Metabolomics
Encyclopedia
Metabolomics is the scientific study of chemical processes involving metabolite
s. Specifically, metabolomics is the "systematic study of the unique chemical fingerprints that specific cellular processes leave behind", the study of their small-molecule metabolite
profiles. The metabolome
represents the collection of all metabolites in a biological cell, tissue, organ or organism, which are the end products of cellular processes. Thus, while mRNA gene expression
data and proteomic
analyses do not tell the whole story of what might be happening in a cell, metabolic profiling can give an instantaneous snapshot of the physiology of that cell. One of the challenges of systems biology
and functional genomics
is to integrate proteomic
, transcriptomic, and metabolomic information to give a more complete picture of living organisms.
The concept that individuals might have a “metabolic profile” that could be reflected in the makeup of their biological fluids was introduced by Roger Williams in the late 1940s, who used paper chromatography to suggest characteristic metabolic patterns in urine and saliva were associated with diseases such as schizophrenia. However, it was only through technological advancements in the 1960s and 1970s that it became feasible to quantitatively (as opposed to qualitatively) measure metabolic profiles. The term “metabolic profile” was introduced by Horning, et al. in 1971 after they demonstrated that gas chromatography-mass spectrometry
(GC-MS) could be used to measure compounds present in human urine and tissue extracts. The Horning group, along with that of Linus Pauling and Arthur Robinson led the development of GC-MS methods to monitor the metabolites present in urine through the 1970s.
Concurrently, NMR spectroscopy, which was discovered in the 1940s, was also undergoing rapid advances. In 1974, Seeley et al. demonstrated the utility of using NMR to detect metabolites in unmodified biological samples. This first study on muscle highlighted the value of NMR in that it was determined that 90% of cellular ATP is complexed with magnesium. As sensitivity has improved with the evolution of higher magnetic field strengths and magic-angle spinning, NMR continues to be a leading analytical tool to investigate metabolism. Recent efforts to utilize NMR for metabolomics have been largely driven by the laboratory of Dr. Jeremy Nicholson at Birkbeck College, University of London and later at Imperial College London
. In 1984, Nicholson showed 1H NMR spectroscopy could potentially be used to diagnose and treat diabetes mellitus, and later pioneered the application of pattern recognition methods to NMR spectroscopic data.
In 2005, the first metabolomics web database, METLIN
, for characterizing human metabolites was developed in the Siuzdak laboratory at The Scripps Research Institute
and contained over 5,000 metabolites and tandem mass spectral data. , METLIN
contains over 40,000 metabolites as well as the largest repository of tandem mass spectrometry data in metabolomics.
On 23 January 2007, the Human Metabolome Project, led by Dr. David Wishart of the University of Alberta, Canada, completed the first draft of the human metabolome, consisting of a database of approximately 2500 metabolites, 1200 drugs and 3500 food components. Similar projects have been underway in several plant species, most notably Medicago truncatula and Arabidopsis thaliana for several years.
As late as mid-2010, metabolomics was still considered an "emerging field". Further, it was noted that further progress in the field depended in large part, through addressing otherwise "irresolvable technical challenges", by technical evolution of mass spectrometry
instrumentation.
; like the transcriptome and the proteome, the metabolome is dynamic, changing from second to second. Although the metabolome can be defined readily enough, it is not currently possible to analyse the entire range of metabolites by a single analytical method. The first metabolite database(called METLIN
) for searching m/z values from mass spectrometry data was developed by scientists at The Scripps Research Institute
in 2005. In January 2007, scientists at the University of Alberta
and the University of Calgary
completed the first draft of the human metabolome. They catalogued approximately 2500 metabolite
s, 1200 drug
s and 3500 food components that can be found in the human body, as reported in the literature. This information, available at the Human Metabolome Database (www.hmdb.ca) and based on analysis of information available in the current scientific literature, is far from complete. In contrast, much more is known about the metabolomes of other organisms. For example, over 50,000 metabolites have been characterized from the plant kingdom, and many thousands of metabolites have been identified and/or characterized from single plants.
. Within the context of metabolomics, a metabolite is usually defined as any molecule less than 1 kDa in size. However, there are exceptions to this depending on the sample and detection method. For example, macromolecules such as lipoprotein
s and albumin
are reliably detected in NMR-based metabolomics studies of blood plasma. In plant-based metabolomics, it is common to refer to "primary" and "secondary" metabolites. A primary metabolite is directly involved in the normal growth, development, and reproduction. A secondary metabolite
is not directly involved in those processes, but usually has important ecological
function. Examples include antibiotics and pigment
s. By contrast, in human-based metabolomics, it is more common to describe metabolites as being either endogenous
(produced by the host organism) or exogenous
. Metabolites of foreign substances such as drugs are termed xenometabolites.
