Publications
380 results found
Sumner LW, Amberg A, Barrett D, et al., 2007, Proposed minimum reporting standards for chemical analysis, METABOLOMICS, Vol: 3, Pages: 211-221, ISSN: 1573-3882
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- Citations: 2925
Griffin JL, Nicholls AW, Daykin CA, et al., 2007, Standard reporting requirements for biological samples in metabolomics experiments: mammalian/in vivo experiments, METABOLOMICS, Vol: 3, Pages: 179-188, ISSN: 1573-3882
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- Citations: 56
Griffin JL, Salek RM, 2007, Metabolomic applications to neuroscience: more challenges than chances?, Expert Rev Proteomics, Vol: 4, Pages: 435-437
Nasrallah FA, Griffin JL, Balcar VJ, et al., 2007, Understanding your inhibitions: modulation of brain cortical metabolism by GABA(B) receptors., J Cereb Blood Flow Metab, Vol: 27, Pages: 1510-1520, ISSN: 0271-678X
Although the impact of neuronal excitation on the functional activity of brain is well understood, the nature of functional responses to inhibitory modulation is far from clear. In this work, we investigated the effects of modulation of the metabotropic GABA(B) receptor on brain metabolism using a targeted neuropharmacological, (1)H/(13)C nuclear magnetic resonance spectroscopy, and metabolomic approach. While agonists at GABA(B) receptors (Baclofen and SKF 97541) generally decreased metabolic activity, mild agonist action could also stimulate metabolism. Less potent antagonists (CGP 35348, Phaclofen) significantly decreased metabolic activity, while more potent antagonists (CGP 52432 and SCH 50911) had opposite, stimulatory, effects. Examination of the data by principal components analysis showed clear divisions of the effects into excitatory and inhibitory components. GABAergic modulation can, therefore, have stimulatory, inhibitory, or even neutral net effects on metabolic activity in brain tissue. This is consistent with GABAergic activity being context dependent, and this conclusion should be taken into account when evaluating functional imaging data involving modulation of neuronal inhibition.
Pears MR, Rubtsov D, Mitchison HM, et al., 2007, Strategies for data analyses in a high resolution <sup>1</sup>H NMR based metabolomics study of a mouse model of Batten disease, Metabolomics, Vol: 3, Pages: 121-136, ISSN: 1573-3882
Using an NMR based approach, employing both solution state and high resolution magic angle spinning (HR MAS) 1H NMR spectroscopy, in conjunction with an array of statistical methods, we report cerebral metabolic de.cits in a mouse model of Batten disease (Cln3 null mutant mice). Batten disease is the most common progressive neurodegenerative disorder of childhood and is caused by mutations in the Cln3 gene. In particular, brain tissue from Cln3 mice was characterised by increased concentrations of glutamine, myo-inositol, scyllo-inositol, aspartate and lactate, alongside decreased concentrations of N-acetyl-L-aspartate (NAA), N-acetyl-L-glutamate (NAG), c-amino butyric acid (GABA), glutamate and creatine. Accompanying changes in lipid deposition were also detected in intact cortical tissue by HR MAS 1H NMR spectroscopy. To realise the true potential of metabolomic datasets necessitates a comprehensive analysis of the data, such that useful biological information can be extracted and used to generate hypotheses which can be further tested and re.ned. We found that using a combination of univariate and multivariate analyses, a maximal number of metabolic de.cits were successfully identi.ed. In particular the complementary nature of the statistical approaches allowed the de.nition of changes which were relative, absolute or simply a change in variance, allowing a greater understanding of the disease processes detected. © 2007 Springer Science+Business Media, LLC.
