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PubMed1986

Lysine and protein metabolism in young women. Subdivision based on the novel use of multiple stable isotopic labels.

A multitracer stable isotope study of lysine kinetics was carried out in fasted adult female volunteers to determine whether a multicompartmental model that partitions protein synthesis and breakdown into at least two types of tissue components can be constructed from plasma and breath data. Five female subjects, maintained on formula diets, received L-[13C1]lysine (27 mumol/kg) as an i.v. bolus and L-[15N2]lysine (27 mumol/kg) as an oral bolus 4 h postprandially. Plasma and breath samples were collected for 6 h. On an alternate day, subjects received NaH13CO3 (10 mumol/kg) as an i.v. bolus and breath samples were collected for 6 h. Plasma tracer lysine levels were determined by gas chromatography-mass spectrometry isotope ratiometry, and breath 13CO2 levels were measured by mass spectrometric gas isotope ratiometry. The tracer data could be fitted to a mammillary multicompartmental model that consisted of a lysine central compartment and slow- and fast-exchanging peripheral compartments containing 37, 38, and 324 mumol/kg, respectively. The rates of lysine oxidation, incorporation into protein, and release by protein breakdown were 21, 35, and 56 mmol/kg/h, respectively, in the fast-exchanging compartment, whereas the rates of protein synthesis and breakdown in the slow compartment were both 53 mmol/kg/min. These values corresponded to a whole-body lysine flux of 106 mmol/kg/h. The kinetic parameters were in excellent agreement with reported values obtained by constant-infusion methods. The measurements indicated that it will be possible to detect changes in amino acid pool sizes and protein synthesis and breakdown associated with the mobilization of protein stores from plasma and breath measurements in multitracer stable isotope experiments.

Administration, Oral↗
PubMed1991

Uniform 13C isotope labeling of proteins with sodium acetate for NMR studies: application to human carbonic anhydrase II.

Uniform double labeling of proteins for NMR studies can be prohibitively expensive, even with an efficient expression and purification scheme, due largely to the high cost of [13C6, 99%]glucose. We demonstrate here that uniformly (greater than 95%) 13C and 15N double-labeled proteins can be prepared for NMR structure/function studies by growing cells in defined media containing sodium [1,2-13C2, 99%]acetate as the sole carbon source and [15N, 99%]ammonium chloride as the sole nitrogen source. In addition, we demonstrate that this labeling scheme can be extended to include uniform carbon isotope labeling to any desired level (below 50%) by utilizing media containing equal amounts of sodium [1-13C, 99%]acetate and sodium [2-13C, 99%]acetate in conjunction with unlabeled sodium acetate. This technique is less labor intensive and more straightforward than labeling using isotope-enriched algal hydrolysates. These labeling schemes have been used to successfully prepare NMR quantities of isotopically enriched human carbonic anhydrase II. The activity and the 1H NMR spectra of the protein labeled by this technique are the same as those obtained from the protein produced from media containing labeled glucose; however, the cost of the sodium [1,2-13C2, 99%]acetate growth media is considerably less than the cost of the [13C6, 99%]glucose growth media. We report here the first published 13C and 15N NMR spectra of human carbonic anhydrase II as an important step leading to the assignment of this 29-kDa zinc metalloenzyme.

Acetates↗
PubMed1995

A novel method for selective isotope labeling of bacterially expressed proteins.

A novel method for isotope labeling in selected amino acids is presented for use with the T7 RNA polymerase system. The protocol is illustrated with the DNA-binding domain from the E2 protein of bovine papillomavirus, BPV-1. On addition of rifampicin, protein expression occurs exclusively from the gene controlled by the T7 promoter. Since the bacteria are now dedicated to the production of E2 protein, labeling with specific amino acids is efficiently performed. For example, 10 mg/l of 15N-labeled phenylalanine is shown to be sufficient for incorporation of the label, without scrambling, and without the use of an auxotrophic strain.

DNA-Binding Proteins↗
PubMed2005

Ab initio modeling of amide I coupling in antiparallel beta-sheets and the effect of 13C isotopic labeling on infrared spectra.