The metabolome
forms a large network of metabolic reactions, where outputs from one enzymatic chemical reaction
are inputs to other chemical reactions. Such systems have been described as hypercycle
s.
Metabonomics is defined as "the quantitative measurement of the dynamic multiparametric metabolic response of living systems to pathophysiological stimuli or genetic modification". The word origin is from the Greek meta meaning change and nomos meaning a rule set or set of laws. This approach was pioneered by Jeremy Nicholson at Imperial College London
and has been used in toxicology, disease diagnosis and a number of other fields. Historically, the metabonomics approach was one of the first methods to apply the scope of systems biology to studies of metabolism.
There has been some disagreement over the exact differences between 'metabolomics' and 'metabonomics'. The difference between the two terms is not related to choice of analytical platform: although metabonomics is more associated with NMR spectroscopy
and metabolomics with mass spectrometry
-based techniques, this is simply because of usages amongst different groups that have popularized the different terms. While there is still no absolute agreement, there is a growing consensus that 'metabolomics' places a greater emphasis on metabolic profiling at a cellular or organ level and is primarily concerned with normal endogenous metabolism. 'Metabonomics' extends metabolic profiling to include information about perturbations of metabolism caused by environmental factors (including diet and toxins), disease processes, and the involvement of extragenomic influences, such as gut microflora. This is not a trivial difference; metabolomic studies should, by definition, exclude metabolic contributions from extragenomic sources, because these are external to the system being studied. However, in practice, within the field of human disease research there is still a large degree of overlap in the way both terms are used, and they are often in effect synonymous.
data. For mass spectrometry data, software is available that identifies molecules that vary in subject groups on the basis of mass and sometimes retention time depending on the experimental design. The first comprehensive software to analyze global mass spectrometry-based metabolomics datasets was developed by the Siuzdak laboratory at The Scripps Research Institute
in 2006. This software, called XCMS, is freely available, has over 20,000 downloads since its inception in 2006, and is one of the most widely cited mass spectrometry-based metabolomics software programs in scientific literature. Other popular metabolomics programs for mass spectral analysis are MZmine, MetAlign, MathDAMP, which also compensate for retention time deviation during sample analysis. LCMStats is another R package for detailed analysis of liquid chromatography mass spectrometry(LCMS)data and is helpful in identification of co-eluting ions especially isotopologues from a complicated metabolic profile. It combines xcms package functions and can be used to apply many statistical functions for correcting detector saturation
using coates correction and creating heat plots. Metabolomics data may also be analyzed by statistical projection (chemometrics
) methods such as principal components analysis
and partial least squares regression
.
http://dbkgroup.org/dave_files/AnalystMetabolicFingerprinting2006.pdf
*Bundy JG, Davey MP, Viant, MR. 2009. Environmental Metabolomics: A Critical Review and Future Perspectives. Metabolomics 5: 3-21.
Metabolite
Metabolites are the intermediates and products of metabolism. The term metabolite is usually restricted to small molecules. A primary metabolite is directly involved in normal growth, development, and reproduction. Alcohol is an example of a primary metabolite produced in large-scale by industrial...
s. Specifically, metabolomics is the "systematic study of the unique chemical fingerprints that specific cellular processes leave behind", the study of their small-molecule metabolite
Metabolite
Metabolites are the intermediates and products of metabolism. The term metabolite is usually restricted to small molecules. A primary metabolite is directly involved in normal growth, development, and reproduction. Alcohol is an example of a primary metabolite produced in large-scale by industrial...
profiles. The metabolome
Metabolome
Metabolome refers to the complete set of small-molecule metabolites to be found within a biological sample, such as a single organism...
represents the collection of all metabolites in a biological cell, tissue, organ or organism, which are the end products of cellular processes. Thus, while mRNA gene expression
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
data and proteomic
Proteomics
Proteomics is the large-scale study of proteins, particularly their structures and functions. Proteins are vital parts of living organisms, as they are the main components of the physiological metabolic pathways of cells. The term "proteomics" was first coined in 1997 to make an analogy with...
analyses do not tell the whole story of what might be happening in a cell, metabolic profiling can give an instantaneous snapshot of the physiology of that cell. One of the challenges of systems biology
Systems biology
Systems biology is a term used to describe a number of trends in bioscience research, and a movement which draws on those trends. Proponents describe systems biology as a biology-based inter-disciplinary study field that focuses on complex interactions in biological systems, claiming that it uses...
and functional genomics
Functional genomics
Functional genomics is a field of molecular biology that attempts to make use of the vast wealth of data produced by genomic projects to describe gene functions and interactions...
is to integrate proteomic
Proteomics
Proteomics is the large-scale study of proteins, particularly their structures and functions. Proteins are vital parts of living organisms, as they are the main components of the physiological metabolic pathways of cells. The term "proteomics" was first coined in 1997 to make an analogy with...
