Mass Spectrometry-Based Methods for the Determination of Sulfur and Related Metabolite Concentrations in Cell Extracts

Godat, Emmanuel; Madalinski, Geoffrey; Muller, Ludovic; Heilier, Jean-François; Labarre, Jean; Junot, Christophe

doi:10.1016/s0076-6879(10)73002-0

Cited by 14 publications

(12 citation statements)

References 55 publications

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“…Another explanation could simply be the fact that GSH is a competitive inhibitor of ␥-GCS with respect to Glu (32). Considering that the K m for Glu is 1.2 mM (32) and that in yeast Glu intracellular concentration is more than 20 mM (45,46), doubling GSH concentration from 3 to 6 mM may not significantly decrease ␥-GCS activity. We thus suggest that the notion that ␥-GCS would be the "rate-limiting enzyme" of the GSH synthesis pathway, a notion mainly based on in vitro data (3), should be reconsidered, at least in yeast, in the light of in vivo data.…”

Section: Discussionmentioning

confidence: 99%

Glutathione Degradation Is a Key Determinant of Glutathione Homeostasis

Baudouin‐Cornu

Lagniel

Kumar

et al. 2012

Journal of Biological Chemistry

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Background: Intracellular concentration of glutathione, an essential sulfur compound, is tightly controlled. Results: In yeast, glutathione degradation is faster than previously published, and glutathione intracellular concentration does not affect its synthesis. Conclusion: Glutathione degradation is a key determinant of glutathione homeostasis. Significance: This work challenges notions on glutathione synthesis and degradation, which were considered as established.

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Section: Discussionmentioning

confidence: 99%

Glutathione Degradation Is a Key Determinant of Glutathione Homeostasis

Baudouin‐Cornu

Lagniel

Kumar

et al. 2012

Journal of Biological Chemistry

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“…All quantification was performed with two different types of pentafluorophenyl columns (C18-PFP; 2.1mm × 150mm, 3μm particle, by Mac-Mode Analytical [Chadds Ford, PA], and Epic-PFP; 2.1mm × 150mm,3μm particle, by ES Industries [West Berlin, NJ]) (Godat et al 2010; Yang et al 2010). Material from a single extraction was used for both column types.…”

Section: Methodsmentioning

confidence: 99%

Validation of a dual LC–HRMS platform for clinical metabolic diagnosis in serum, bridging quantitative analysis and untargeted metabolomics

2013

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Mass spectrometry-based metabolomics is a rapidly growing field in both research and diagnosis. Generally, the methodologies and types of instruments used for clinical and other absolute quantification experiments are different from those used for biomarkers discovery and untargeted analysis, as the former requires optimal sensitivity and dynamic range, while the latter requires high resolution and high mass accuracy. We used a Q-TOF mass spectrometer with two different types of pentafluorophenyl (PFP) stationary phases, employing both positive and negative ionization, to develop and validate a hybrid quantification and discovery platform using LC-HRMS. This dual-PFP LC-MS platform quantifies over 50 clinically relevant metabolites in serum (using both MS and MS/MS acquisitions) while simultaneously collecting high resolution and high mass accuracy full scans to monitor all other co-eluting non-targeted analytes. We demonstrate that the linearity, accuracy, and precision results for the quantification of a number of metabolites, including amino acids, organic acids, acylcarnitines and purines/pyrimidines, meets or exceeds normal bioanalytical standards over their respective physiological ranges. The chromatography resolved highly polar as well as hydrophobic analytes under reverse-phase conditions, enabling analysis of a wide range of chemicals, necessary for untargeted metabolomics experiments. Though previous LC-HRMS methods have demonstrated quantification capabilities for various drug and small molecule compounds, the present study provides an HRMS quant/qual platform tailored to metabolic disease; and covers a multitude of different metabolites including compounds normally quantified by a combination of separate instrumentation.