Broom KA, Anthony DC, Lowe JP, et al., 2007, MRI and MRS alterations in the preclinical phase of murine prion disease: association with neuropathological and behavioural changes., Neurobiol Dis, Vol: 26, Pages: 707-717, ISSN: 0969-9961
Prion diseases are fatal chronic neurodegenerative diseases. Previous qualitative magnetic resonance imaging (MRI) and spectroscopy (MRS) studies report conflicting results in the symptomatic stages of the disease, but little work has been carried out during the earlier stages of the disease. Here we have used the murine ME7 model of prion disease to quantitatively investigate MRI and MRS changes during the period prior to the onset of overt clinical signs (20+ weeks) and have correlated these with pathological and behavioural abnormalities. Using in vivo MRI, at the later stages of the preclinical period (18 weeks) the diffusion of tissue water was significantly reduced, coinciding with significant microglial activation and behavioural hyperactivity. Using in vivo MRS, we found early (12 weeks) decreases in the ratio of N-acetyl aspartate to both choline (NAA/Cho) and creatine (NAA/Cr) in the thalamus and hippocampus, which were associated with early behavioural deficits. Ex vivo MRS of brain extracts confirmed and extended these findings, showing early (8-12 weeks) decreases in both the neuronal metabolites NAA and glutamate, and the metabolic metabolites lactate and glucose. Increases in the glial metabolite myo-inositol were observed at later stages when microglial and astrocyte activation is substantial. These changes in MRI and MRS signals, which precede overt clinical signs of disease, could provide insights into the pathogenesis of this disease and may enable early detection of pathology.
Salek RM, Maguire ML, Bentley E, et al., 2007, A metabolomic comparison of urinary changes in type 2 diabetes in mouse, rat, and human., Physiol Genomics, Vol: 29, Pages: 99-108
Type 2 diabetes mellitus is the result of a combination of impaired insulin secretion with reduced insulin sensitivity of target tissues. There are an estimated 150 million affected individuals worldwide, of whom a large proportion remains undiagnosed because of a lack of specific symptoms early in this disorder and inadequate diagnostics. In this study, NMR-based metabolomic analysis in conjunction with multivariate statistics was applied to examine the urinary metabolic changes in two rodent models of type 2 diabetes mellitus as well as unmedicated human sufferers. The db/db mouse and obese Zucker (fa/fa) rat have autosomal recessive defects in the leptin receptor gene, causing type 2 diabetes. 1H-NMR spectra of urine were used in conjunction with uni- and multivariate statistics to identify disease-related metabolic changes in these two animal models and human sufferers. This study demonstrates metabolic similarities between the three species examined, including metabolic responses associated with general systemic stress, changes in the TCA cycle, and perturbations in nucleotide metabolism and in methylamine metabolism. All three species demonstrated profound changes in nucleotide metabolism, including that of N-methylnicotinamide and N-methyl-2-pyridone-5-carboxamide, which may provide unique biomarkers for following type 2 diabetes mellitus progression.
Hodson MP, Dear GJ, Roberts AD, et al., 2007, A gender-specific discriminator in Sprague-Dawley rat urine: The deployment of a metabolic profiling strategy for biomarker discovery and identification, ANALYTICAL BIOCHEMISTRY, Vol: 362, Pages: 182-192, ISSN: 0003-2697
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- Citations: 42
Griffin JL, Kauppinen RA, 2007, A metabolomics perspective of human brain tumours., FEBS J, Vol: 274, Pages: 1132-1139, ISSN: 1742-464X
During the past decade or so, a wealth of information about metabolites in various human brain tumour preparations (cultured cells, tissue specimens, tumours in vivo) has been accumulated by global profiling tools. Such holistic approaches to cellular biochemistry have been termed metabolomics. Inherent and specific metabolic profiles of major brain tumour cell types, as determined by proton nuclear magnetic resonance spectroscopy ((1)H MRS), have also been used to define metabolite phenotypes in tumours in vivo. This minireview examines the recent advances in the field of human brain tumour metabolomics research, including advances in MRS and mass spectrometry technologies, and data analysis.
