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At least 19 recordsLinked to original sources
PubMed2005

Liquid chromatography-mass spectrometry and 15N metabolic labeling for quantitative metabolic profiling.

Metabolomics, i.e., the global analysis of cellular metabolites, is becoming a powerful tool for gaining insights into biological functions in the postgenomic context. However, absolute quantitation of endogenous metabolites in biological media remains an issue, and available technologies for the analysis of metabolome still lack robustness and accuracy. We describe here a new method based on liquid chromatography-mass spectrometry and (15)N uniform metabolic labeling of Saccharomyces cerevisiae for accurate and absolute quantitation of nitrogen-containing cell metabolites in metabolic profiling experiments. As a proof of concept study, eight sulfur metabolites involved in the glutathione biosynthesis pathway (i.e., cysteine, homocysteine, methionine, gamma-glutamylcysteine, cystathionine, reduced and oxidized forms of glutathione, and S-adenosylhomocysteine) were simultaneously quantified. The analytical method has been validated by studies of stability, selectivity, precision, and linearity and by the determination of the limits of detection and quantification. It was then applied to the analysis of extracts from cadmium-treated yeasts. In these conditions, the intracellular concentrations of most of the metabolites involved in the glutathione biosynthesis pathway were increased when compared to control extracts. These data correlate with previous proteomic results and also underline the importance of glutathione in cadmium detoxication.

Cadmium↗
PubMed1991

Metabolic labeling of Dirofilaria immitis third- and fourth-stage larvae and their excretory-secretory products.

Infective third-stage larvae of Dirofilaria immitis were collected from Aedes aegypti and cultured in vitro to the fourth stage. Larval proteins were labeled metabolically using [35S]cysteine and methionine in different media and for different lengths of time. Labeled proteins in the excretory-secretory component and the larval homogenates were evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions and by 2-dimensional gel electrophoresis. Numerous proteins ranging from 14 to greater than 200 kDa were identified from both the excretory-secretory components and the larval homogenates. Both fractions demonstrated shared and unique proteins. Using timed labeling, age- and stage-specific proteins were identified; at least 2 proteins of approximately 20.5 and 22 kDa were associated in time with the molt from the third to fourth stage. Two proteins of the same molecular weight were specifically recognized by immune dog sera, but not by sera of their infected nonimmune cohorts.

Animals↗
PubMed1993

The glycosylphosphatidylinositol anchor of the trypomastigote-specific Tc-85 glycoprotein from Trypanosoma cruzi. Metabolic-labeling and structural studies.

The Tc-85 glycoprotein, specific for the infective stage of Trypanosoma cruzi, is anchored via glycosylphosphatidylinositol. The protein was purified from parasites, labeled metabolically with palmitic acid, by immunoprecipitation with the H1A10 monoclonal antibody or by affinity column chromatography on wheat germ agglutinin. Antisera to the soluble form of the variant surface glycoprotein of Trypanosoma brucei brucei cross-reacted with Tc-85 when the immunoprecipitate was analysed by Western blotting. The reaction was intensified upon previous incubation of the glycoprotein with phosphatidylinositol-specific phospholipase C. Such recognition was abolished when the cyclic phosphate was opened by mild acid treatment. The lipid cleaved by phospholipase C digestion, was identified as 1-O-hexadecylglycerol by reverse-phase thin-layer chromatography. The glycan core was deaminated and chemically labeled by reduction with NaB3H4. The labeled glycoprotein was exhaustively treated with pronase and dephosphorylated with 50% HF. Although microheterogeneity of the oligosaccharide moiety was apparent, by thin layer chromatography, a main spot coincident with Man(alpha 1-2) Man(alpha 1-6) Man(alpha 1-4) anhydromannitol was shown, consistent with the conserved core structure of all glycosylphosphatidylinositol anchors analysed to date.

Animals↗
PubMed2003

Metabolic labeling of C. elegans and D. melanogaster for quantitative proteomics.

