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

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

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

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

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

A simple, reliable and sensitive liquid chromatography-isotope dilution mass spectrometry (LC-ID/MS) was developed and validated for quantification of olanzapine in human plasma. Plasma samples (50 microL) were extracted with tert-butyl methyl ether and isotope-labeled internal standard (olanzapine-D3) was used. The chromatographic separation was performed on XBridge Shield RP 18 (100 mm x 2.1 mm, 3.5 microm, Waters). An isocratic program was used at a flow rate of 0.4 m x min(-1) with mobile phase consisting of acetonitrile and ammonium buffer (pH 8). The protonated ions of analytes were detected in positive ionization by multiple reactions monitoring (MRM) mode. The plasma method, with a lower limit of quantification (LLOQ) of 0.1 ng x mL(-1), demonstrated good linearity over a range of 0.1 - 30 ng x mL(-1) of olanzapine. Specificity, linearity, accuracy, precision, recovery, matrix effect and stability were evaluated during method validation. The validated method was successfully applied to analyzing human plasma samples in bioavailability study.
Antipsychotic Agents ; blood ; pharmacokinetics ; Benzodiazepines ; blood ; pharmacokinetics ; Biological Availability ; Chromatography, Liquid ; methods ; Humans ; Indicator Dilution Techniques ; Isotope Labeling ; Reproducibility of Results ; Sensitivity and Specificity ; Tandem Mass Spectrometry ; methods

To explore the preparation method of liposome-mediated 99m-technetium-labeled antisense oligonucleotides of c-myc mRNA and lay foundations for antisense imaging and treatment, antisense oligonucleotides (DNA) with 15 bases and di-functional chelate, hydrazino nicotinamide derivatives, were synthesized. After DNA combined with chelate, they were labeled with 99m-technetium to form compounds, 99mTc-chelate-DNA (99mTc-DNA), and were purified through Sep-Pak reverse column(C18, Waters) by using methanol and water as eluent. The leaching curve was made; the labeling efficiency was calculated. The products were then encapsulated with cation liposome to form liposome-mediated 99mTc-DNA. The radiochemical purity and stability of the liposome-mediated 99mTc-DNA were tested through strip chromatography. The labeling efficiency was 63.37% +/- 3.51% at the radioactive concentration of 1480 MBq, 62.52% +/- 3.69% at that of 740 MBq, 59.82% +/- 5.12% at that of 592 MBq. There were no significant differences between these labeling efficiencies. The radiochemical purity was 96.47% +/- 3.01%. The liposome-mediated radiolabeled antisense oligonucleotides were stable after incubation with water or serum. Therefore liposome-mediated radiolabeled antisense oligonucleotides could be obtained through hydrazino nicotinamide derivatives as di-functional chelate and liposome as vector.
DNA, Antisense ; chemical synthesis ; Drug Compounding ; methods ; Drug Stability ; Isotope Labeling ; methods ; Liposomes ; Proto-Oncogene Proteins c-myc ; genetics ; RNA, Messenger ; genetics ; Technetium

BACKGROUNDBacterial infection can pose a substantial diagnostic dilemma. (99m)Tc-labeled ciprofloxacin (CPF) was developed as a biologically active radiopharmaceutical to diagnose infection. In the present research, we studied the biodistribution and imaging properties of infection tracer (99m)Tc-CPF in a mouse model of infection.

METHODSCPF was labeled with (99m)Tc and the radiochemical purity and labeling rate were measured. A mouse model of infection was established. We then determined the biodistribution of (99m)Tc-CPF and conducted the whole body scintigraphy of the animal model.

RESULTS(99m)Tc-Ciprotech was stable for at least 6 hours at room temperature. The labeling rate of CPF by (99m)Tc was over 90%. Clearance of radioactivity mainly occurred in the liver and kidney, and the clearance from blood was rapid. Both biodistribution and imaging results showed higher uptake of (99m)Tc-CPF at sites of infection. The infectious tissue/normal tissue ratio peak was 4.30 at 4 hours after injection.

CONCLUSIONS(99m)Tc-CPF is a sensitive radiopharmaceutical for scintigraphy of infectious lesions and it is easy to prepare.

Animals ; Anti-Infective Agents ; chemistry ; pharmacokinetics ; Bacterial Infections ; diagnosis ; Ciprofloxacin ; chemistry ; pharmacokinetics ; Disease Models, Animal ; Isotope Labeling ; methods ; Mice ; Mice, Inbred BALB C ; Organotechnetium Compounds ; chemistry ; Tissue Distribution

AIMTo develop a rapid and sensitive method for monosaccharide composition analysis.

METHODSGlycoprotein was first hydrolyzed to monosaccharides, which were subsequently reacetylated (amino monosaccharides), tagged with 2-aminopyridine and then separated and monitored in selected ion mode by CapGCC-LC/MS. The use of tetradeuterium labeled-aminopyridyl-monosaccharides prepared by tagging monosacahrides with hexadeuterium labeled 2-aminopyridine as internal standards improved the linearity and reproducibility in quantification.

RESULTSThis method was successfully applied to monosaccharide composition analysis of model glycoproteins, fetuin and erythropoietin down to 1 pmol monosaccharides.

CONCLUSIONThis method has been shown to be highly sensitive and is applicable to monosaccharide composition analysis of glycoproteins.

Animals ; Chromatography, Liquid ; methods ; Erythropoietin ; chemistry ; Glycoproteins ; chemistry ; Hydrolysis ; Isotope Labeling ; Monosaccharides ; analysis ; Sensitivity and Specificity ; Spectrometry, Mass, Electrospray Ionization ; alpha-Fetoproteins ; chemistry