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Results for “Isotope Labeling”

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PubMed2006

A further study on the combined use of internal standard and isotope-labeled derivatization reagent for expansion of linear dynamic ranges in liquid chromatography-electrospray mass spectrometry.

The combined use of a so-called internal standard and the isotope-labeled derivatization reagent for the quantification of analytes for liquid chromatography-mass spectrometry (LC/MS) was further studied. The sample solution (containing the analytes and an internal standard) was derivatized with the light form of the derivatization reagent, 7-(N,N-dimethylaminosulfonyl)-4-(aminoethyl)piperazino-2,1,3-benzoxadiazole (DBD-PZ-NH(2)) or 7-(N,N-dimethylaminosulfonyl)-4-piperazino-2,1,3-benzoxadiazole (DBD-PZ). A standard solution of the analytes (containing an internal standard) was derivatized with the isotope (d(6))-labeled derivatization reagent, DBD-PZ-NH(2) (D) or DBD-PZ (D), and served as the isotope-labeled internal standards. The peak heights of the targeted analytes derivatives in the sample solution were corrected using those of the internal standard and the heavy form derivatives of the standards, and the calibration curves were constructed. The curve bending of the calibration curves caused by the ion suppression at the ion source was suppressed and the linear dynamic ranges of the calibration curves were expanded. The derivatives of DBD-PZ-NH(2) were about 10 times more sensitively detected than those of DBD-PZ derivatives and, therefore, DBD-PZ-NH(2) might be suitable for sensitive detection.

Chromatography, Liquid↗

PubMed1994

Site-directed isotope labelling and FTIR spectroscopy of bacteriorhodopsin.

Insight into integral membrane proteins function is presently limited by the difficulty of producing three-dimensional crystals. In addition, X-ray structures of proteins normally do not provide information about the protonation state and structural changes of individual residues. We report here the first use of site-directed isotope labelling and Fourier transform infrared (FTIR) difference spectroscopy to detect structural changes at the level of single residues in an integral membrane protein. Two site-directed isotope labeled (SDIL) tyrosine analogues of bacteriorhodopsin were produced which exhibit normal activity. FTIR spectroscopy shows that out of 11 tyrosines, only Tyr 185 is structurally active during the early photocycle and may be part of a proton wire.

Bacteriorhodopsins↗

PubMed2002

Using stable-isotope-labeled proteins for hydrogen exchange studies in complex mixtures.

The use of mass spectrometry to measure hydrogen exchange rates for individual proteins in complex mixtures is described. Incorporation of stable-isotope-labeled (SIL) amino acids into a protein of interest during overexpression in bacteria produced distinctive isotope patterns in mass spectra of peptic peptides from the labeled protein. The isotope pattern was used as a signature for peptides originating from the SIL protein. In addition, stable-isotope labeling simplified identification of the peptic peptides by providing partial amino acid composition information. Despite the complex isotope patterns associated with SIL peptides, hydrogen exchange rates could still be measured for peptides from SIL protein and were found to be the same as exchange rates for unlabeled protein. Hydrogen exchange in a single protein of interest was measured in a complex mixture of proteins, a bacterial cell lysate. This methodology, which includes easy recognition of peptic peptides from the protein(s) of interest during hydrogen exchange studies in heterogeneous systems, will permit analysis of structural properties and dynamics of large protein complexes and complex protein systems.

Cytosol↗

PubMed2002

Direct detection of isotopically labeled metabolites bound to a protein microarray using a charge-coupled device.

A charge-coupled device (CCD) was used to quantitatively detect isotope-labeled ligands bound to a protein microarray. Protein microarrays with protein dots, 10-50 microm in diameter, were fabricated on an aluminized Mylar film using an electrospray deposition technique. Proteins in dots were immobilized by cross-linking in glutaraldehyde vapor. After contact with solutions of isotope-labeled metabolites, the protein microarrays were washed, dried and placed face down onto the surface of a standard B/W video CCD chip with the protective window removed. We show here that such a simple inexpensive CCD detector can be used to quantify distribution of 14C and other radioactive isotopes on microarrays.