, transcriptomic, and metabolomic information to give a more complete picture of living organisms.
Origins
The idea that biological fluids reflect the health of an individual has existed for a long time. Ancient Chinese doctors used ants for the evaluation of urine of patients to detect whether the urine contained high levels of glucose, and hence detect diabetes. In the Middle Ages, “urine charts” were used to link the colours, tastes and smells of urine to various medical conditions, which are metabolic in origin.The concept that individuals might have a “metabolic profile” that could be reflected in the makeup of their biological fluids was introduced by Roger Williams in the late 1940s, who used paper chromatography to suggest characteristic metabolic patterns in urine and saliva were associated with diseases such as schizophrenia. However, it was only through technological advancements in the 1960s and 1970s that it became feasible to quantitatively (as opposed to qualitatively) measure metabolic profiles. The term “metabolic profile” was introduced by Horning, et al. in 1971 after they demonstrated that gas chromatography-mass spectrometry
Gas chromatography-mass spectrometry
Gas chromatography–mass spectrometry is a method that combines the features of gas-liquid chromatography and mass spectrometry to identify different substances within a test sample. Applications of GC-MS include drug detection, fire investigation, environmental analysis, explosives investigation,...
(GC-MS) could be used to measure compounds present in human urine and tissue extracts. The Horning group, along with that of Linus Pauling and Arthur Robinson led the development of GC-MS methods to monitor the metabolites present in urine through the 1970s.
Concurrently, NMR spectroscopy, which was discovered in the 1940s, was also undergoing rapid advances. In 1974, Seeley et al. demonstrated the utility of using NMR to detect metabolites in unmodified biological samples. This first study on muscle highlighted the value of NMR in that it was determined that 90% of cellular ATP is complexed with magnesium. As sensitivity has improved with the evolution of higher magnetic field strengths and magic-angle spinning, NMR continues to be a leading analytical tool to investigate metabolism. Recent efforts to utilize NMR for metabolomics have been largely driven by the laboratory of Dr. Jeremy Nicholson at Birkbeck College, University of London and later at Imperial College London
Imperial College London
Imperial College London is a public research university located in London, United Kingdom, specialising in science, engineering, business and medicine...
. In 1984, Nicholson showed 1H NMR spectroscopy could potentially be used to diagnose and treat diabetes mellitus, and later pioneered the application of pattern recognition methods to NMR spectroscopic data.
In 2005, the first metabolomics web database, METLIN
METLIN
The METLIN Metabolomics Database is a repository of metabolite information as well as tandem mass spectrometry data. The information is provided to facilitate metabolomics experiments.METLIN is a metabolomics database containing over 42,000 structures...
, for characterizing human metabolites was developed in the Siuzdak laboratory at The Scripps Research Institute
The Scripps Research Institute
The Scripps Research Institute is an American medical research facility that focuses on research in the basic biomedical sciences. Headquartered in La Jolla, California, with a sister facility in Jupiter, Florida, the institute is home to 3,000 scientists, technicians, graduate students, and...
and contained over 5,000 metabolites and tandem mass spectral data. , METLIN
METLIN
The METLIN Metabolomics Database is a repository of metabolite information as well as tandem mass spectrometry data. The information is provided to facilitate metabolomics experiments.METLIN is a metabolomics database containing over 42,000 structures...
contains over 40,000 metabolites as well as the largest repository of tandem mass spectrometry data in metabolomics.
On 23 January 2007, the Human Metabolome Project, led by Dr. David Wishart of the University of Alberta, Canada, completed the first draft of the human metabolome, consisting of a database of approximately 2500 metabolites, 1200 drugs and 3500 food components. Similar projects have been underway in several plant species, most notably Medicago truncatula and Arabidopsis thaliana for several years.
As late as mid-2010, metabolomics was still considered an "emerging field". Further, it was noted that further progress in the field depended in large part, through addressing otherwise "irresolvable technical challenges", by technical evolution of mass spectrometry
Mass spectrometry
Mass spectrometry is an analytical technique that measures the mass-to-charge ratio of charged particles.It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and...
instrumentation.
Metabolome
Metabolome refers to the complete set of small-molecule metabolites (such as metabolic intermediates, hormones and other signaling molecules, and secondary metabolites) to be found within a biological sample, such as a single organism. The word was coined in analogy with transcriptomics and proteomicsProteomics
Proteomics is the large-scale study of proteins, particularly their structures and functions. Proteins are vital parts of living organisms, as they are the main components of the physiological metabolic pathways of cells. The term "proteomics" was first coined in 1997 to make an analogy with...
