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PubMed2005

ZoomQuant: an application for the quantitation of stable isotope labeled peptides.

The main goal of comparative proteomics is the quantitation of the differences in abundance of many proteins between two different biological samples in a single experiment. By differentially labeling the peptides from the two samples and combining them in a single analysis, relative ratios of protein abundance can be accurately determined. Protease catalyzed (18)O exchange is a simple method to differentially label peptides, but the lack of robust software tools to analyze the data from mass spectra of (18)O labeled peptides generated by common ion trap mass spectrometers has been a limitation. ZoomQuant is a stand-alone computational tool that analyzes the mass spectra of (18)O labeled peptides from ion trap instruments and determines relative abundance ratios between two samples. Starting with a filtered list of candidate peptides that have been successfully identified by Sequest, ZoomQuant analyzes the isotopic forms of the peptides using high-resolution zoom scan spectrum data. The theoretical isotope distribution is determined from the peptide sequence and is used to deconvolute the peak areas associated with the unlabeled, partially labeled, and fully labeled species. The ratio between the labeled and unlabeled peptides is then calculated using several different methods. ZoomQuant's graphical user interface allows the user to view and adjust the parameters for peak calling and quantitation and select which peptides should contribute to the overall abundance ratio calculation. Finally, ZoomQuant generates a summary report of the relative abundance of the peptides identified in the two samples.

Animals↗
PubMed2005

Quantitative analysis of the low molecular weight serum proteome using 18O stable isotope labeling in a lung tumor xenograft mouse model.

With advancements in the analytical technologies and methodologies in proteomics, there is great interest in biomarker discovery in biofluids such as serum and plasma. Current hypotheses suggest that the low molecular weight (LMW) serum proteome possesses an archive of clipped and cleaved protein fragments that may provide insight into disease development. Though these biofluids represent attractive samples from which new and more accurate disease biomarkers may be found, the intrinsic person-to-person variability in these samples complicates their discovery. Mice are one of the most extensively used animal models for studying human disease because they represent a highly controllable experimental model system. In this study, the LMW serum proteome was compared between xenografted tumor-bearing mice and control mice by differential labeling utilizing trypsin-mediated incorporation of the stable isotope of oxygen, 18O. The digestates were combined, fractionated by strong cation exchange chromatography, and analyzed by nanoflow reversed-phase liquid chromatography coupled online with tandem mass spectrometry, resulting in the identification of 6003 proteins identified by at least a single, fully tryptic peptide. Almost 1650 proteins were identified and quantitated by two or more fully tryptic peptides. The methodology adopted in this work provides the means for future quantitative measurements in comparative animal models of disease and in human disease cohorts.

Animals↗
PubMed2002

Growth and isotopic labelling of adherent cells in small volumes of medium.

The in vivo labelling of viral or cellular components is usually conducted through the addition of radioactively labelled precursors to the culture medium. A limiting factor for isotope use is often the cost of isotope purchase and disposal. Therefore, significant savings can be achieved if the smallest possible volume of medium is employed. However, in the case of adherent cells growing in tissue culture dishes or multi-well plates, surface tension causes a very uneven distribution of the liquid due to the formation of a meniscus at the edge of the petri. This prevents the use of very small volumes of medium for cell growth and labelling because the cells at the center of the petri dish would dry out and die, especially after longer incubation periods. In this communication, we describe a technique whereby cells are grown in an area surrounded by a hydrophobic ring of Teflon, which greatly improves the distribution of the medium by eliminating the concave meniscus. This translates into a dramatic improvement in the condition of the cells, as well as the efficiency of labelling of phosphoproteins, such as the Simian Virus 40 large tumor antigen with 32P-orthophosphate or labelling of the cellular DNA with 3[H]thymidine. The technique is useful for any application where growth of cells in small volumes of medium is required.

Animals↗
PubMed2000

Tracer methodology in whole body and organ balance metabolic studies: plasma sampling is required. A study in post-absorptive rats using isotopically labeled arginine, phenylalanine, valine and leucine.

