Stable isotope ¹³C labeling is an important tool to analyze cellular metabolic flux. The ¹³C distribution in intracellular metabolites can be detected via mass spectrometry and used as a constraint in intracellular metabolic flux calculations. Then, metabolic flux analysis algorithms can be employed to obtain the flux distribution in the corresponding metabolic reaction network. However, in addition to carbon, other elements such as oxygen in the nature also have natural stable isotopes (e.g., ¹⁷O, ¹⁸O). This makes the isotopic information of elements other than the ¹³C marker interspersed in the isotopic distribution measured by the mass spectrometry, especially that of the molecules containing many other elements, which leads to large errors. Therefore, it is essential to correct the mass spectrometry data before performing metabolic flux calculations. In this paper, we proposed a method for construction of correction matrix based on Python language for correcting the measurement errors due to natural isotope distribution. The method employed a basic power method for constructing the correction matrix with simple structure and easy coding implementation, which can be directly applied to data pre-processing in ¹³C metabolic flux analysis. The correction method was then applied to the intracellular metabolic flux analysis of ¹³C-labeled Aspergillus niger. The results showed that the proposed method was accurate and effective, which can serve as a reliable data correction method for accurate microbial intracellular metabolic flux analysis.
Metabolic Flux Analysis ; Isotope Labeling/methods* ; Carbon Isotopes/metabolism* ; Mass Spectrometry/methods* ; Metabolic Networks and Pathways

OBJECTIVETo evaluate the iron utilization in children using single stable isotopes tracer.

METHODS57 children aged from 10 to 12 from a primary school of Beijing in 2010 were selected, 30 of them were boys and 27 were girls. All the subjects were given 5 ml artificial enriched ⁵⁷FeSO₄ twice per day within 5 days, and the total amount of ⁵⁷Fe was 30 mg. 5 ml blood were taken at 1 day before and 14 days after test, and all the feces during the test were collected. The samples were detected by AAS and MC-ICP-MS after pre-treatment to determine the content and abundances of iron in samples, then the iron utilization in whole blood were calculated.

RESULTSThe blood volume of male and female subjects 14 days after test were (3.19 ± 0.41) and (3.15 ± 0.29) ml respectively, and there was no significantly difference (t = 1.13, P > 0.05) between them; The amount of ⁵⁷Fe intake by male and female subjects were (27.46 ± 0.25) and (27.29 ± 0.15) mg (t = 1.13, P > 0.05); The amount of ⁵⁷Fe in blood were (5.92 ± 0.71) and (6.30 ± 0.65) mg respectively (t = 2.29, P < 0.05); The iron utilization in whole blood at 14 days of male and female subjects were (20.41 ± 2.03)% and (22.04 ± 0.80)% respectively, male subjects were significantly lower than females (t = 2.51, P < 0.05).

CONCLUSIONSingle stable isotopes tracer can be used in iron utilization evaluation in children, and the iron utilization in whole blood of female children is higher than males.

Biological Availability ; Child ; Female ; Humans ; Iron ; blood ; Isotope Labeling ; methods ; Male

The stable isotope labeling by amino acids in culture (SILAC) based quantitative proteomics serves as a gold standard because of the high accuracy and throughput for protein identifications and quantification. In this study, we discussed the application of SILAC technology in mammal model, and developed quantitative internal standard for comparative proteomics of disease model. The C57BL/6J mice fed by special diet containing the 13C6-Lysine and bred F2 generation. We identified and analyzed total proteins of 9 mice tissues of F2 generation, including brain, lung, heart, stomach, intestine, liver, spleen, kidney, and muscle. Quantitative analysis information could evaluate the mice and different tissues' labeling efficiency. Liver was the most efficient, brain the least, and the labeling efficiency were 96.34%±0.90% and 92.62%±1.98% respectively. The average of the labeling efficiency of F2 generation was 95.80%±0.64%, which met the international standard (≥ 95%) for SILAC quantitative proteomics effective study. SILAC technology was successfully extended to mammalian model system, which will provide powerful tools for the mechanism study of the pathophysiology process with mouse model.
Amino Acids ; chemistry ; Animals ; Diet ; veterinary ; Isotope Labeling ; Lysine ; chemistry ; Mice ; Mice, Inbred C57BL ; Proteins ; chemistry ; Proteomics ; methods

OBJECTIVETo analysis the molecular interaction network of 14-3-3 sigma in non small cell lung cancer (NSCLC) cells.

METHODSEstablished stable over-expressed 14-3-3 sigma protein PG cells, MTT assay was used to assess the growth rate of PG cells. Though stable isotope labeling by amino acids in cell culture (SILAC) and Mass spectrometry (MS) technology, to identify difference expressed proteins caused by over expressed 14-3-3 sigma. The protein expressed >2 or <0.5 times was termed as the differential protein. By searching Human protein reference database (HPRD) and Kyoto encyclopedia of genes and genomes (KEGG), established the molecular interaction network of tumor suppressor gene 14-3-3 sigma.