Moussa CE-H, Rae C, Bubb WA, et al., 2007, Inhibitors of glutamate transport modulate distinct patterns in brain metabolism., J Neurosci Res, Vol: 85, Pages: 342-350, ISSN: 0360-4012
High affinity uptake of glutamate plays a major role in the termination of excitatory neurotransmission. Identification of the ramifications of transporter function is essential to understand the diseases in which defective excitatory amino acid transporters (EAAT) have been implicated. In this work we incubated Guinea pig cortical tissue slices with [3-(13)C]pyruvate and major currently available glutamate uptake inhibitors and studied the resultant metabolic sequelae by (13)C and (1)H NMR spectroscopy using a multivariate statistical approach. Perturbation of glutamate uptake produced significant effects on metabolic flux through the Krebs cycle, and on glutamate/glutamine cycling rates, with this effect accounting for 76% of the variation in the total data set. The effects of all inhibitors were separable from each other along three major principal components. The competitive inhibitor L-CCG III ((2S,1'S,2'R)-2-carboxycyclopropyl)glycine) differed most from the other inhibitors, showing negative weightings on both the first and second principal components, whereas the EAAT2-specific inhibitor dihydrokainate (DHK) showed metabolic patterns similar to that of anti-endo-3,4-methanopyrolidine dicarboxylate but separate from those of DL-threo-beta-benzyloxyaspartate (TBOA) and L-trans-pyrrolidine-2,4-dicarboxylate (L-tPDC). This indicates that different inhibition mechanisms or different colocalisation of the separate transporter subtypes with glutamate receptors can produce significantly different metabolic and functional outcomes for the brain.
Griffin JL, Kauppinen RA, 2007, Tumour metabolomics in animal models of human cancer., J Proteome Res, Vol: 6, Pages: 498-505, ISSN: 1535-3893
Multinuclear Nuclear Magnetic Resonance Spectroscopy (MRS) and mass spectrometry (MS) are the key analytical techniques used in an increasing manner to explore tumor metabolite profiles. Recent work has revealed that metabolite profiles in various tumor preparations (i.e., cultured cells, tissue specimens, and tumors in vivo) show strong correlations with tumor type, proliferation, metabolic activity, and cell death. These data are regarded as highly promising for tumor diagnosis as well as assessment of prognosis and treatment response in a clinical setting. In this pursuit, animal models of human cancer have played a central role. In this short account, we review the potentials of MRS and MS techniques for animal tumor metabolomic work, as well as highlight some interesting applications of these techniques for various animal tumor types.
Griffin JL, Scott J, Nicholson JK, 2007, The influence of pharmacogenetics on fatty liver disease in the Wistar and Kyoto rats: A combined transcriptomic and metabonomic study, JOURNAL OF PROTEOME RESEARCH, Vol: 6, Pages: 54-61, ISSN: 1535-3893
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- Citations: 35
Sansone SA, Nikolau B, van Ommen B, et al., 2007, The metabolomics standards initiative, Nature Biotechnology, Vol: 25, Pages: 846-848
Fiehn O, Robertson D, Griffin J, et al., 2007, The metabolomics standards initiative (MSI), Metabolomics, Vol: 3, Pages: 175-178
Atherton HJ, Bailey NJ, Zhang W, et al., 2006, A combined 1H-NMR spectroscopy- and mass spectrometry-based metabolomic study of the PPAR-alpha null mutant mouse defines profound systemic changes in metabolism linked to the metabolic syndrome., Physiol Genomics, Vol: 27, Pages: 178-186
The mobilization of triacylglycerides from storage in adipocytes to the liver is a vital response to the fasting state in mammalian metabolism. This is accompanied by a rapid translational activation of genes encoding mitochondrial, microsomal, and peroxisomal beta-oxidation in the liver, in part under the regulation of peroxisome proliferator-activated receptor-alpha (PPAR-alpha). A failure to express PPAR-alpha results in profound metabolic perturbations in muscle tissue as well as the liver. These changes represent a number of deficits that accompany diabetes, dyslipidemia, and the metabolic syndrome. In this study, the metabolic role of PPAR-alpha has been investigated in heart, skeletal muscle, liver, and adipose tissue of PPAR-alpha null mice at 1 mo of age using metabolomics. To maximize the coverage of the metabolome in these tissues, (1)H-NMR spectroscopy, magic angle spinning (1)H-NMR spectroscopy, gas chromatography-mass spectrometry, and liquid chromatography-mass spectrometry were used to examine metabolites in aqueous tissue extracts and intact tissue. The data were analyzed by the multivariate approaches of principal components analysis and partial least squares. Across all tissues, there was a profound decrease in glucose and a number of amino acids, including glutamine and alanine, and an increase in lactate, demonstrating that a failure to express PPAR-alpha results in perturbations in glycolysis, the citric acid cycle, and gluconeogenesis. Furthermore, despite PPAR-alpha being weakly expressed in adipose tissue, a profound metabolic perturbation was detected in this tissue.