; like the transcriptome and the proteome, the metabolome is dynamic, changing from second to second. Although the metabolome can be defined readily enough, it is not currently possible to analyse the entire range of metabolites by a single analytical method. The first metabolite database(called METLIN
METLIN
The METLIN Metabolomics Database is a repository of metabolite information as well as tandem mass spectrometry data. The information is provided to facilitate metabolomics experiments.METLIN is a metabolomics database containing over 42,000 structures...
) for searching m/z values from mass spectrometry data was developed by scientists at The Scripps Research Institute
The Scripps Research Institute
The Scripps Research Institute is an American medical research facility that focuses on research in the basic biomedical sciences. Headquartered in La Jolla, California, with a sister facility in Jupiter, Florida, the institute is home to 3,000 scientists, technicians, graduate students, and...
in 2005. In January 2007, scientists at the University of Alberta
University of Alberta
The University of Alberta is a public research university located in Edmonton, Alberta, Canada. Founded in 1908 by Alexander Cameron Rutherford, the first premier of Alberta and Henry Marshall Tory, its first president, it is widely recognized as one of the best universities in Canada...
and the University of Calgary
University of Calgary
The University of Calgary is a public research university located in Calgary, Alberta, Canada. Founded in 1966 the U of C is composed of 14 faculties and more than 85 research institutes and centres.More than 25,000 undergraduate and 5,500 graduate students are currently...
completed the first draft of the human metabolome. They catalogued approximately 2500 metabolite
Metabolite
Metabolites are the intermediates and products of metabolism. The term metabolite is usually restricted to small molecules. A primary metabolite is directly involved in normal growth, development, and reproduction. Alcohol is an example of a primary metabolite produced in large-scale by industrial...
s, 1200 drug
Drug
A drug, broadly speaking, is any substance that, when absorbed into the body of a living organism, alters normal bodily function. There is no single, precise definition, as there are different meanings in drug control law, government regulations, medicine, and colloquial usage.In pharmacology, a...
s and 3500 food components that can be found in the human body, as reported in the literature. This information, available at the Human Metabolome Database (www.hmdb.ca) and based on analysis of information available in the current scientific literature, is far from complete. In contrast, much more is known about the metabolomes of other organisms. For example, over 50,000 metabolites have been characterized from the plant kingdom, and many thousands of metabolites have been identified and/or characterized from single plants.
Metabolites
Metabolites are the intermediates and products of metabolismMetabolism
Metabolism is the set of chemical reactions that happen in the cells of living organisms to sustain life. These processes allow organisms to grow and reproduce, maintain their structures, and respond to their environments. Metabolism is usually divided into two categories...
. Within the context of metabolomics, a metabolite is usually defined as any molecule less than 1 kDa in size. However, there are exceptions to this depending on the sample and detection method. For example, macromolecules such as lipoprotein
Lipoprotein
A lipoprotein is a biochemical assembly that contains both proteins and lipids water-bound to the proteins. Many enzymes, transporters, structural proteins, antigens, adhesins, and toxins are lipoproteins...
s and albumin
Albumin
Albumin refers generally to any protein that is water soluble, which is moderately soluble in concentrated salt solutions, and experiences heat denaturation. They are commonly found in blood plasma, and are unique to other blood proteins in that they are not glycosylated...
are reliably detected in NMR-based metabolomics studies of blood plasma. In plant-based metabolomics, it is common to refer to "primary" and "secondary" metabolites. A primary metabolite is directly involved in the normal growth, development, and reproduction. A secondary metabolite
Secondary metabolite
Secondary metabolites are organic compounds that are not directly involved in the normal growth, development, or reproduction of an organism. Unlike primary metabolites, absence of secondary metabolities does not result in immediate death, but rather in long-term impairment of the organism's...
is not directly involved in those processes, but usually has important ecological
Ecology
Ecology is the scientific study of the relations that living organisms have with respect to each other and their natural environment. Variables of interest to ecologists include the composition, distribution, amount , number, and changing states of organisms within and among ecosystems...
function. Examples include antibiotics and pigment
Pigment
A pigment is a material that changes the color of reflected or transmitted light as the result of wavelength-selective absorption. This physical process differs from fluorescence, phosphorescence, and other forms of luminescence, in which a material emits light.Many materials selectively absorb...
s. By contrast, in human-based metabolomics, it is more common to describe metabolites as being either endogenous
Endogenous
Endogenous substances are those that originate from within an organism, tissue, or cell. Endogenous retroviruses are caused by ancient infections of germ cells in humans, mammals and other vertebrates...