Automation↗

PubMed2002

Use of stable isotopically labeled tracers to measure very low density lipoprotein-triglyceride turnover.

Tracer methods for VLDL-TG kinetics vary in their ability to account for the effect of tracer recycling, which can influence the calculation of VLDL-TG fractional catabolic rates (FCRs). We evaluated a novel approach, involving stable isotopically labeled glycerol or palmitate tracers in conjunction with compartmental modeling, for measuring VLDL-TG kinetics in normolipidemic human subjects. When administered as a bolus simultaneously, both tracers provided identical VLDL-TG FCRs when the data were analyzed by a compartmental model that accounted for hepatic lipid tracer recycling, but not by non-compartmental analysis. The model-derived FCR was greater than that determined using a non-compartmental approach, and was 2- to 3-fold higher than that usually reported by using a bolus of radioactive [3H]glycerol. When palmitate tracer was given as a constant infusion, VLDL-TG turnover appeared 5-fold slower, because tracer recycling through hepatic lipid pools could not be resolved with the infusion protocol. We conclude that accounting for tracer recycling, particularly the contribution of hepatic glycerolipid pools, is essential to accurately measure VLDL-TG kinetics, and that bolus injection of stable isotopically labeled glycerol or palmitate tracers in conjunction with compartmental modeling analysis offers a reliable approach for measuring VLDL-TG kinetics.

Adult↗

PubMed1998

Comparison of isotopic and non-isotopic labelling for in situ hybridisation of various mRNA targets with cRNA probes.

In situ hybridisation methods to localise messenger ribonucleic acid (mRNA) targets in tissue sections or cell preparations using riboprobes can be successful with either isotopic or non-isotopic labelling. Investigators often wish to decide which labelling method provides the maximum specificity, sensitivity and resolution, with minimum nonspecific background. In this study we compared isotopic (35S) and non-isotopic (digoxigenin) labelling, using a variety of probes and paraffin-embedded tissues. The targets were human beta-actin and von Willebrand Factor mRNAs in archival human tissues; and mRNAs for two closely related trefoil factor family (TFF) peptides, TFF2 and TFF3, in rat duodenum. Patterns of localisation with both isotopic and non-isotopic probes were broadly similar for each target. The 35S labelling provided good contrast and sensitive detection under darkfield illumination, but the cellular or subcellular resolution of the target was less precise than that obtained with the digoxigenin-labelled probes in transmitted light. Digoxigenin labelling in individual cells was more clearly demonstrated, but occasionally the contrast of positive staining with background was poor. The sensitivity of each method appeared to be similar for these high-abundance targets, therefore the choice between isotopic and non-isotopic labels is dependent upon the aim of the study and the cellular resolution required.

Animals↗

PubMed2006

In vacuo isotope coded alkylation technique (IVICAT); an N-terminal stable isotopic label for quantitative liquid chromatography/mass spectrometry proteomics.

We present a new isotopic labeling strategy to modify the N-terminal amino group of peptides in a quantifiable reaction without the use of expensive reagents or solvents. The In Vacuo Isotope Coded Alkylation Technique (IVICAT) is a methylation reaction, carried out at low pressure (<100 mTorr), that results in a stable quaternary trimethylammonium group, thus adding a permanent positive charge at the N-terminus of peptides without modifying the epsilon-amino groups of lysine. The methylation reaction increases the signal intensity of modified peptides in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and liquid chromatography (LC)/MS and the isotopic peak pair differs by 9 mass units which can be easily resolved by either instrument. N-terminally trimethylated peptides exhibit collision-induced dissociation (CID) mass spectra that differ from their unmodified analogues by an enhanced b-ion series in MS2 spectra due to the fixed positive charge. Using LC/MS/MS with an LTQ mass analyzer for quantification, the experimentally determined ratios of H9- to D9-trimethyl-labeled peptides of beta-casein provided accurate estimates of the actual ratios with low % error. IVICAT labeling also accurately quantified proteins in rat kidney inner medullary collecting duct cell types, as judged by comparison with relative quantification by subsequent immunoblotting experiments. IVICAT labeling, when used in conjunction with the new proteomics software QUIL, can accurately report relative protein abundances and increase the sequence coverage of proteins of tissue proteomes.