A crucial issue in comparative proteomics is the accurate quantification of differences in protein expression levels. To achieve this, several methods have been developed in which proteins are labeled with stable isotopes either in vivo via metabolic labeling or in vitro by protein derivatization. Although metabolic labeling is the only way to obtain labeling of all proteins, it has thus far only been applied to single- celled organisms and cells in culture. Here we describe quantitative 15N metabolic labeling of the multicellular organisms Caenorhabditis elegans, a nematode, and Drosophila melanogaster, the common fruit fly, achieved by feeding them on 15N-labeled Escherichia coli and yeast, respectively. The relative abundance of individual proteins obtained from different samples can then be determined by mass spectrometry (MS). The applicability of the method is exemplified by the comparison of protein expression levels in two C. elegans strains, one with and one without a germ line. The methodology described provides tools for accurate quantitative proteomic studies in these model organisms.

Animals↗
PubMedDate not supplied

Metabolic labeling of human primary retinal pigment epithelial cells for accurate comparative proteomics.

Metabolic labeling was evaluated, using both 13C6-Arg and 13C6, 15N2-Lys amino acids, for a primary human retinal pigment epithelial cell (hRPE) culture prepared from an autopsy eye of an 81 year old donor. Satisfactory incorporation (>90%) was achieved with both stable isotope labeled amino acids after four passages (roughly 7 population doublings). The degree of incorporation was found to be efficient with both amino acids as well as in different proteins. The presence of 10% whole serum in the culture medium did not interfere with the incorporation of the exogenous stable isotope labeled amino acids. Metabolic labeling of these human primary retinal pigment epithelial cells was further tested to quantify protein ratios between proliferating and resting cells using a combination of 2-DG and MALDI-TOF-TOF/MS analysis. Using computational data processing and analysis, we obtained accurate protein ratio measurement for every single identified protein (156 proteins) in the 2-Dg array. Of these 156 proteins, 12 proteins were found significantly increased in dividing versus resting cells by at least a factor of 1.5 while 13 other proteins were found increased in resting versus dividing cells by at least the same fold. Most of these differentially expressed proteins are directly involved in cell proliferation, protein synthesis, and actin-remodeling and differentiation.

Aged↗
PubMed2000

Combining enhanced metabolic labeling with immunoblotting to detect interactions of endogenous cellular proteins.

Metabolic labeling, immunoblotting and two-dimensional isoelectric focusing/SDS-PAGE are powerful techniques for characterizing endogenously expressed cellular proteins and their interactions. We achieved improved resolution and sensitivity for the detection of metabolically labeled proteins separated on two-dimensional gels by electroblotting the proteins onto polyvinylidene difluoride or nitrocellulose membranes and detecting the 35S signal on a bio-image analyzer. We obtained independent detection of specific proteins from the same blot by subsequent rehydration of the membrane and immunoblot analysis. The combination of these enhanced detection techniques with immunoprecipitation and two-dimensional electrophoresis on precast minigels provides a simple, sensitive method for detecting interactions between endogenous proteins in the cell.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Radiolabeling revisited: metabolic labeling with (35)S-methionine inhibits cell cycle progression, proliferation, and survival.

Metabolic labeling of cells with low-energy beta-emitting radioisotopes such as [(35)S]methionine is often used to follow the biosynthesis, maturation, and degradation of proteins in vivo. Such techniques have generally been assumed to be relatively nonperturbing to the cell. The results presented here indicate that metabolic labeling of cells with [(35)S]methionine under standard experimental conditions can inhibit cell progression into mitosis, cause cell cycle arrest, inhibit cell proliferation in both short-term and colony-forming assays, alter cell morphology, and induce apoptosis over the course of several days. These results thus suggest the need for caution in interpretation of studies using such methods, especially if the experiments rely on the normal progression of the cell cycle or are intended to monitor events occurring in a normally proliferating cell.

Animals↗
PubMed2006

Comparative proteomic analysis of metabolically labelled proteins from Plasmodium falciparum isolates with different adhesion properties.