(produced by the host organism) or exogenous
Exogenous
Exogenous refers to an action or object coming from outside a system. It is the opposite of endogenous, something generated from within the system....
. Metabolites of foreign substances such as drugs are termed xenometabolites.
The metabolome
Metabolome
Metabolome refers to the complete set of small-molecule metabolites to be found within a biological sample, such as a single organism...
forms a large network of metabolic reactions, where outputs from one enzymatic chemical reaction
Chemical reaction
A chemical reaction is a process that leads to the transformation of one set of chemical substances to another. Chemical reactions can be either spontaneous, requiring no input of energy, or non-spontaneous, typically following the input of some type of energy, such as heat, light or electricity...
are inputs to other chemical reactions. Such systems have been described as hypercycle
Hypercycle
Hypercycle may refer to:* Hypercycle , a line of equal distance in hyperbolic geometry* Hypercycle , a kind of reaction network prominent in a theory of the self-organization of matter, see Manfred Eigen and quasispecies model...
s.
Metabonomics
Metabonomics is defined as "the quantitative measurement of the dynamic multiparametric metabolic response of living systems to pathophysiological stimuli or genetic modification". The word origin is from the Greek meta meaning change and nomos meaning a rule set or set of laws. This approach was pioneered by Jeremy Nicholson at Imperial College London
Imperial College London
Imperial College London is a public research university located in London, United Kingdom, specialising in science, engineering, business and medicine...
and has been used in toxicology, disease diagnosis and a number of other fields. Historically, the metabonomics approach was one of the first methods to apply the scope of systems biology to studies of metabolism.
There has been some disagreement over the exact differences between 'metabolomics' and 'metabonomics'. The difference between the two terms is not related to choice of analytical platform: although metabonomics is more associated with NMR spectroscopy
NMR spectroscopy
Nuclear magnetic resonance spectroscopy, most commonly known as NMR spectroscopy, is a research technique that exploits the magnetic properties of certain atomic nuclei to determine physical and chemical properties of atoms or the molecules in which they are contained...
and metabolomics with mass spectrometry
Mass spectrometry
Mass spectrometry is an analytical technique that measures the mass-to-charge ratio of charged particles.It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and...
-based techniques, this is simply because of usages amongst different groups that have popularized the different terms. While there is still no absolute agreement, there is a growing consensus that 'metabolomics' places a greater emphasis on metabolic profiling at a cellular or organ level and is primarily concerned with normal endogenous metabolism. 'Metabonomics' extends metabolic profiling to include information about perturbations of metabolism caused by environmental factors (including diet and toxins), disease processes, and the involvement of extragenomic influences, such as gut microflora. This is not a trivial difference; metabolomic studies should, by definition, exclude metabolic contributions from extragenomic sources, because these are external to the system being studied. However, in practice, within the field of human disease research there is still a large degree of overlap in the way both terms are used, and they are often in effect synonymous.
Separation methods
- Gas chromatography, especially when interfaced with mass spectrometry (GC-MSGas chromatography-mass spectrometryGas chromatography–mass spectrometry is a method that combines the features of gas-liquid chromatography and mass spectrometry to identify different substances within a test sample. Applications of GC-MS include drug detection, fire investigation, environmental analysis, explosives investigation,...
), is one of the most widely used and powerful methods. It offers very high chromatographic resolution, but requires chemical derivatization for many biomolecules: only volatile chemicals can be analysed without derivatization. (Some modern instruments allow '2D' chromatography, using a short polar column after the main analytical column, which increases the resolution still further.) Some large and polar metabolites cannot be analysed by GCGas-liquid chromatographyGas chromatography , is a common type of chromatography used in analytical chemistry for separating and analysing compounds that can be vaporized without decomposition. Typical uses of GC include testing the purity of a particular substance, or separating the different components of a mixture...
.
- High performance liquid chromatographyHigh performance liquid chromatographyHigh-performance liquid chromatography , HPLC, is a chromatographic technique that can separate a mixture of compounds and is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of the mixture.HPLC typically utilizes different types of stationary...
(HPLC). Compared to GCGas-liquid chromatographyGas chromatography , is a common type of chromatography used in analytical chemistry for separating and analysing compounds that can be vaporized without decomposition. Typical uses of GC include testing the purity of a particular substance, or separating the different components of a mixture...
, HPLC has lower chromatographic resolution, but it does have the advantage that a much wider range of analytes can potentially be measured.
- Capillary electrophoresisCapillary electrophoresisCapillary electrophoresis , also known as capillary zone electrophoresis , can be used to separate ionic species by their charge and frictional forces and hydrodynamic radius. In traditional electrophoresis, electrically charged analytes move in a conductive liquid medium under the influence of an...