BACKGROUND AND AIMS: Radioactive and stable amino acid isotopes are frequently used in metabolic research. Blood cells contain amino acid transporters, which may influence tracer distribution in blood. The aim of this study was to determine whether plasma or whole blood specific activity or enrichment of amino acid tracers should be used in the calculation of whole body and organ production rates. METHODS: Seven male Wistar rats were infused with L-[2,3-(3)H]-Arginine, L-[2, 6-(3)H]-Phenylalanine, L-[3,4-(3)H]-Valine, and [L-[4,5-(3)H]-Leucine. Whole body and portal drained visceral, hepatic and renal production rates of arginine, phenylalanine, valine and leucine were determined in plasma and in whole blood. RESULTS: Amino acid tracers that equilibrate well between plasma and blood cells (for instance phenylalanine, valine and leucine) yield similar whole body production rates when whole blood or plasma is sampled. Also, organ production rates measured using these amino acid tracers are consistent. However, a discrepancy exists between the whole body production rate and the sum of PDV, hepatic and renal production rates. When tracers are used that do not equilibrate well between plasma and blood cells (for instance arginine) the use of whole blood specific activity in the calculations yield overestimations of whole body and organ production rates. CONCLUSION: From our data we recommend plasma sampling and strongly advise against whole blood sampling in metabolic organ balance studies in which amino acid tracers are used.

Amino Acids↗
PubMed2006

Chemoenzymatic synthesis of stable isotope labeled UDP-N-[2H]-acetyl-glucosamine and [2H]-acetyl-chitooligosaccharides.

Labeled UDP-GlcNAc and chitooligosaccharides should be powerful tools for studies of N-acetylglucosaminyltransferase such as chitin synthases. We describe here a rapid, inexpensive and a common strategie for the chemoenzymatic synthesis of uridine 5'-diphospho-N-[(2)H]-acetyl-glucosamine and the chemical preparation of N-[(2)H]-acetyl chitooligosaccharides (from 2 to 5 mers). Deuterated UDP-GlcNAc analogue was tested as chitin synthase substrate and it exhibited an incorporation level in chitin as the natural substrate. Deuterium labeling of carbohydrates present different advantages: it is a stable isotope and allows glycosyltransferase mechanism studies by a mass spectrometry approach.

Acetylation↗
PubMed1996

Pharmacokinetics of stable isotopically labeled L-histidine in humans and the assessment of in vivo histidine ammonia lyase activities.

The pharmacokinetics of L-histidine in humans has been investigated to evaluate the in vivo histidine ammonia lyase system for the conversion of L-histidine to urocanic acid. Two healthy volunteers (subjects A and B) received a single 100-mg oral dose of L-[3,3-2H2,1',3'-15N2]histidine. Blood and urine samples were obtained over 24 hr after the administration and analyzed by stable isotope dilution ms. Labeled L-histidine was rapidly absorbed, and a maximum plasma concentration of L-histidine was observed at 30 min (1057.6 ng/ml) in subject A and at 60 min (1635.6 ng/ml) in subject B after oral administration. Pharmacokinetic parameters were calculated based on a two-compartment model. Labeled L-histidine in subject A (t1/2 = 1.0 hr) was eliminated approximately twice faster than that in subject B (t1/2 = 1.9 hr). Total body clearances were 70.0 liters/hr in subject A and 30.0 liters/hr in subject B. The low ratios of the renal clearance to the total body clearance (1.04% for subject A and 0.43% for subject B) indicated that most of L-histidine was eliminated via the nonrenal processes. L-Histidine was rapidly metabolized to urocanic acid. Maximum plasma concentrations of urocanic acid were 59.61 ng/ml at 30 min for subject A and 46.10 ng/ml at 60 min for subject B. The slope of the plot of urinary excretion rate of urocanic acid vs. the plasma concentration of unchanged L-histidine was demonstrated to reflect the metabolic clearance of L-histidine to urocanic acid. The method of evaluating the in vivo human histidine ammonia lyase activities discussed in this study offers a significant value with regard to the biochemical and clinical elucidations of the heterogeneity of histidinemia.

Adult↗
PubMed2004

FTIR difference spectroscopy in combination with isotope labeling for identification of the carbonyl modes of P700 and P700+ in photosystem I.