The virulence of Plasmodium falciparum relates in part to the cytoadhesion characteristics of parasitized erythrocytes but the molecular basis of the different qualitative and quantitative binding phenotypes is incompletely understood. This paucity of information is due partly to the difficulty in working with membrane proteins, the variant nature of these surface antigens and their relatively low abundance. To address this two-dimensional (2D) protein profiles of closely related, but phenotypically different laboratory strains of P. falciparum have been characterized using proteomic approaches. Since the mature erythrocyte has no nucleus and no protein synthesis capability, metabolic labelling of proteins was used to selectively identify parasite proteins and increase detection sensitivity.A small number of changes (less than 10) were observed between four different P. falciparum laboratory strains with distinctive cytoadherence properties using metabolic labelling, with more parasite protein changes found in trophozoite iRBCs than ring stage. The combination of metabolic labelling and autoradiography can therefore be used to identify parasite protein differences, including quantitative ones, and in some cases to obtain protein identifications by mass spectrometry. The results support the suggestion that the membrane protein profile may be related to cytoadherent properties of the iRBCs. Most changes between parasite variants were differences in iso-electric point indicating differential protein modification rather than the presence or absence of a specific peptide.

Animals↗
PubMed1984

Metabolic labelling and characterisation of S-antigens, the heat-stable, strain-specific antigens of Plasmodium falciparum.

The C-10 clone of Plasmodium falciparum was metabolically labelled with [3H]glycine. Analysis by sodium dodecylsulphate/polyacrylamide gel electrophoresis (SDS/PAGE) revealed that S-antigens were the only significantly labelled products released into culture supernatants by the end of the asexual cycle. This result indicates conclusively that heat-stable, strain-specific antigens (the 'S-antigens') emanate from the parasite and not the host. The S-antigen of clone C-10 was resolved on SDS/PAGE as two labelled products with molecular weights of 156000 +/- 5000 and 130000 +/- 2000. The two components were differentially susceptible to proteolysis with trypsin. In other experiments, boiled plasma from Aotus monkeys infected with a different isolate of P. falciparum was partially purified by isoelectric focusing and the fraction containing S-antigen was iodinated by the Iodogen procedure. Analysis by SDS/PAGE of immunoprecipitated material revealed an iodinated component corresponding to the lower molecular weight band of the metabolically labelled S-antigen.

Animals↗
PubMed1992

Mucin changes in ileoanal pouches monitored by metabolic labelling and histochemistry.

The pattern of mucin synthesis and secretion in mucosal biopsies from the proximal ileum, distal ileum, rectum and pouch before and after ileostomy closure was monitored in patients undergoing restorative proctocolectomy by metabolic labelling with [3H]glucosamine and [35S]sulphate and compared with the mucin histochemistry in each patient. Metabolic labelling clearly demonstrated a reduction in sulphation associated with the rectal mucosa in colitis. Significant differences in the turnover of isolated secreted mucin between proximal and distal ileum and rectum were also found, as was a metaplastic change towards a colonic-type mucosa in the pouch before and after ileostomy closure relative to the proximal and distal ileum, and rectum. This technique can be used to monitor colonic metaplasia in the pouch mucosa and is suitable for comparative studies where mucin changes are implicated.

Adult↗
PubMed1981

Metabolic labeling of lutropin with [35S]sulfate.

Chemical analyses have previously detected sulfate linked to the oligosaccharides of lutropin isolated from bovine and human pituitaries. To determine whether lutropin could be metabolically labeled with sulfate, isolated bovine and rat pituitaries were incubated with [35S]sulfate. In both species, two major labeled products were immunoprecipitated with antisera specific to lutropin subunits. Incorporation into the subunits occurred posttranslationally since it was not blocked by cycloheximide, which did, however, block the incorporation of radiolabeled methionine. Metabolic labeling with [35S]sulfate provides a valuable approach for examining the biosynthetic processing of lutropin and the physiological role of sulfate in this hormone.

Animals↗
PubMed2006

Metabolic labeling of plant cell cultures with K(15)NO3 as a tool for quantitative analysis of proteins and metabolites.