(CE). CE has a higher theoretical separation efficiency than HPLC, and is suitable for use with a wider range of metabolite classes than is GC. As for all electrophoretic techniques, it is most appropriate for charged analytes.
Detection methods
- Mass spectrometryMass spectrometryMass spectrometry is an analytical technique that measures the mass-to-charge ratio of charged particles.It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and...
(MS) is used to identify and to quantify metabolites after separation by GC, HPLCHigh-performance liquid chromatographyHigh-performance liquid chromatography , HPLC, is a chromatographic technique that can separate a mixture of compounds and is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of the mixture.HPLC typically utilizes different types of stationary...
(LC-MSLiquid chromatography-mass spectrometryLiquid chromatography–mass spectrometry is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry. LC-MS is a powerful technique used for many applications which has very high...
), or CECapillary electrophoresisCapillary electrophoresis , also known as capillary zone electrophoresis , can be used to separate ionic species by their charge and frictional forces and hydrodynamic radius. In traditional electrophoresis, electrically charged analytes move in a conductive liquid medium under the influence of an...
. GC-MS is the most 'natural' combination of the three, and was the first to be developed. In addition, mass spectral fingerprint libraries exist or can be developed that allow identification of a metabolite according to its fragmentation pattern. MS is both sensitive (although, particularly for HPLC-MS, sensitivity is more of an issue as it is affected by the charge on the metabolite, and can be subject to ion suppression artifacts) and can be very specific. There are also a number of studies which use MS as a stand-alone technology: the sample is infused directly into the mass spectrometer with no prior separation, and the MS serves to both separate and to detect metabolites.
- Surface-based mass analysis has seen a resurgence in the past decade, with new MS technologies focused on increasing sensitivity, minimizing background, and reducing sample preparation. The ability to analyze metabolites directly from biofluids and tissues continues to challenge current MS technology, largely because of the limits imposed by the complexity of these samples, which contain thousands to tens of thousands of metabolites. Among the technologies being developed to address this challenge is Nanostructure-Initiator MS (NIMS), a desorption/ ionization approach that does not require the application of matrix and thereby facilitates small-molecule (i.e., metabolite) identification. MALDI is also used however, the application of a MALDI matrix can add significant background at <1000 Da that complicates analysis of the low-mass range (i.e., metabolites). In addition, the size of the resulting matrix crystals limits the spatial resolution that can be achieved in tissue imaging. Because of these limitations, several other matrix-free desorption/ionization approaches have been applied to the analysis of biofluids and tissues. Secondary ion mass spectrometrySecondary ion mass spectrometrySecondary ion mass spectrometry is a technique used in materials science and surface science to analyze the composition of solid surfaces and thin films by sputtering the surface of the specimen with a focused primary ion beam and collecting and analyzing ejected secondary ions...
(SIMS) was one of the first matrix-free desorption/ionization approaches used to analyze metabolites from biological samples. SIMS uses a high-energy primary ion beam to desorb and generate secondary ions from a surface. The primary advantage of SIMS is its high spatial resolution (as small as 50 nm), a powerful characteristic for tissue imaging with MS. However, SIMS has yet to be readily applied to the analysis of biofluids and tissues because of its limited sensitivity at >500 Da and analyte fragmentation generated by the high-energy primary ion beam. Desorption electrospray ionizationDesorption electrospray ionizationDesorption electrospray ionization is an ambient ionization technique that can be used in mass spectrometry for chemical analysis. It is an atmospheric pressure ion source that ionizes gases, liquids and solids in open air under ambient conditions. It was developed in 2004 by Professor Graham...
(DESI) is a matrix-free technique for analyzing biological samples that uses a charged solvent spray to desorb ions from a surface. Advantages of DESI are that no special surface is required and the analysis is performed at ambient pressure with full access to the sample during acquisition. A limitation of DESI is spatial resolution because “focusing” the charged solvent spray is difficult. However, a recent development termed laser ablation ESI (LAESI) is a promising approach to circumvent this limitation.
- Nuclear magnetic resonanceNuclear magnetic resonanceNuclear magnetic resonance is a physical phenomenon in which magnetic nuclei in a magnetic field absorb and re-emit electromagnetic radiation...
(NMR) spectroscopy. NMR is the only detection technique which does not rely on separation of the analytes, and the sample can thus be recovered for further analyses. All kinds of small molecule metabolites can be measured simultaneously - in this sense, NMR is close to being a universal detector. The main advantages of NMR are high analytical reproducibility and simplicity of sample preparation. Practically, however, it is relatively insensitive compared to mass spectrometry-based techniques.
