical derivatization for gas chromatography–mass spectrometry-based multi-targeted profiling of the major phytohormones

J. Chromatography A 993, 89-102 (2003)

Claudia Birkemeyer, Ania Kolasa and Joachim Kopka

ABA

Cytokinin

IAA

SA

MJA

GA

Anal. Chem., 77 (10), 3090 -3100, 2005

Automated 20 kpsi RPLC-MS and MS/MS with Chromatographic Peak Capacities of 1000-1500 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

MBioS 478/578 Bioinformatics Mark Lange

Lecture 23: Metabolomics Technology

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