Griffin JL, Nicholls AW, 2006, Metabolomics as a functional genomic tool for understanding lipid dysfunction in diabetes, obesity and related disorders., Pharmacogenomics, Vol: 7, Pages: 1095-1107, ISSN: 1462-2416
With the rise of systems biology, a number of approaches have been developed to globally profile a tier of organization in a cell, tissue or organism. Metabolomics is an approach that attempts to profile all the metabolites in a biological matrix. One of the major challenges of this approach, as with other 'omic' technologies, is that the metabolome is context-dependent and will vary with pathology, developmental stage and environmental factors. Thus, the possibility of globally profiling the metabolome of an organism is a genuine analytical challenge, as by definition this must also take into consideration all relevant factors that influence metabolism. Despite these challenges, the approach has already been applied to understand the metabolism in a range of animal models, and has more recently started to be projected into the clinical situation. In this review, the technologies currently being used in metabolomics will be assessed prior to examining their use to study diseases related to the metabolic syndrome, including Type II diabetes, obesity, cardiovascular disease and fatty liver disease.
Griffin JL, 2006, Understanding mouse models of disease through metabolomics., Curr Opin Chem Biol, Vol: 10, Pages: 309-315, ISSN: 1367-5931
Metabolomics is widely applicable to a number of fields including toxicology, plant metabolism and functional genomics. In the area of functional genomics, a number of studies have demonstrated the potential of this approach, which combines high-throughput metabolite profiling with computer-assisted pattern recognition approaches. In this review, recent applications of metabolomics to understanding mouse models of disease are considered. This includes studies on the impact of mouse strain on disease models, as well as metabolic profiling of cardiovascular, metabolic and neurodegenerative diseases. This versatile tool is set to increase in popularity as functional genomic approaches produce more mouse models for phenotyping.
Rae C, Moussa CE-H, Griffin JL, et al., 2006, A metabolomic approach to ionotropic glutamate receptor subtype function: a nuclear magnetic resonance in vitro investigation., J Cereb Blood Flow Metab, Vol: 26, Pages: 1005-1017, ISSN: 0271-678X
A range of behaviours are elucidated via ionotropic glutamate receptors (iGluR). In this work, we examined the acute activation of iGluRs by a range of receptor ligands and effectors to see whether distinguishable metabolic sequelae were elucidated by the activity. We used a guinea-pig brain cortical tissue slice model using targeted receptor ligands ((RS)-(tetrazol-5-yl)glycine (TZG), (5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801, dizocilpine), cis-4-[phosphomethyl]-piperidine-2-carboxylic acid (CGS 19755), (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, (2S, 3S, 4S)-2-carboxy-4-(1-methylethenyl)-3-pyrrolidineacetic acid (kainate) and D-serine (D-Ser), as well as compounds (quinolinic acid and kynurenic acid (KynA)) involved in some neuroinflammatory responses. The data were derived using 13C and 1H NMR spectroscopy, and analysed by metabolomic approaches and multivariate statistics. The metabolic effects of agonists at the three major classes of iGluR were easily separated from each other using this method. The classical N-methyl-D-aspartate receptor agonist TZG and the antagonist CGS 19755 produced excitatory and inhibitory metabolic responses, respectively, while the blocker MK-801 resulted in a significant decrease in net metabolism and produced the largest decrease in all metabolite pool sizes seen by any glutamatergic ligand we have studied. Quinolinic acid and KynA produced similar acute metabolic responses, which were unlike those to TZG or CGS 19755, but similar to that of D-Ser. D-Ser was highly stimulatory of net flux into the Krebs cycle. These data show that the metabolic response to iGluR perturbation in vitro is a sensitive discriminator of function.