- Although NMR and MS are the most widely used techniques, other methods of detection that have been used include ion-mobility spectrometry, electrochemical detection (coupled to HPLC) and radiolabel (when combined with thin-layer chromatography).
Statistical methods
The data generated in metabolomics usually consist of measurements performed on subjects under various conditions. These measurements may be digitized spectra, or a list of metabolite levels. In its simplest form this generates a matrix with rows corresponding to subjects and columns corresponding to metabolite levels. Several statistical programs are currently available for analysis of both NMR and mass spectrometryMass spectrometry
Mass spectrometry is an analytical technique that measures the mass-to-charge ratio of charged particles.It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and...
data. For mass spectrometry data, software is available that identifies molecules that vary in subject groups on the basis of mass and sometimes retention time depending on the experimental design. The first comprehensive software to analyze global mass spectrometry-based metabolomics datasets was developed by the Siuzdak laboratory at The Scripps Research Institute
The Scripps Research Institute
The Scripps Research Institute is an American medical research facility that focuses on research in the basic biomedical sciences. Headquartered in La Jolla, California, with a sister facility in Jupiter, Florida, the institute is home to 3,000 scientists, technicians, graduate students, and...
in 2006. This software, called XCMS, is freely available, has over 20,000 downloads since its inception in 2006, and is one of the most widely cited mass spectrometry-based metabolomics software programs in scientific literature. Other popular metabolomics programs for mass spectral analysis are MZmine, MetAlign, MathDAMP, which also compensate for retention time deviation during sample analysis. LCMStats is another R package for detailed analysis of liquid chromatography mass spectrometry(LCMS)data and is helpful in identification of co-eluting ions especially isotopologues from a complicated metabolic profile. It combines xcms package functions and can be used to apply many statistical functions for correcting detector saturation
Dead time
For detection systems that record discrete events, such as particle and nuclear detectors, the dead time is the time after each event during which the system is not able to record another event....
using coates correction and creating heat plots. Metabolomics data may also be analyzed by statistical projection (chemometrics
Chemometrics
Chemometrics is the science of extracting information from chemical systems by data-driven means. It is a highly interfacial discipline, using methods frequently employed in core data-analytic disciplines such as multivariate statistics, applied mathematics, and computer science, in order to...
) methods such as principal components analysis
Principal components analysis
Principal component analysis is a mathematical procedure that uses an orthogonal transformation to convert a set of observations of possibly correlated variables into a set of values of uncorrelated variables called principal components. The number of principal components is less than or equal to...
and partial least squares regression
Partial least squares regression
Partial least squares regression is a statistical method that bears some relation to principal components regression; instead of finding hyperplanes of maximum variance between the response and independent variables, it finds a linear regression model by projecting the predicted variables and the...
.
Key applications
- ToxicityToxicityToxicity is the degree to which a substance can damage a living or non-living organisms. Toxicity can refer to the effect on a whole organism, such as an animal, bacterium, or plant, as well as the effect on a substructure of the organism, such as a cell or an organ , such as the liver...
assessment/toxicologyToxicologyToxicology is a branch of biology, chemistry, and medicine concerned with the study of the adverse effects of chemicals on living organisms...
. Metabolic profiling (especially of urine or blood plasma samples) can be used to detect the physiological changes caused by toxic insult of a chemical (or mixture of chemicals). In many cases, the observed changes can be related to specific syndromes, e.g. a specific lesion in liver or kidney. This is of particular relevance to pharmaceutical companies wanting to test the toxicity of potential drugMedicationA pharmaceutical drug, also referred to as medicine, medication or medicament, can be loosely defined as any chemical substance intended for use in the medical diagnosis, cure, treatment, or prevention of disease.- Classification :...
candidates: if a compound can be eliminated before it reaches clinical trialClinical trialClinical trials are a set of procedures in medical research and drug development that are conducted to allow safety and efficacy data to be collected for health interventions...
s on the grounds of adverse toxicity, it saves the enormous expense of the trials.
- Functional genomicsFunctional genomicsFunctional genomics is a field of molecular biology that attempts to make use of the vast wealth of data produced by genomic projects to describe gene functions and interactions...