RESULTSThe growth rate of over-expressed 14-3-3 sigma PG cell was obviously slower down compared to vector PG cells. A database including 147 differential protein was established. And a molecular interaction network of 14-3-3 sigma containing 26 protein was constructed.In this network, the expression of CSNK2A1 (casein kinase II subunit alpha), involved in numerous cellular processes, such as cell cycle progression, apoptosis and transcription, was the most significantly increased. A DNA repair protein, MEN1 (Menin) which functions as a transcriptional regulator was the most significantly decreased.

CONCLUSIONAfter stable transfected with 14-3-3 sigma gene, growth rate of PG cells was inhibited, the proteins associated with cell cycle, DNA damage repair mechanisms were significantly changed, and constructed the molecular interaction network.

14-3-3 Proteins ; genetics ; Amino Acids ; Biomarkers, Tumor ; genetics ; Carcinoma, Non-Small-Cell Lung ; genetics ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Exoribonucleases ; genetics ; Humans ; Isotope Labeling ; methods ; Lung Neoplasms ; genetics ; Mass Spectrometry ; Transfection

OBJECTIVEUsing the stable isotopes as the internal standard of microdialysis technology to establish a new method to study the whole and local brain dynamics of nicotine percutaneous preparations.

METHODUsing th healthy rats as experimental animals, administrating nicotine in abdominal transdermal way, then sample in the blood and brain simultaneously by microdialysis which use deuterium nicotine (DL-nicotine) as internal standard. Detecting the samples by LC-MS/MS method.

RESULTThe configuration process in blood and brain both conforms to 2 compartments model, t1/2 is 29.38 min, t1/2beta is 208.51 min, AUC(0-infinity) is 152 127.10 microg x min x L(-1) in the blood t1/2 is 86.64 min, t1/2beta is 386.00 min, AUC(0-infinity) is 152 820.90 microg x min x L(-1) in the brain.

CONCLUSIONDl-nicotine can be used as internal standard of nicotine to correcte the recovery; Stable isotopes internal standard microdialysis technology can be used for studing the whole and the local pharmacokinetic of nicotine and also provide new ideas and methods to studing the process of new drug delivery system.

Animals ; Brain ; metabolism ; Brain Chemistry ; Deuterium ; chemistry ; Isotope Labeling ; methods ; Male ; Microdialysis ; methods ; Nicotine ; blood ; pharmacokinetics ; Rats ; Rats, Sprague-Dawley

To evaluate the reagent 2-methoxy-4,5-dihydro-1H-imidazole used for isotopic labeling in quantitative proteomics, we synthesized 2-methoxy-4,5-dihydro-1H-imidazole and its tetradeuterated analog in three steps. Prior to tryptic cleavage, bovine serum albumin (BSA) was reduced and alkylated. Tryptic peptides were derivatized with an equal volume of either DO or D4 and D4-derivatized peptides were mixed with at variable ratio (from 10:1 to 1:5) prior to MS and MS/MS analysis. We used matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and Electro spray ionization-mass spectrometry (ESI-MS) to evaluate the quantitative capability of labeling. The specificity of the reagent is excellent: only lysine side chains were modified among tryptic peptides. MALDI and ESI ionization modes not only could achieve the quantification of differentially expressed proteins but also facilitate the de novo sequencing. This side-chain modification can be used for quantitative analysis with proteomic strategies involving liquid chromatography. Reverse phase liquid chromatography (RPLC) kept a good resolution, and the introduction of D atoms did not introduce a variation of retention time between heavy and light peptides in RPLC.
Imidazoles ; chemistry ; Isotope Labeling ; methods ; Lysine ; chemistry ; Peptides ; analysis ; chemistry ; Proteomics ; methods ; Serum Albumin, Bovine ; chemistry ; Spectrometry, Mass, Electrospray Ionization ; methods ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; methods

OBJECTIVETo detect the changes of heat shock protein(HSP) expression in human hepatocellular carcinoma HepG2 cells after treated by quercetin through a proteomics strategy termed SILAC (stable isotope labeling by amino acids in cell culture)-MS (mass spectrometry).

METHODSHepG2 cells cultured in d3-labeled DMEM medium were passaged for more than ten generations to reach an enough high labeling ratio. MTT assay was used to assess the inhibitory effect of quercetin on proliferation of HepG2 cells. In SILAC, total protein was extracted from control HepG2 cells and those treated by 50 µmol/L quercetin for 48 h, and then mixed to a 1:1 ratio. After in-gel digestion and idenfication by LC-MS/MS analysis, quantification informations of changed proteins were acquired by searching on Mascot 2.0 program (MatrixScience Ltd., London) against SWISS-PROT protein database. To ensure a high confidence level for identification, those peptides with Mascot scores below the threshold value were excluded from analysis and not included in the list of quantified proteins (P < 0.01). Protein abundance was calculated as ratios of the peak intensity of the fragment ions from the labeled versus the unlabeled peptides. RT-PCR was uesd to verify the reliability of HSPs changes by quercetin treatment from the SILAC-MS results.