Kirschenlohr HL, Griffin JL, Clarke SC, et al., 2006, Erratum: Proton NMR analysis of plasma is a weak predictor of coronary artery disease (Nature Medicine (2006) 12, (705-710)), Nature Medicine, Vol: 12, ISSN: 1078-8956
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- Citations: 3
Usher-Smith JA, Fraser JA, Bailey PSJ, et al., 2006, The influence of intracellular lactate and H+ on cell volume in amphibian skeletal muscle., J Physiol, Vol: 573, Pages: 799-818, ISSN: 0022-3751
The combined effects of intracellular lactate and proton accumulation on cell volume, Vc, were investigated in resting Rana temporaria striated muscle fibres. Intracellular lactate and H+ concentrations were simultaneously increased by exposing resting muscle fibres to extracellular solutions that contained 20-80 mm sodium lactate. Cellular H+ and lactate entry was confirmed using pH-sensitive electrodes and 1H-NMR, respectively, and effects on Vc were measured using confocal microscope xz-scanning. Exposure to extracellular lactate up to 80 mm produced significant changes in pH and intracellular lactate (from a pH of 7.24 +/- 0.03, n = 8, and 4.65 +/- 1.07 mm, n = 6, respectively, in control fibres, to 6.59 +/- 0.03, n = 4, and 26.41 +/- 0.92 mm, n = 3, respectively) that were comparable to those observed following fatiguing stimulation (6.30-6.70 and 18.04 +/- 1.78 mm, n = 6, respectively). Yet, the increase in intracellular osmolarity expected from such an increase in intracellular lactate did not significantly alter Vc. Simulation of these experimental results, modified from the charge difference model of Fraser & Huang, demonstrated that such experimental manoeuvres produced changes in intracellular [H+] and [lactate] comparable to those observed during muscle fatigue, and accounted for this paradoxical conservation of Vc through balancing negative osmotic effects resulting from the net cation efflux that would follow a titration of intracellular membrane-impermeant anions by the intracellular accumulation of protons. It demonstrated that with established physiological values for intracellular buffering capacity and the permeability ratio of lactic acid and anionic lactate, P(LacH): P(Lac-), this would provide a mechanism that precisely balanced any effect on cell volume resulting from lactate accumulation during exercise.
Kirschenlohr HL, Griffin JL, Clarke SC, et al., 2006, Proton NMR analysis of plasma is a weak predictor of coronary artery disease., Nat Med, Vol: 12, Pages: 705-710, ISSN: 1078-8956
Multivariate analysis of 1H-NMR spectra of blood sera was reported previously to predict angiographically defined advanced coronary artery disease (CAD) with >90% accuracy and specificity. The analysis depended mainly on the major lipid regions of the spectra, but many variables, including gender and drug treatment, affect lipid composition and are potential confounders. We have determined the predictive power of the same methodology for angiographically defined CAD using plasma samples from groups of male patients, classified by statin treatment, who had normal coronary arteries (NCAs) or CAD. Predictions for NCA and CAD groups were only 80.3% correct for patients not treated with statins and 61.3% for treated patients, compared with random correct predictions of 50%. A confidence limit of >99% was achieved for 36.2% of predictions for untreated groups and 6.2% for treated groups. Detection of CAD by 1H-NMR with >99% confidence was therefore very weak compared with angiography.