Strategies for robust quantitative comparison between different biological samples are of high importance in experiments that address biological questions beyond the establishment of protein lists. Here, we propose the use of 15N-KNO3 as the only nitrogen source in Arabidopsis cell cultures in order to achieve a metabolically fully labeled cell population. Proteins from such metabolically labeled culture are distinguishable from unlabeled protein populations by a characteristic mass shift that depends on the amino acid composition of the tryptic peptide analyzed. In addition, the metabolically labeled cell extracts are also suitable for comparative quantitative analysis of nitrogen-containing cellular metabolic complement. Protein extracts from unlabeled and from standardized 15N-labeled cells were combined into one sample for joined analytical processing. This has the advantage of (i) reduced experimental variability and (ii) immediate relative quantitation at the level of single extracted peptide and metabolite spectra. Together ease and accuracy of relative quantitation for profiling experiments is substantially improved. The metabolic labeling strategy has been validated by mixtures of protein extracts and metabolite extracts from the same cell cultures in known ratios of labeled to unlabeled extracts (1:1, 1:4, and 4:1). We conclude that saturating metabolic 15N-labeling provides a robust and affordable integrative strategy to answer questions in quantitative proteomics and nitrogen focused metabolomics.

Journal Article↗
PubMed2001

Quantitative analysis of bacterial and mammalian proteomes using a combination of cysteine affinity tags and 15N-metabolic labeling.

We describe the combined use of 15N-metabolic labeling and a cysteine-reactive biotin affinity tag to isolate and quantitate cysteine-containing polypeptides (Cys-polypeptides) from Deinococcus radiodurans as well as from mouse B16 melanoma cells. D. radiodurans were cultured in both natural isotopic abundance and 15N-enriched media. Equal numbers of cells from both cultures were combined and the soluble proteins extracted. This mixture of isotopically distinct proteins was derivatized using a commercially available cysteine-reactive reagent that contains a biotin group. Following trypsin digestion, the resulting modified peptides were isolated using immobilized avidin. The mixture was analyzed by capillary reversed-phase liquid chromatography (LC) online with ion trap mass spectrometry (MS) as well as Fourier transform ion cyclotron resonance (FTICR) MS. The resulting spectra contain numerous pairs of Cyspolypeptides whose mass difference corresponds to the number of nitrogen atoms present in each of the peptides. Designation of Cys-polypeptide pairs is also facilitated by the distinctive isotopic distribution of the 15N-labeled peptides versus their 14N-labeled counterparts. Studies with mouse B16 cells maintained in culture allowed the observation of hundreds of isotopically distinct pairs of peptides by LC-FTICR analysis. The ratios of the areas of the pairs of isotopically distinct peptides showed the expected 1:1 labeling of the 14N and 15N versions of each peptide. An additional benefit from the present strategy is that the 15N-labeled peptides do not display significant isotope-dependent chromatographic shifts from their 14N-labeled counterparts, therefore improving the precision for quantitating peptide abundances. The methodology presented offers an alternate, cost-effective strategy for conducting global, quantitative proteomic measurements.

Animals↗
PubMed1983

Identification of surface and internal antigens from spontaneously released Plasmodium falciparum merozoites by radio-iodination and metabolic labelling.

Spontaneously released merozoites from synchronous Plasmodium falciparum cultures were isolated in the presence of protease blocker. 1-5 X 10(10) merozoites were obtained in each experiment. The isolated merozoites possessed a thick surface coat and about 80% were invasive to human erythrocytes although they did not subsequently develop into ring stages. Tests using several analytical methods showed the merozoite preparations to be free of any erythrocyte contamination. Six labelled proteins were identified after surface radio-iodination, the largest with a molecular weight of 82 000. All six proteins were precipitated with various immune sera. Four other proteins with molecular weights of 200 000 and 160 000-145 000 (a triplet) were identified by precipitation with the same immune sera after metabolically labelling the merozoites. The six surface proteins were not prominent in the metabolically labelled preparations. Using these methods it is possible to identify and differentiate between surface and internal merozoite antigens.

Animals↗
PubMed2006

High-throughput quantitation of metabolically labeled anionic glycoconjugates by scintillation proximity assay utilizing binding to cationic dyes.