RESULTSAfter passaged for ten generations, the d3-labeling ratio was above 95%. MTT showed that quercetin inhibited the proliferation of HepG2 cells obviously, with a IC(50) close to 50 µmol/L, and in a dose-dependent and time-dependent manner. The MS showed that the expression of almost all heat shock family proteins was down-regulated a lot. The expression of HSP90 exposed to quercetin for 48 h was decreased to 49.3% of the normal HepG2 cells, and the expression of HSP70 was decreased to 43.6% of the normal Hep G2 cells. Quantitation information showed that the expression of HSP90α, HSP76, HSP60 and HSP27 was declined to 59.3%, 44.2%, 51.3% and 62.6%, respectively. Those results demonstrated that the quantification for changed protiens by SILAC-MS was correct.

CONCLUSIONSQuercetin can exert a significant inhibitory effect on whole expression of heat shock proteins in HepG2 cells. We suppose this maybe one of the pathways through which quercetin plays an important anti-tumor role. SILAC-MS is a reliale technique and can be used to quantify the changes of whole protein spectrum in HepG2 cells before and after treatment with some exogeneous factors.

Amino Acids ; Antineoplastic Agents, Phytogenic ; pharmacology ; Cell Culture Techniques ; Cell Proliferation ; drug effects ; HSP70 Heat-Shock Proteins ; metabolism ; HSP90 Heat-Shock Proteins ; metabolism ; Hep G2 Cells ; Humans ; Isotope Labeling ; Mass Spectrometry ; methods ; Proteomics ; Quercetin ; pharmacology

DNA stable-isotope probing (DNA-SIP) is a recently developed method with which the incorporation of stable isotope from a labeled substrate is used to identify the function of microorganisms in the environment. The technique has now been used in conjunction with metagenomics to establish links between microbial identity and particular metabolic functions. The combination of DNA-SIP and metagenomics not only permits the detection of rare low-abundance species from metagenomic libraries but also facilitates the detection of novel enzymes and bioactive compounds. We summarize recent progress in SIP-metagenomic techniques and applications and discuss prospects for this combined approach in environmental microbiology and biotechnology.
Animals ; DNA ; genetics ; DNA Probes ; chemistry ; genetics ; metabolism ; DNA, Bacterial ; chemistry ; genetics ; metabolism ; Humans ; Isotope Labeling ; methods ; Metagenomics ; methods ; Molecular Probe Techniques ; Sequence Analysis, DNA ; methods

The paper is to report the study of pharmacokinetics of transdermal administered nicotine in the brain of freely moving rat by using microdialysis with stable labeled isotope as internal standard. The pharmacokinetic behavior of nicotine in Sprague Dawley rat brain was investigated after intranasal administration (3.75 mg). Brain fluid samples were collected by intracerebral microdialysis with DL-nicotine as internal standard. Concentrations of nicotine and DL-nicotine in the sample were measured by HPLC-MS/MS. Main pharmacokinetic parameters were calculated and analyzed by Das 2.0 pharmacokinetic software. The recovery of nicotine and the delivery of DL-nicotine were the same. The fate of absorption and distribution was two compartment model and the values of t1/2alpha was 170.31 min, t1/2beta was 263.30 min and the AUC(0-infinity) was 2.75 x 10(5) microg x L(-1) min separately. DL-nicotine can be used to calibrate the recovery of nicotine, and the new method of stable isotope microdialysis can be used to study the pharmacokinetics of freely moving rat. It will make sense for the treatment of addiction of tobacco and provide a new thought for the research of pharmacokinetics-pharmacodynamic combination.
Administration, Cutaneous ; Administration, Intranasal ; Animals ; Area Under Curve ; Brain ; metabolism ; Chromatography, High Pressure Liquid ; Deuterium ; Female ; Isotope Labeling ; methods ; Male ; Microdialysis ; methods ; Nicotine ; administration & dosage ; pharmacokinetics ; Rats ; Rats, Sprague-Dawley ; Tandem Mass Spectrometry

Amine-specific isobaric tagging (iTRAQ) reagents, as a new class of isobaric reagent, were developed in 2004, which, combined with liquid chromatography and tandem mass spectrometry (LC-MS/MS) , have been applied to the identification and quantification of proteins in a wide range of biological samples, including bacteria, yeasts, human tissues, cells, and fluids. As a new method of quantitative proteomics, the technique of iTRAQ allows for the quantitative analysis of four samples simultaneously and displays its advantages of high-flux, food reproducibility, and high sensitivity; it also provides a potential technological platform for studying the mechanisms of the development and progression of prostate cancer.
Humans ; Isotope Labeling ; methods ; Male ; Prostatic Neoplasms ; metabolism ; Proteomics ; methods