Isotopic substitution with 13C on the amide C=O has become an important means of determining localized structural information about peptide conformations with vibrational spectroscopy. Various approaches to the modeling of the interactions between labeled amide sites, specifically for antiparallel two-stranded, beta-forming peptides, were investigated, including different force fields [dipole-dipole interaction vs density functional theory (DFT) treatments], basis sets, and sizes of model peptides used for ab initio calculations, as well as employing models of solvation. For these beta-sheet systems the effect of the relative positions of the 13C isotopic labels in each strand on their infrared spectra was investigated. The results suggest that the interaction between labeled amide groups in different strands can be used as an indicator of local beta-structure formation, because coupling between close-lying C=O groups on opposing chains leads to the largest frequency shifts, yet some alternate placements can lead to intensity enhancements. The basic character of the coupling interaction between labeled modes on opposing strands is independent of changes in peptide length, water solvent environment, twisting of the sheet structure, and basis set used in the calculations, although the absolute frequencies and detailed coupling magnitudes change under each of these perturbations. In particular, two strands of three amides each contain the basic interactions needed to simulate larger sheets, with the only exception that the C=O groups forming H-bonded rings at the termini can yield different coupling values than central ones of the same structure. Spectral frequencies and intensities were modeled ab initio by DFT primarily at the BPW91/6-31G** level for pairs of three, four, and six amide strands. Comparison to predictions of a classical coupled oscillator model show qualitative but not quantitative agreement with these DFT results.

Amides↗
PubMed2006

Synthesis of the isotope-labeled derivatization reagent for carboxylic acids, 7-(N,N-dimethylaminosulfonyl)-4-(aminoethyl)piperazino-2,1,3-benzoxadiazole (d6) [DBD-PZ-NH2 (D)], and its application to the quantification and the determination of relative amount of fatty acids in rat plasma samples by high-performance liquid chromatography/mass spectrometry.

The isotope-labeled benzofurazan derivatization reagent for carboxylic acids, 7-(N,N-dimethylaminosulfonyl)-4-(aminoethyl)piperazino-2,1,3-benzoxadiazole (d6) [DBD-PZ-NH2 (D)] was synthesized. DBD-PZ-NH2 (D) was used for the accurate quantification of fatty acids by liquid chromatography/mass spectrometry (LC/MS). The standard fatty acids were derivatized with DBD-PZ-NH2 (D) to the stable isotope-labeled compounds for the fatty acids derivatives of DBD-PZ-NH2 and used for the internal standards. The obtained calibration curves for fatty acids were linear over the range 0.1-200 microM (r2 > 0.999). Fatty acids in plasma samples were determined after derivatization with DBD-PZ-NH2 and analyzed by LC/MS using standard fatty acid DBD-PZ-NH2 (D) derivatives as internal standards. Furthermore, the relative amounts of fatty acids in two plasma samples were determined after derivatization with DBD-PZ-NH2 and DBD-PZ-NH2) (D). The isotope-labeled derivatization reagent was useful for accurate quantification and the determination of relative amounts of the metabolites in biological samples having the target functional group.

Animals↗
PubMed1985

Formation and metabolism of nitrosamines in vivo, monitored by 14N-stable isotope labelling.

Microsomal metabolism of N-nitrosodimethylamine entails release of molecular nitrogen; the extent is determined by 15N stable isotope labelling and mass-spectrometric isotope ratio measurements. Exhalation of labelled nitrogen by rats treated with 15N-dimethylamine and nitrite or 15N-nitrite alone indicates that nitrogen may arise from nitrite via two pathways: either directly from nitrosation of primary amines or from secondary and tertiary amines with subsequent enzymic N-demethylation. The overall yield of nitrosamine formation, N-demethylation and nitrogen-release represent about 0.3-6% or the administered dose of dimethylamine (1.1 mmol/kg), depending upon the dose of nitrite (0.55-2.2 mmol/kg). 15N-stable isotope labelling and mass-spectrometric isotope ratio measurements are powerful tools for assessment of endogenous nitrosamine formation from nitrite. One hundred nmol of labelled nitrogen are easily detectable in vivo; with further methodological refinement the limit of detection may be lowered by two orders of magnitude.