. Metabolomics can be an excellent tool for determining the phenotypePhenotypeA phenotype is an organism's observable characteristics or traits: such as its morphology, development, biochemical or physiological properties, behavior, and products of behavior...
caused by a genetic manipulation, such as gene deletion or insertion. Sometimes this can be a sufficient goal in itself—for instance, to detect any phenotypic changes in a genetically-modified plant intended for human or animal consumption. More exciting is the prospect of predicting the function of unknown geneGeneA gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
s by comparison with the metabolic perturbations caused by deletion/insertion of known genes. Such advances are most likely to come from model organismModel organismA model organism is a non-human species that is extensively studied to understand particular biological phenomena, with the expectation that discoveries made in the organism model will provide insight into the workings of other organisms. Model organisms are in vivo models and are widely used to...
s such as Saccharomyces cerevisiaeSaccharomyces cerevisiaeSaccharomyces cerevisiae is a species of yeast. It is perhaps the most useful yeast, having been instrumental to baking and brewing since ancient times. It is believed that it was originally isolated from the skin of grapes...
and Arabidopsis thalianaArabidopsis thalianaArabidopsis thaliana is a small flowering plant native to Europe, Asia, and northwestern Africa. A spring annual with a relatively short life cycle, arabidopsis is popular as a model organism in plant biology and genetics...
. The Cravatt laboratoryBenjamin Cravatt IIIBenjamin Franklin Cravatt is a professor in and chair of the Department of Chemical Physiology at The Scripps Research Institute in La Jolla, California...
at The Scripps Research InstituteThe Scripps Research InstituteThe Scripps Research Institute is an American medical research facility that focuses on research in the basic biomedical sciences. Headquartered in La Jolla, California, with a sister facility in Jupiter, Florida, the institute is home to 3,000 scientists, technicians, graduate students, and...
has recently applied this technology to mammalMammalMammals are members of a class of air-breathing vertebrate animals characterised by the possession of endothermy, hair, three middle ear bones, and mammary glands functional in mothers with young...
ian systems, identifying the N-acyltaurines as previously uncharacterized endogenous substrates for the enzyme fatty acid amide hydrolase (FAAH) and the monoalkylglycerol ethers (MAGEs) as endogenous substrates for the uncharacterized hydrolaseHydrolaseIn biochemistry, a hydrolase is an enzyme that catalyzes the hydrolysis of a chemical bond. For example, an enzyme that catalyzed the following reaction is a hydrolase:-Nomenclature:...
KIAA1363.
- NutrigenomicsNutrigenomicsNutrigenomics is the study of the effects of foods and food constituents on gene expression. It is about how our DNA is transcribed into mRNA and then to proteins and provides a basis for understanding the biological activity of food components...
is a generalised term which links genomics, transcriptomics, proteomics and metabolomics to human nutrition. In general a metabolome in a given body fluid is influenced by endogenous factors such as age, sex, body composition and genetics as well as underlying pathologies. The large bowel microflora are also a very significant potential confounder of metabolic profiles and could be classified as either an endogenous or exogenous factor. The main exogenous factors are diet and drugs. Diet can then be broken down to nutrients and non- nutrients. Metabolomics is one means to determine a biological endpoint, or metabolic fingerprint, which reflects the balance of all these forces on an individual's metabolism.
Environmental Metabolomics
- Environmental Metabolomics is the application of metabolomics to characterise the interactions of organisms with their environment. This approach has many advantages for studying organism–environment interactions and for assessing organism function and health at the molecular level. As such, metabolomics is finding an increasing number of applications in the environmental sciences, ranging from understanding organismal responses to abiotic pressures, to investigating the responses of organisms to other biota. These interactions can be studied from individuals to populations, which can be related to the traditional fields of ecophysiology and ecology, and from instantaneous effects to those over evolutionary time scales, the latter enabling studies of genetic adaptation.
Sources and notes
- Tomita M., Nishioka T. (2005), Metabolomics: The Frontier of Systems Biology, Springer, ISBN 4-431-25121-9
- Wolfram Weckwerth W. (2006), Metabolomics: Methods And Protocols (Methods in Molecular Biology), Humana Press, ISBN 1-58829-561-3
- Dunn, W.B. and Ellis, D.I. (2005), Metabolomics: current analytical platforms and methodologies. Trends in Analytical Chemistry 24(4), 285-294.
http://dbkgroup.org/dave_files/AnalystMetabolicFingerprinting2006.pdf
- Claudino, W.M., Quatronne, A., Pestrim, M., Biganzoli, L., Bertini and Di Leo, A.(2007) Metabolomics: Available Results, Current Research Projects in Breast Cancer, and Future *Applications. J Clin Oncol May 14; [Epub ahead of print]. http://lab.bcb.iastate.edu/projects/plantmetabolomics/
- Ellis, D.I., Dunn, W.B., Griffin, J.L., Allwood, J.W. and Goodacre, R. (2007) Metabolic Fingerprinting as a Diagnostic Tool. Pharmacogenomics, 8(9), 1243-1266. http://dbkgroup.org/dave_files/Pharmacogenomics.pdf
*Bundy JG, Davey MP, Viant, MR. 2009. Environmental Metabolomics: A Critical Review and Future Perspectives. Metabolomics 5: 3-21.