Room temperature, light induced (P700(+)-P700) Fourier transform infrared (FTIR) difference spectra have been obtained using photosystem I (PS I) particles from Synechocystis sp. PCC 6803 that are unlabeled, uniformly (2)H labeled, and uniformly (15)N labeled. Spectra were also obtained for PS I particles that had been extensively washed and incubated in D(2)O. Previously, we have found that extensive washing and incubation of PS I samples in D(2)O does not alter the (P700(+)-P700) FTIR difference spectrum, even with approximately 50% proton exchange. This indicates that the P700 binding site is inaccessible to solvent water. Upon uniform (2)H labeling of PS I, however, the (P700(+)-P700) FTIR difference spectra are considerably altered. From spectra obtained using PS I particles grown in D(2)O and H(2)O, a ((1)H-(2)H) isotope edited double difference spectrum was constructed, and it is shown that all difference bands associated with ester/keto carbonyl modes of the chlorophylls of P700 and P700(+) downshift 4-5/1-3 cm(-1) upon (2)H labeling, respectively. It is also shown that the ester and keto carbonyl modes of the chlorophylls of P700 need not be heterogeneously distributed in frequency. Finally, we find no evidence for the presence of a cysteine mode in our difference spectra. The spectrum obtained using (2)H labeled PS I particles indicates that a negative difference band at 1698 cm(-1) is associated with at least two species. The observed (15)N and (2)H induced band shifts strongly support the idea that the two species are the 13(1) keto carbonyl modes of both chlorophylls of P700. We also show that a negative difference band at approximately 1639 cm(-1) is somewhat modified in intensity, but unaltered in frequency, upon (2)H labeling. This indicates that this band is not associated with a strongly hydrogen bonded keto carbonyl mode of one of the chlorophylls of P700.

Carbon Monoxide↗
PubMed1982

Quantitation of methadone enantiomers in humans using stable isotope-labeled [2H3]-, [2H5]-, and [2H8]Methadone.

A new technique for simultaneous stereoselective kinetic studies of methadone enantiomers was developed using three deuterium-labeled forms of methadone and GLC-chemical-ionization mass spectrometry. A racemic mixture (1:1) of (R)-(-)-[2H5]methadone (l-form) and (S)-(R)-[2H3]methadone (d-form) was administered orally in place of a single daily dose of unlabeled (+/-)-[2H0]methadone in long-term maintenance patients. Racemic (+/-)-[2H8]methadone was used as an internal standard for the simultaneous quantitation of [2H0]-, [2H3]-, and [2H5]methadone in plasma and urine. A newly developed extraction procedure, using a short, disposable C18 reversed-phase cartridge and improved chemical-ionization procedures employing ammonia gas, resulted in significant reduction of the background impurities contributing to the ions used for isotopic abundance measurements. These improvements enabled the measurement of labeled plasma methadone levels for 120 hr following a single dose. This methodology was applied to the study of methadone kinetics in two patients; in both patients, the analgesically active l-enantiomer of the drug had a longer plasma elimination half-life and a smaller area under the plasma disappearance curve than did the inactive d-form.

Adult↗
PubMed1999

Production of large quantities of isotopically labeled protein in Pichia pastoris by fermentation.

Heterologous expression in Pichia pastoris has many of the advantages of eukaryotic expression, proper folding and disulfide bond formation, glycosylation, and secretion. Contrary to other eukaryotic systems, protein production from P. pastoris occurs in simple minimal defined media making this system attractive for production of labeled proteins for NMR analysis. P. pastoris is therefore the expression system of choice for NMR of proteins that cannot be refolded from inclusion bodies or that require post-translational modifications for proper folding or function. The yield of expressed proteins from P. pastoris depends critically on growth conditions, and attainment of high cell densities by fermentation has been shown to improve protein yields by 10-100-fold. Unfortunately, the cost of the isotopically enriched fermentation media components, particularly 15NH4OH, is prohibitively high. We report fermentation methods that allow for both 15N-labeling from (15NH4)2SO4 and 13C-labeling from 13C-glucose or 13C-glycerol of proteins produced in Pichia pastoris. Expression of an 83 amino acid fragment of thrombomodulin with two N-linked glycosylation sites shows that fermentation is more cost effective than shake flask growth for isotopic enrichment.