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“…Liquid chromatography-mass spectrometry (LC-MS) analysis used 20-ml injection volumes. Chromatographic separation (2.1 · 250 mm, 5 mm Discovery HS-F5 column; Supelco) used a water-to-acetonitrile gradient (Godat et al 2010) and was followed by detection on an LTQ-Orbitrap XL hybrid mass spectrometer equipped with an IonMax electrospray ionization source (Thermo Fisher Scientific, Waltham, MA). For the G307S data set, a fourfold dilution series of a mixture of 17 metabolite standards was added to a pooled cell extract that contained equal volumes from each experimental sample, and was then used for metabolite identification and calibration.…”

Section: Metabolite Measurementsmentioning

confidence: 99%

“…Equal numbers of cells from log-phase cultures were harvested 12 hr after inoculation. Metabolite extraction combined previously described methods (Canelas et al 2008;Boer et al 2010;Godat et al 2010) as follows: 8.0 · 10 8 (G307S data set) or 1.9 · 10 9 (V320A data set) cells were pelleted by centrifugation at 3200 · g. The cell pellets were resuspended with 9.5 ml of their spent medium supernatant, then quenched with 20 ml 280°methanol. The cells were pelleted at 4000 · g at 210°in a rotor (Sorvall SS-34), prechilled to 280°, and then resuspended with 1.0 ml of 4°extraction solvent [0.1% perchloric acid with 400 mM glycine-1-13 C, 15 N (Sigma 299340) and 20 mM isotopically labeled methionine-13 C 5 , 15 N (Sigma 608106)].…”

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confidence: 99%

Surrogate Genetics and Metabolic Profiling for Characterization of Human Disease Alleles

et al. 2012

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Cystathionine-b-synthase (CBS) deficiency is a human genetic disease causing homocystinuria, thrombosis, mental retardation, and a suite of other devastating manifestations. Early detection coupled with dietary modification greatly reduces pathology, but the response to treatment differs with the allele of CBS. A better understanding of the relationship between allelic variants and protein function will improve both diagnosis and treatment. To this end, we tested the function of 84 CBS alleles previously sequenced from patients with homocystinuria by ortholog replacement in Saccharomyces cerevisiae. Within this clinically associated set, 15% of variant alleles were indistinguishable from the predominant CBS allele in function, suggesting enzymatic activity was retained. An additional 37% of the alleles were partially functional or could be rescued by cofactor supplementation in the growth medium. This large class included alleles rescued by elevated levels of the cofactor vitamin B6, but also alleles rescued by elevated heme, a second CBS cofactor. Measurement of the metabolite levels in CBS-substituted yeast grown with different B6 levels using LC-MS revealed changes in metabolism that propagated beyond the substrate and product of CBS. Production of the critical antioxidant glutathione through the CBS pathway was greatly decreased when CBS function was restricted through genetic, cofactor, or substrate restriction, a metabolic consequence with implications for treatment.T HE first complete human genome sequence seeded the defining challenge of human genetics for the foreseeable future: interpreting the impact of variations in the sequences of individual human genomes. Comparative genome sequencing reveals an average of one single-nucleotide change per 1200 bp between any two individuals. In the absence of strong Mendelian inheritance and linkage, confirming that any human genotype actually caused a phenotype is a significant challenge given the approximately 3 million genetic variants per person. Indeed, 4000 traits of medical interest show evidence for inheritance but lack a clear determinant (Online Mendelian Inheritance in Man 2012). Next-generation sequencing within small pedigrees (Ng et al. 2010a,b;Fan et al. 2011), or a more narrowly defined clinical phenotype (Schubert et al. 1997), can sometimes disentangle the underlying contribution of a gene to disease. In this work we have taken an approach that complements both increased sequencing capacity and expanded phenotypic description. We used surrogate genetics to assay directly the function of allelic variants and then evaluate their potential contribution to phenotypes of clinical importance.Homocystinuria, elevated levels of the sulfur-containing metabolite homocystine in the urine, illustrates several Reference numbers for publicly available data; GenBank: L14577.1 (CBS); dbSNP: rs17849313 (A69P), rs2229413 (P70L), rs11700812 (R369P); SGD: YGR155W (CYS4) and YDR232W (HEM1 challenges inherent to elucidating the molecular bases of human geneti...

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Mass Spectrometry-Based Methods for the Determination of Sulfur and Related Metabolite Concentrations in Cell Extracts

Cited by 14 publications

References 55 publications

Glutathione Degradation Is a Key Determinant of Glutathione Homeostasis

Glutathione Degradation Is a Key Determinant of Glutathione Homeostasis

Validation of a dual LC–HRMS platform for clinical metabolic diagnosis in serum, bridging quantitative analysis and untargeted metabolomics

Surrogate Genetics and Metabolic Profiling for Characterization of Human Disease Alleles

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