Prieto P, Baird AW, Blaauboer BJ, et al., 2006, The assessment of repeated dose toxicity in vitro: a proposed approach. The report and recommendations of ECVAM workshop 56., Pages: 315-341, ISSN: 0261-1929
Dunkley TPJ, Hester S, Shadforth IP, et al., 2006, Mapping the Arabidopsis organelle proteome., Proc Natl Acad Sci U S A, Vol: 103, Pages: 6518-6523, ISSN: 0027-8424
A challenging task in the study of the secretory pathway is the identification and localization of new proteins to increase our understanding of the functions of different organelles. Previous proteomic studies of the endomembrane system have been hindered by contaminating proteins, making it impossible to assign proteins to organelles. Here we have used the localization of organelle proteins by the isotope tagging technique in conjunction with isotope tags for relative and absolute quantitation and 2D liquid chromatography for the simultaneous assignment of proteins to multiple subcellular compartments. With this approach, the density gradient distributions of 689 proteins from Arabidopsis thaliana were determined, enabling confident and simultaneous localization of 527 proteins to the endoplasmic reticulum, Golgi apparatus, vacuolar membrane, plasma membrane, or mitochondria and plastids. This parallel analysis of endomembrane components has enabled protein steady-state distributions to be determined. Consequently, genuine organelle residents have been distinguished from contaminating proteins and proteins in transit through the secretory pathway.
Griffin JL, Blenkiron C, Valonen PK, et al., 2006, High-resolution magic angle spinning 1H NMR spectroscopy and reverse transcription-PCR analysis of apoptosis in a rat glioma., Anal Chem, Vol: 78, Pages: 1546-1552, ISSN: 0003-2700
The functional genomic approaches of transcriptomics, proteomics and metabolomics aim to measure the mRNA, protein or metabolite complement of a cell, tissue or organism. In this study we have investigated the compatibility of transcriptional analysis, using Reverse Transcription (RT)-PCR, and metabolite analysis, by high-resolution magic angle spinning (HRMAS) 1H NMR spectroscopy, in BT4C rat glioma following the induction of programmed cell death. The metabolite and transcriptional changes that accompanied apoptosis were examined at 0, 4 and 8 days of ganciclovir/thymidine kinase gene therapy. Despite the high spinning speeds employed during HRMAS 1H NMR spectroscopy of one-half of the tumor samples, RT-PCR analysis of the pro-apoptotic transcripts Bcl-2, BAK-1, caspase-9 and FAS was possible, producing similar results to those detected in the unspun half of the tumors. Furthermore, the expression of FAS was inversely correlated with some of the key metabolic changes across the time period examined including the increases CH=CH and CH=CHCH2 lipid resonances which accompany apoptosis. This study demonstrates how combined transcriptomic and metabolomic studies of tumors can be used to understand the molecular events that accompany well documented metabolic perturbations during cell death processes.
Tsang TA, Woodman B, McLoughlin GA, et al., 2006, Metabolic characterization of the R6/2 transgenic mouse model of Huntington's disease by high-resolution MAS <SUP>1</SUP>H NMR spectroscopy, JOURNAL OF PROTEOME RESEARCH, Vol: 5, Pages: 483-492, ISSN: 1535-3893
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- Citations: 101
Griffin JL, 2006, The Cinderella story of metabolic profiling: does metabolomics get to go to the functional genomics ball?, Philos Trans R Soc Lond B Biol Sci, Vol: 361, Pages: 147-161, ISSN: 0962-8436
To date most global approaches to functional genomics have centred on genomics, transcriptomics and proteomics. However, since a number of high-profile publications, interest in metabolomics, the global profiling of metabolites in a cell, tissue or organism, has been rapidly increasing. A range of analytical techniques, including 1H NMR spectroscopy, gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), Fourier Transform mass spectrometry (FT-MS), high performance liquid chromatography (HPLC) and electrochemical array (EC-array), are required in order to maximize the number of metabolites that can be identified in a matrix. Applications have included phenotyping of yeast, mice and plants, understanding drug toxicity in pharmaceutical drug safety assessment, monitoring tumour treatment regimes and disease diagnosis in human populations. These successes are likely to be built on as other analytical and bioinformatic approaches are developed to fully exploit the information obtained in metabolic profiles. To assist in this process, databases of metabolomic data will be necessary to allow the passage of information between laboratories. In this prospective review, the capabilities of metabolomics in the field of medicine will be assessed in an attempt to predict the impact this 'Cinderella approach' will have at the 'functional genomic ball'.