Amino Acid Sequence↗
PubMed2001

Kinetics of folate turnover in pregnant women (second trimester) and nonpregnant controls during folic acid supplementation: stable-isotopic labeling of plasma folate, urinary folate and folate catabolites shows subtle effects of pregnancy on turnover of folate pools.

To investigate the effects of pregnancy on folate metabolism, we conducted an 84-d study in second-trimester (gestational wk 14-25) pregnant women (n = 6) and nonpregnant controls (n = 6) with stable-isotopic tracer methods. All subjects were fed a diet containing approximately 272 nmol/d (120 microg/d) folate from food, along with supplemental folic acid that contained 15% [3',5'-(2)H(2)] folic acid ([(2)H(2)]folic acid) during d 1--41 and that was unlabeled during d 42--84 to yield a constant total folate intake of 1.02 or 1.93 micromol/d (450 or 850 microg/d). Isotopic enrichment of plasma folate, urinary folate and the urinary folate catabolites para-aminobenzoylglutamate (pABG) and para-acetamidobenzoylglutamate (ApABG) was determined at intervals throughout the study. The labeling of pABG and ApABG reflected that of tissue folate pools from which the catabolites originate. After the intake of labeled folic acid was terminated on d 41, labeling of urinary folate exhibited a biphasic exponential decline with distinct fast and slow components. In contrast, during d 42--84, the enrichment of urinary pABG and ApABG exhibited primarily monophasic exponential decline, and plasma folate underwent little decline of labeling during this period. Pregnant women and controls did not differ in estimates of body folate pool size and most aspects of the excretion of labeled urinary folate and catabolites, rates of decline of excretion, and areas under the curves for folate and catabolite excretion. Pregnant women, however, tended to have a slower rate of decline of pABG than ApABG and higher enrichment at d 42 of ApABG and pABG. These data support and extend our previous findings indicating that pregnancy (gestational wk 14--26) causes subtle changes in folate metabolism but does not elicit substantial increases in the rate or extent of folate turnover at these moderately high folate intakes.

4-Aminobenzoic Acid↗
PubMed1997

Tyrosine and tryptophan structure markers in hemoglobin ultraviolet resonance Raman spectra: mode assignments via subunit-specific isotope labeling of recombinant protein.

Phenyl-deuterated tyrosine (Tyr-d4) and indole-deuterated tryptophan (Trp-d5) have been selectively incorporated into hemoglobin (Hb) by expressing the gene in auxotrophic strains of Escherichia coli. Ultraviolet resonance Raman (UVRR) spectra, using 229-nm excitation, show that difference features characteristic of the Hb quaternary R --> T transition are not perturbed by the incorporation of the isotopes. All the UVRR bands between 800 and 1700 cm-1 are assigned to either Tyr or Trp except for the 1511 cm-1 band, which had been thought to arise from the Trp 2 x W18 overtone. This band does not shift upon Trp or Tyr labeling but does shift 5 cm-1 in D2O, suggesting assignment to a histidine (His) residue. Its intensification in the T-state is consistent with His protonation. The alpha- and beta-subunits were selectively labeled, by reconstitution of labeled subunits with unlabeled subunits, to make isotope hybrids. Selective Tyr labeling identified the alpha subunits as the locus of the Y8a upshift observed in Hb, supporting the previous inference that this shift is associated with the T-state H-bond involving the interfacial Tyr alpha42 [Rodgers, Su, Subramaniam, & Spiro (1992) J. Am. Chem. Soc. 114, 3697]. Selective Trp labeling showed the Trp alpha14 contributions to the T - R difference spectrum to be negligible and confirmed Trp beta37 as the locus of the W3 difference signal, and probably of the remaining Trp signals as well. The observed downshift of W17 and upshift of Wd5 in the T-state are consistent with a stronger T-state H-bond between Trp beta37 and Asp alpha94; the resulting excitation profile red shift accounts for the dominance of the Trp beta37 contribution to the T - R difference UVRR spectrum.

Crystallography, X-Ray↗
PubMed2006

Identification and quantification of protein carbonylation using light and heavy isotope labeled Girard's P reagent.