Animals↗
PubMed2000

Strategies for metabolic flux analysis in plants using isotope labelling.

Flux measurements through metabolic pathways generate insights into the integration of metabolism, and there is increasing interest in using such measurements to quantify the metabolic effects of mutation and genetic manipulation. Isotope labelling provides a powerful approach for measuring metabolic fluxes, and it gives rise to several distinct methods based on either dynamic or steady-state experiments. We discuss the application of these methods to photosynthetic and non-photosynthetic plant tissues, and we illustrate the different approaches with an analysis of the pathways interconverting hexose phosphates and triose phosphates. The complicating effects of the pentose phosphate pathway and the problems arising from the extensive compartmentation of plant cell metabolism are considered. The non-trivial nature of the analysis is emphasised by reference to invalid deductions in earlier work. It is concluded that steady-state isotopic labelling experiments can provide important information on the fluxes through primary metabolism in plants, and that the combination of stable isotope labelling with detection by nuclear magnetic resonance is particularly informative.

Biotechnology↗
PubMed2005

Bacterial expression and one-step purification of an isotope-labeled heterotrimeric G-protein alpha-subunit.

Heterologous expression systems are often employed to generate sufficient quantities of isotope-labeled proteins for high-resolution NMR studies. Recently, the interaction between the prodomain region of subtilisin and an active, mutant form of the mature enzyme has been exploited to develop a cleavable affinity tag fusion system for one-step generation and purification of full-length soluble proteins obtained by inducible prokaryotic expression. As a first step towards applying high-resolution NMR methods to study heterotrimeric G-protein alpha-subunit (G(alpha)) conformation and dynamics, the utility of this subtilisin prodomain fusion system for expressing and purifying an isotope-labeled G(alpha) chimera (approximately 40 kDa polypeptide) has been tested. The results show that a prodomain fused G(alpha) chimera can be expressed to levels approaching 6-8 mg/l in minimal media and that the processed, mature protein exhibits properties similar to those of G(alpha) isolated from natural sources. To assay for the functional integrity of the purified G(alpha) chimera at NMR concentrations and probe for changes in the structure and dynamics of G(alpha) that result from activation, 15N-HSQC spectra of the GDP/Mg2+ bound form of G(alpha) obtained in the absence and presence of aluminum fluoride, a well known activator of the GDP bound state, have been acquired. Comparisons of the 15N-HSQC spectra reveals a number of changes in chemical shifts of the 1HN, 15N crosspeaks that are discussed with respect to expected changes in the protein conformation associated with G(alpha) activation.

GTP-Binding Protein alpha Subunits, Gi-Go↗
PubMed2005

Efficient uniform isotope labeling of Abl kinase expressed in Baculovirus-infected insect cells.

This report shows for the first time the efficient uniform isotope labeling of a recombinant protein expressed using Baculovirus-infected insect cells. The recent availability of suitable media for (15)N- and (13)C/(15)N-labeling in insect cells, the high expression of Abl kinase in these labeling media and a suitable labeling protocol made it possible to obtain a (1)H-(15)N-HSQC spectrum for the catalytic domain of Abl kinase of good quality and with label incorporation rates > 90%. The presented isotope labeling method should be applicable also to further proteins where successful expression is restricted to the Baculovirus expression system.

Animals↗
PubMed2006

Isotope-labeled cross-linkers and Fourier transform ion cyclotron resonance mass spectrometry for structural analysis of a protein/peptide complex.