Amino Acid Sequence↗

PubMed1997

Isolation and quantitation of isotopically labeled amino acids from biological samples.

To face the problem of simultaneous isolation and quantitation of isotopically labeled amino acids in biological samples, two semi-preparative chromatographic methods were developed. One method was especially designed to isolate radioactively labeled amino acids for which we used derivatization with the fluorophore o-phthaldialdehyde (OPA), which is known to be easy and reliable. Isolation of amino acids labeled with stable isotopes required another approach as we wanted to use isotope ratio mass spectroscopy (IRMS), which can only be performed on pure, non-derivatized amino acids. Because the OPA probe cannot be removed after isolation of the derivative, we used 9-fluorenylmethylchloroformate (FMOC) instead. This probe is linked to an amino acid via a peptide bond which can easily be broken by gas-phase acid hydrolysis (103% recovery after 5 h at 150 degrees C: S.D=3.5%, n=14). Run time (injection to injection) was 60 min for the OPA method and 75 min for the FMOC method. Both fluorescence and UV absorbance detection can be employed. The coefficient of variation (C.V.) for peak area measurement was below 2% for most OPA amino acids and below 3% for most FMOC amino acids. At maximum, a total of 1000 microl could be injected, representing approximately 200 microl of deproteinized plasma. The methods were linear up to injection of 0.5 micromol of all amino acids (OPA: r2=0.995-0.999; FMOC: r2=0.992-0.999). The C.V. of the IRMS measurement within the range which can be isolated maximally in one chromatographic run (50-500 nmol), was less than 3% above 100 nmol, indicating that chromatographic isolation fulfils the needs of the IRMS determination. The resulting methods are suitable for the isolation and quantitation of micromolar amounts of labeled amino acids from biological samples.

Amino Acids↗

PubMed2006

Isotope labeling and theoretical study of the formation of a3* ions from protonated tetraglycine.

Extensive 13C, 15N, and 2H labeling of tetraglycine was used to investigate the b3+ --> a3* reaction during low-energy collision-induced dissociation (CID) in a quadrupole ion-trap mass spectrometer. The patterns observed with respect to the retention or elimination of the isotope labels demonstrate that the reaction pathway involves elimination of CO and NH3. The ammonia molecule includes 2 H atoms from amide or amino positions, and one from an alpha-carbon position. The loss of NH3 does not involve elimination of the N-terminal amino group but, instead, the N atom of the presumed oxazolone ring in the b3+ ion. The CO molecule eliminated is the carbonyl group of the same oxazolone ring, and the alpha-carbon H atom is transferred from the amino acid adjacent to the oxazolone ring. Quantum chemical calculations indicate a multistep reaction cascade involving CO loss on the b3 --> a3 pathway and loss of NH=CH2 from the a3 ion to form b2. In the postreaction complex of b2 and NH=CH2, the latter can be attacked by the N-terminal amino group of the former. The product of this attack, an isomerized a3 ion, can eliminate NH3 from its N-terminus to form a3*. Calculations suggest that the ammonia and a3* species can form various ion-molecule complexes, and NH3 can initiate relay-type mobilization of the oxazolone H atoms from alpha-carbon positions to form a new oxazolone isomer. This multiple-step reaction scheme clearly explains the isotope labeling results, including unexpected scrambling of H atoms from alpha-carbon positions.

Computer Simulation↗

PubMed1977

Use of 13C in biosynthetic studies. The labelling pattern in tenellin enriched from isotope-labelled acetate, methionine, and phenylalanine.