Protein carbonyls are one of the most widely studied markers of oxidative stress. Determining increases in the concentration of protein carbonyls known to be associated with neurodegenerative diseases, heart disease, cancer and ageing. Identification of carbonylation sites in oxidized proteins has been a challenge. Even though recent advances in proteomics has facilitate the identification of carbonylation sites in oxidized proteins, confident identification remains a challenge due to the complicated nature of oxidative damage and the wide range of oxidative modifications. Here, we report the development of a multiplexing strategy that facilitates confident carbonylated peptide identification through a combination of heavy and light isotope coding and a multi-step filtering process. This procedure involves (1) labeling aliquots of oxidized proteins with heavy and light forms of Girard's reagent P (GPR) and combining them in a 1:1 ratio along with (2) LC/MS and MALDI-MS/MS analysis. The filtering process uses LC/MS and MALDI-MS/MS data to rule out false positives by rejecting peptide doublets that do not appear with the correct concentration ratio, retention time, tag number, or resolution. This strategy was used for the identification of heavily oxidized transferrin peptides and resulted in identification 13 distinct peptides. The competency of the method was validated in a complex mixture using oxidized transferrin in a yeast lysate as well as oxidized yeast. Twenty-five percent of the peptides identified in a pure oxidized sample of transferrin were successfully identified from the complex mixture. Analysis of yeast proteome stressed with hydrogen peroxide using this multiplexing strategy resulted in identification of 41 carbonylated peptides from 36 distinct proteins. Differential isotope coding of model peptides at different concentrations followed by mixing at different ratios was used to establish the linear dynamic range for quantification of carbonylated peptides using light and heavy forms of GPR.

Amino Acid Sequence↗
PubMed1994

Non-isotopic labeling of DNA by newly developed hapten-containing platinum compounds.

A newly developed reagent was tested for its applicability in in situ hybridization and in reversed hybridization of DNA fragments generated by PCR amplification. This Platinum-complex, designated universal linkage system (ULS), equipped, for instance, with biotin or fluorescein as hapten, enables versatile nonenzymatic "one step" labeling of genomic, cloned or amplified DNA. Here we demonstrate direct in situ detection of integrated human papilloma virus (HPV) DNA in cervical carcinoma cells using DNA probes labeled with fluorescein-ULS. In cervical smears the presence of HPV or Chlamydia trachomatis was assessed by PCR. To analyze the amplified DNA, a reversed hybridization assay was developed. Immobilized probes were incubated with amplimers that were labeled post-amplification through the action of the biotinylated (BIO)-ULS complex. This novel type of nonradioactive analysis appeared to be as sensitive as its isotopic or colorimetric equivalents. This labeling procedure is simple, versatile and can be included as a universal hapten linkage system in any PCR test or in situ hybridization assay aiming at the detection and identification of DNA or RNA molecules.

Biotin↗
PubMed1989

Estradiol-17 beta determined in plasma by gas chromatography-mass spectrometry with selected ion monitoring of mixed silyl ether-perfluoroacyl ester derivatives and use of various stable-isotope-labeled internal standards.

A highly specific method is described for measuring estradiol-17 beta (E2) in plasma by gas chromatography-mass spectrometry (GC-MS) associated with stable isotope dilution. A mixed derivative, E2-3-trimethylsilyl ether-17-heptafluorobutyrate (E2-3-TMS-17-HFB), was found to have excellent analytical properties. The specificity of the derivatization procedure exploits a unique feature of estrogens: the selective exchange of a phenolic perfluoroacyl ester for a trialkylsilyl ether. No significant differences in E2 concentration could be ascribed to the use of 2H- or 13C-labeled analogs, thus ruling out interferences from possible isotope exchange commonly attributed to deuterated compounds. Precision is closely similar to that for methods in which the more common E2-3, 17-bis(trimethylsilyl) ether and E2-3, 17-bis(heptafluorobutyrate) derivatives are used. Sensitivity and specificity of the mixed 3-TMS-17-HFB derivative allow adequate determinations of E2, even in plasma from males, in 2-mL samples. Interlaboratory mean concentrations of E2 obtained by routine immunoassays were consistently higher than the target values estimated by GC-MS, particularly at concentrations less than 100 pmol/L.

Carbon Isotopes↗