For structural studies of proteins and their complexes, chemical cross-linking combined with mass spectrometry presents a promising strategy to obtain structural data of protein interfaces from low quantities of proteins within a short time. We explore the use of isotope-labeled cross-linkers in combination with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry for a more efficient identification of cross-linker containing species. For our studies, we chose the calcium-independent complex between calmodulin and a 25-amino acid peptide from the C-terminal region of adenylyl cyclase 8 containing an "IQ-like motif." Cross-linking reactions between calmodulin and the peptide were performed in the absence of calcium using the amine-reactive, isotope-labeled (d0 and d4) cross-linkers BS3 (bis[sulfosuccinimidyl]suberate) and BS2G (bis[sulfosuccinimidyl]glutarate). Tryptic in-gel digestion of excised gel bands from covalently cross-linked complexes resulted in complicated peptide mixtures, which were analyzed by nano-HPLC/nano-ESI-FTICR mass spectrometry. In cases where more than one reactive functional group, e.g., amine groups of lysine residues, is present in a sequence stretch, MS/MS analysis is a prerequisite for unambiguously identifying the modified residues. MS/MS experiments revealed two lysine residues in the central alpha-helix of calmodulin as well as three lysine residues both in the C-terminal and N-terminal lobes of calmodulin to be cross-linked with one single lysine residue of the adenylyl cyclase 8 peptide. Further cross-linking studies will have to be conducted to propose a structural model for the calmodulin/peptide complex, which is formed in the absence of calcium. The combination of using isotope-labeled cross-linkers, determining the accurate mass of intact cross-linked products, and verifying the amino acid sequences of cross-linked species by MS/MS presents a convenient approach that offers the perspective to obtain structural data of protein assemblies within a few days.

Adenylyl Cyclases↗
PubMed1982

Pharmacokinetic equivalence of stable-isotope-labeled and unlabeled drugs. Phenobarbital in man.

Stable isotope labeling of drugs has been used in human metabolism studies because it eliminates the risk of radiation exposure accompanying use of radioactive tracers. The labeled drug can be measured by gas-chromatographic mass spectrometry (GCMS). However, if reliable pharmacokinetic data are to be obtained, one has to be certain the rate of metabolism of labeled and unlabeled drug is the same, i.e., there is no kinetic isotope effect. To evaluate this for phenobarbital (PB), three humans were infused with a 1:1 mixture of phenobarbital and 1,3-15N2-2-13C-PB. Serum was collected at regular intervals. Concentrations of labeled and unlabeled phenobarbital were determined by GCMS. Within each subject, there was no trend for concentrations of labeled phenobarbital to be higher or lower than concentrations of unlabeled phenobarbital (P greater than 0.90 for all three subjects). There was no difference in the zero time intercepts, distribution and elimination time constants and half-lives, volumes of distribution and central compartment, or clearance of the two forms of phenobarbital. Thus, no isotope effect was found. Published data on other labeled drugs and the likelihood of encountering an isotope effect based on type of isotope and its location in the molecule are discussed.

Adult↗
PubMed2003

Amino-acid-type selective isotope labeling of proteins expressed in Baculovirus-infected insect cells useful for NMR studies.

Culture conditions for successful amino-acid-type selective isotope labeling of proteins expressed in Baculovirus-infected insect cells are described. The method was applied to the selective labeling of the catalytic domain of c-Abl kinase with (15)N-phenylalanine, (15)N-glycine, (15)N-tyrosine or (15)N-valine. For the essential amino acids phenylalanine, tyrosine and valine high (15)N-label incorporation rates of >/=90% and approximately the expected number of resonances in the HSQC spectra were observed, which was not the case for the non-essential amino acid glycine. The method should be applicable to amino-acid-type selective isotope labeling of other recombinant proteins which have not been amenable to NMR analysis.

Amino Acid Sequence↗
PubMed2003

Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry.

Quantitative proteome profiling using stable isotope protein tagging and automated tandem mass spectrometry (MS/MS) is an emerging technology with great potential for the functional analysis of biological systems and for the detection of clinical diagnostic or prognostic marker proteins. Owing to the enormous complexity of proteomes, their comprehensive analysis is an as-yet-unresolved technical challenge. However, biologically or clinically important information can be obtained if specific, information-rich protein classes, or sub-proteomes, are isolated and analyzed. Glycosylation is the most common post-translational modification. Here we describe a method for the selective isolation, identification and quantification of peptides that contain N-linked carbohydrates. It is based on the conjugation of glycoproteins to a solid support using hydrazide chemistry, stable isotope labeling of glycopeptides and the specific release of formerly N-linked glycosylated peptides via peptide- N-glycosidase F (PNGase F). The recovered peptides are then identified and quantified by MS/MS. We applied the approach to the analysis of plasma membrane proteins and proteins contained in human blood serum.