The biogenetic origin of the carbon atoms in tenellin has been established by adding 13C-enriched compounds to cultures of Beauveria bassiana, and determining the isotopic distribution in the metabolite by 13C nuclear magnetic resonance spectrometry. Tenellin is formed by condensation of an acetate-derived polyketide chain with a phenylpropanoid unit that may be phenylalanine. Alternate carbon atoms of the polyketide chain were labelled with sodium [1(-13C)]- and [2-(13C]-acetate; sodium [1,2-(13C)]acetate was incorporated as intact two-carbon units, the presence of which in tenellin was apparent from coupling between adjacent 13C-enriched carbons. Substituent methyl groups of the polyketide-derived alkenyl chain were labelled with L-[Me-13C]methionine. The labelling patterns from DL-[carboxy-13C]phenylalanine and DL-[alpha-13C]phenylalanine indicated a rearrangement of the propanoid component at some stage in the synthesis. The mass spectrum of tenellin from cultures administered L-[15N]phenylalanine showed isotopic enrichment similar to that obtained with 13C- or 14C-labelled phenylalanine. During incorporation of L-[carboxy-14C, beta-3H]phenylalanine 96% of the tritium label was lost, discounting the possibility of a 1,2-hydride shift during biosynthesis of the metabolite.

Acetates↗

PubMed2006

One-step purification of bacterially expressed recombinant transducin alpha-subunit and isotopically labeled PDE6 gamma-subunit for NMR analysis.

Interactions between the transducin alpha-subunit (Galpha(t)) and the cGMP phosphodiesterase gamma-subunit (PDEgamma) are critical not only for turn-on but also turn-off of vertebrate visual signal transduction. Elucidation of the signaling mechanisms dominated by these interactions has been restrained by the lack of atomic structures for full-length Galpha(t)/PDEgamma complexes, in particular, the signaling-state complex represented by Galpha(t).GTPgammaS/PDEgamma. As a preliminary step in our effort for NMR structural analysis of Galpha(t)/PDEgamma interactions, we have developed efficient protocols for the large-scale production of recombinant Galpha(t) (rGalpha(t)) and homogeneous and functional isotopically labeled PDEgamma from Escherichia coli cells. One-step purification of rGalpha(t) was achieved through cobalt affinity chromatography in the presence of glycerol, which effectively removed the molecular chaperone DnaK that otherwise persistently co-purified with rGalpha(t). The purified rGalpha(t) was found to be functional in GTPgammaS/GDP exchange upon activation of rhodopsin and was used to form a signaling-state complex with labeled PDEgamma, rGalpha(t). GTPgammaS/[U-13C,15N]PDEgamma. The labeled PDEgamma sample yielded a well-resolved 1H-15N HSQC spectrum. The methods described here for large-scale production of homogeneous and functional rGalpha(t) and isotope-labeled PDEgamma should support further NMR structural analysis of the rGalpha(t)/PDEgamma complexes. In addition, our protocol for removing the co-purifying DnaK contaminant may be of general utility in purifying E. coli-expressed recombinant proteins.

Amino Acid Sequence↗

PubMed2006

FTIR spectroscopy combined with isotope labeling and quantum chemical calculations to investigate adsorbed bicarbonate formation following reaction of carbon dioxide with surface hydroxyl groups on Fe2O3 and Al2O3.

FTIR spectroscopy combined with isotope labeling experiments and quantum chemical calculations is used to investigate the adsorption of carbon dioxide on hydroxylated metal oxide surfaces. In particular, transmission FTIR spectra following CO2 adsorption on hydroxylated nanoparticulate Fe2O3, alpha-Al2O3, and gamma-Al2O3 particles at 296 K are reported. As expected, reaction of CO2 with these surfaces results in the formation of adsorbed bicarbonate and carbonate. In this study, the vibrational spectrum of the bicarbonate product is analyzed in detail through the use of isotope labeling experiments and quantum chemical calculations. The experimental and calculated vibrational frequencies of adsorbed HC16O3-, DC16O3-, HC18O3-, HC16O18O2-, and HC18O16O2- indicate that bicarbonate bonds to the surface in a bridged structure. There is some evidence from the mixed isotope experiments that following initial nucleophilic attack of OH, the formation of the final bicarbonate structure involves a proton transfer. On the basis of energetic considerations, the proton transfer mechanism most likely occurs through an intermolecular process involving either coadsorbed hydroxyl groups and/or carbonate.