Griffin JL, 2006, The Potential of Metabonomics in Toxicology, Biological Concepts and Techniques in Toxicology: An Integrated Approach, Pages: 47-60, ISBN: 9780824729790
With recent increased interests in systems biology, a number of analytical approaches have been developed to globally profile a tier of organization in a cell, tissue, or organism. Genomics, the first of the "-omic" technologies to embrace global analysis, describes the genes present in an organism. This approach is notably compared with other -omic approaches in that it is not context dependent, with a genome not being directly influenced by the environment. Large-scale genome projects are now being complemented by other -omic strategies, including transcriptomics, proteomics, and metabolomics or metabonomics to profile all the mRNA, proteins, or small molecule metabolites in a tissue, cell, or organism. One of the major challenges of these "-omic" technologies is that the transcriptome, proteome, and metabolome are all context dependent and will vary with pathology, development stage, and environmental factors. Thus, the possibility of globally profiling the transcriptome, proteome, or metabolome of an organism is a real analytical challenge, because by definition these efforts must also take into consideration all factors that influence metabolism. However, one major advantage that metabonomics has over the other "-omic" approaches is that the analytical approaches are relatively cheap on a per sample basis, suggesting that databases which embrace both environmental and genomic influences on the metabolism of a given cell, tissue, organ, or even organism may be possible.
Sadowski PG, Dunkley TPJ, Shadforth IP, et al., 2006, Quantitative proteomic approach to study subcellular localization of membrane proteins., Nat Protoc, Vol: 1, Pages: 1778-1789
As proteins within cells are spatially organized according to their role, knowledge about protein localization gives insight into protein function. Here, we describe the LOPIT technique (localization of organelle proteins by isotope tagging) developed for the simultaneous and confident determination of the steady-state distribution of hundreds of integral membrane proteins within organelles. The technique uses a partial membrane fractionation strategy in conjunction with quantitative proteomics. Localization of proteins is achieved by measuring their distribution pattern across the density gradient using amine-reactive isotope tagging and comparing these patterns with those of known organelle residents. LOPIT relies on the assumption that proteins belonging to the same organelle will co-fractionate. Multivariate statistical tools are then used to group proteins according to the similarities in their distributions, and hence localization without complete centrifugal separation is achieved. The protocol requires approximately 3 weeks to complete and can be applied in a high-throughput manner to material from many varied sources.
Pears MR, Cooper JD, Mitchison HM, et al., 2005, High resolution 1H NMR-based metabolomics indicates a neurotransmitter cycling deficit in cerebral tissue from a mouse model of Batten disease., J Biol Chem, Vol: 280, Pages: 42508-42514, ISSN: 0021-9258
The neuronal ceroid lipofuscinoses (NCLs) constitute a range of progressive neurological disorders primarily affecting children. Although six of the causative genes have been characterized, the underlying disease pathogenesis for this family of disorders is unknown. Using a metabolomics approach based on high resolution 1H NMR spectroscopy of the cortex, cerebellum, and remaining regions of the brain in conjunction with statistical pattern recognition, we report metabolic deficits associated with juvenile NCL in a Cln3 knock-out mouse model. Tissue from Cln3 null mutant mice aged 1-6 months was characterized by an increased glutamate concentration and a decrease in -amino butyric acid (GABA) concentration in aqueous extracts from the three regions of the brain. These changes are consistent with the reported altered expression of genes involved in glutamate metabolism in older mice and imply a change in neurotransmitter cycling between glutamate/glutamine and the production of GABA. Further variations in myo-inositol, creatine, and N-acetyl-aspartate were also identified. These metabolic changes were distinct from the normal aging/developmental process. Together, these changes represent the first documented pre-symptomatic symptoms of the Cln3 mouse at 1 month of age and demonstrate the versatility of 1H NMR spectroscopy as a tool for phenotyping mouse models of disease.
Lowe JP, Stuckey DJ, Awan FR, et al., 2005, MRS reveals additional hexose <i>N</i>-acetyl resonances in the brain of a mouse model for Sandhoff disease, NMR IN BIOMEDICINE, Vol: 18, Pages: 517-526, ISSN: 0952-3480
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- Citations: 12
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