Amino Acid Sequence↗
PubMed1988

The use of biotinylated DNA probes in parentage testing: non-isotopic labeling and non-toxic extraction.

With a few exceptions DNA probing techniques require the use of radioisotopes and toxic DNA extraction techniques which render the method expensive, potentially hazardous and time-consuming. Most isotopic labeling techniques use the isotope 32P and require 3-10 days to visualize bands after hybridization. An alternative approach is based on the use of non-isotopic detection methods. The available non-isotopic techniques were assessed and their practicality tested. All probes analyzed were tested on samples extracted with a non-toxic extraction procedure using 6 M NaCl as the substitute for phenol and isochloroform. By manipulating probe sizes, blocking agents, selection of membrane and detection system, it is feasible to use non-isotopic labeling and detection in routine parentage testing. Reproducible results were obtained with labeling a variety of DNA probes of various sizes, plasmid and inserts. With an absence of waste disposal costs, probes that are stable for over two years and a staining procedure which takes 3-5 h versus days the technique is well suited for a normal laboratory setting. The next key to the acceptability of DNA testing will be the commercial availability of DNA probes for widespread use.

Biotin↗
PubMed1994

A novel isotope labeling protocol for bacterially expressed proteins.

A novel protocol for isotopically labeling bacterially expressed proteins is presented. This method circumvents problems related to poor cell growth, commonly associated with the use of minimal labeled media, and problems with protein induction encountered, less commonly, when using enriched labeled media. The method involves initially growing the bacterial cells to high optical density in a commercially available enriched labeled medium. Following a suitable growth period, the cells are transferred to a different (minimal) labeled medium, appropriate for induction. The method is demonstrated using the protein melanoma growth stimulating activity (MGSA).

Bacteria↗
PubMed1993

Measurements of energy expenditure using isotope-labelled water (2H2(18)O) during an Arctic expedition.

Estimates of energy expenditure using both isotope-labelled (2H2(18)O) water and dietary intake/body composition changes were made during an attempt by two men (MS and RF) to walk to the North Pole. The isotope-labelled water technique gave mean estimates of daily energy expenditure for the 48-day expedition of 28.05 MJ (MS) and 32.38 MJ (RF), which compared with estimates of 25.66 MJ (MS) and 24.86 MJ (RF) from the intake/body composition measurements. Fluid retention and peripheral oedema probably caused a considerable underestimate of the losses in body energy stores when applying the energy balance method, whereas in the isotope method, uncertainty in the measurements of isotopic background led to minimum errors of -4.9% to +4.0% of the means for MS and -12.7% to +8.2% for RF (95% confidence limits).

Adult↗
PubMedDate not supplied

Quantitative proteome analysis using differential stable isotopic labeling and microbore LC-MALDI MS and MS/MS.

We demonstrate an approach for global quantitative analysis of protein mixtures using differential stable isotopic labeling of the enzyme-digested peptides combined with microbore liquid chromatography (LC) matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS). Microbore LC provides higher sample loading, compared to capillary LC, which facilitates the quantification of low abundance proteins in protein mixtures. In this work, microbore LC is combined with MALDI MS via a heated droplet interface. The compatibilities of two global peptide labeling methods (i.e., esterification to carboxylic groups and dimethylation to amine groups of peptides) with this LC-MALDI technique are evaluated. Using a quadrupole-time-of-flight mass spectrometer, MALDI spectra of the peptides in individual sample spots are obtained to determine the abundance ratio among pairs of differential isotopically labeled peptides. MS/MS spectra are subsequently obtained from the peptide pairs showing significant abundance differences to determine the sequences of selected peptides for protein identification. The peptide sequences determined from MS/MS database search are confirmed by using the overlaid fragment ion spectra generated from a pair of differentially labeled peptides. The effectiveness of this microbore LC-MALDI approach is demonstrated in the quantification and identification of peptides from a mixture of standard proteins as well as E. coli whole cell extract of known relative concentrations. It is shown that this approach provides a facile and economical means of comparing relative protein abundances from two proteome samples.

Chromatography, High Pressure Liquid↗