Rapid, quantitative methods suited to a large number of samples are required for studies into the determination of disease etiology and in the evaluation of drugs and biological agents. This chapter describes an assay for anionic glycoconjugates (GCs), including glycosaminoglycans, which are major gene products of chondrocytes appearing in the extracellular matrix. The assay utilizes the electrostatic interaction between negatively charged sulfate and carboxyl groups of anionic GCs synthesized and secreted by chondrocytes with the cationic dye Alcian blue, immobilized to scintillant-coated 96-well plates. Metabolic labeling with D-[1, 6-3H (N)]-glucosamine allows all anionic GCs, including cartilage-specific and hyperglycosylated variants of fibronectin, to be quantitated. If Na235SO4 is used for the metabolic labeling instead, only glycosaminoglycans and proteoglycans will be quantitated. The samples are counted using a multi-detector instrument for scintillation proximity assays, such as the Wallac 1450 Microbeta Trilux, designed for detection of samples in 96-well plates and, as such, can be a high-throughput system. The bound anionic GCs can be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after quantitation by elution with denaturing buffers. The method can be modified to include predigestion of the sample with a specific lyase, e.g., chondroitinase ABC or testicular hyaluronidase. To separate polyanions from other digested material after ethanol precipitation, the sample can be assayed as described in this chapter for a particular subtype of anionic GC. This assay addresses the need for high-throughput applications in arthritis and other medical and biological problems.

Alcian Blue↗
PubMed2002

Metabolic labelling of membrane microdomains/rafts in Jurkat cells indicates the presence of glycerophospholipids implicated in signal transduction by the CD3 T-cell receptor.

Cell membranes contain sphingolipids and cholesterol, which cluster together in distinct domains called rafts. The outer-membrane leaflet of these peculiar membrane domains contains glycosylphosphatidylinositol-anchored proteins, while the inner leaflet contains proteins implicated in signalling, such as the acylated protein kinase p56(lck) and the palmitoylated adaptator LAT (linker for activation of T-cells). We present here an approach to study the lipid composition of rafts and its change upon T-cell activation. Our method is based on metabolic labelling of Jurkat T-cells with different precursors of glycerophospholipid synthesis, including glycerol and fatty acids with different lengths and degrees of saturation as well as phospholipid polar head groups. The results obtained indicate that lipid rafts isolated by the use of sucrose density-gradient centrifugation after Triton X-100 extraction in the cold, besides sphingolipids and cholesterol, contain unambiguously all classes of glycerophospholipids: phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine and phosphatidylcholine. Fatty acid labelling shows that lipid rafts are labelled preferentially with saturated fatty acids while the rest of the plasma membrane incorporates mostly long-chained polyunsaturated fatty acids. To see whether the raft composition as measured by metabolic labelling of phospholipids is involved in T-cell activation, we investigated the production of sn-1,2-diacylglycerol (DAG) in CD3-activated cells. DAG production occurs within rafts, confirming previous demonstration of protein kinase C translocation into membrane microdomains. Our data demonstrate that raft disorganization by methyl-beta-cyclodextrin impairs both CD3-induced DAG production and changes in cytosolic Ca(2+) concentration. These lines of evidence support the conclusion that the major events in T-cell activation occur within or due to lipid rafts.

Arachidonic Acid↗
PubMed1986

A new metabolic labelling medium for Trichomonas vaginalis and Tritrichomonas foetus using 35S methionine.

A metabolic labelling medium was devised for Trichomonas vaginalis and Tritrichomonas foetus utilizing 35S methionine. T. vaginalis cultured for 24h in the medium took up approximately 27% of the available label and increased greater than two fold in number. Counts per microgram of protein were 32,555 +/- 10% between different strains or identical strains in different labelling runs. T. foetus took up approximately 5% of the available label and increased greater than two fold in 24h. This resulted in specific labelling of 12,704 cpm/ug protein +/- 10% between different runs with the same strain.

Animals↗
PubMed2025

Assessment of mRNA Decay and Calculation of Codon Occurrence to mRNA Stability Correlation Coefficients after 5-EU Metabolic Labeling.

mRNA translation and decay are tightly connected. This chapter describes a method to assess the influence of each codon identity on mRNA stability in cultured cells. The technique involves metabolic labeling of the nascent mRNAs by addition of the nucleoside analog 5-ethynyluridine (5-EU), purification of the RNA at different time-points after chase of the 5-EU, then biotinylation with Click chemistry, pull-down, and sequencing. The transcripts' half-lives are calculated from the expression level of each mRNA at the different time-points. Finally, the method describes the calculation of the Codon occurrence to mRNA Stability correlation Coefficient, or CSC, as a correlation between the codon occurrence in a transcript and the transcript half-life, for each codon.

RNA Stability↗