Journal Article↗

PubMed2005

Quantitative analysis of both protein expression and serine / threonine post-translational modifications through stable isotope labeling with dithiothreitol.

While phosphorylation and O-GlcNAc (cytoplasmic and nuclear glycosylation) are linked to normal and pathological changes in cell states, these post-translational modifications have been difficult to analyze in proteomic studies. We describe advances in beta-elimination / Michael addition-based approaches which allow for mass spectrometry-based identification and comparative quantification of O-phosphate or O-GlcNAc-modified peptides, as well as cysteine-containing peptides for expression analysis. The method (BEMAD) involves differential isotopic labeling through Michael addition with normal dithiothreitol (DTT) (d0) or deuterated DTT (d6), and enrichment of these peptides by thiol chromatography. BEMAD was comparable to isotope-coded affinity tags (ICAT; a commercially available differential isotopic quantification technique) in protein expression analysis, but also provided the identity and relative amounts of both O-phosphorylation and O-GlcNAc modification sites. Specificity of O-phosphate vs. O-GlcNAc mapping is achieved through coupling enzymatic dephosphorylation or O-GlcNAc hydrolysis with differential isotopic labeling. Blocking of cysteine labeling by prior oxidation of a cytosolic lysate from mouse brain allowed specific targeting of serine / threonine post-translational modifications as demonstrated through identification of 21 phosphorylation sites (5 previously reported) in a single mass spectrometry analysis. These results demonstate BEMAD is suitable for large-scale quantitative analysis of both protein expression and serine / threonine post-translational modifications.

Animals↗

PubMed2001

Reducing mass degeneracy in SAR by MS by stable isotopic labeling.

Mass spectrometry (MS) promises to be an invaluable tool for functional genomics, by supporting low-cost, high-throughput experiments. However, large-scale MS faces the potential problem of mass degeneracy---indistinguishable masses for multiple biopolymer fragments (e.g., from a limited proteolytic digest). This paper studies the tasks of planning and interpreting MS experiments that use selective isotopic labeling, thereby substantially reducing potential mass degeneracy. Our algorithms support an experimental--computational protocol called structure-activity relation by mass spectrometry (SAR by MS) for elucidating the function of protein-DNA and protein-protein complexes. SAR by MS enzymatically cleaves a crosslinked complex and analyzes the resulting mass spectrum for mass peaks of hypothesized fragments. Depending on binding mode, some cleavage sites will be shielded; the absence of anticipated peaks implicates corresponding fragments as either part of the interaction region or inaccessible due to conformational change upon binding. Thus, different mass spectra provide evidence for different structure--activity relations. We address combinatorial and algorithmic questions in the areas of data analysis (constraining binding mode based on mass signature) and experiment planning (determining an isotopic labeling strategy to reduce mass degeneracy and aid data analysis). We explore the computational complexity of these problems, obtaining upper and lower bounds. We report experimental results from implementations of our algorithms.

Algorithms↗

PubMed1993

15N- and 13C-labeled media from Anabaena sp. for universal isotopic labeling of bacteriocins: NMR resonance assignments of leucocin A from Leuconostoc gelidum and nisin A from Lactococcus lactis.

A procedure for universal 13C and/or 15N labeling of microbial peptides which are produced by fermentation in complex media and its application to two food-preserving bacteriocins from lactic acid bacteria are described. Isotopic enrichment of nisin A (from Lactococcus lactis) and of leucocin A (from Leuconostoc gelidum) is readily achieved using a soluble peptone derived from enzymatic hydrolysis (pepsin and chymopapain) of Anabaena sp. ATCC 27899 cells grown on sodium [13C]bicarbonate and/or sodium [15N]nitrate as sole carbon and nitrogen sources. Combustion of this peptone followed by mass spectrometric analysis indicates that 45% of the labeled carbon and 65% of the labeled nitrogen added to the Anabaena culture are utilized in the amino acids of the peptone and that the isotopic purity for both 13C and 15N remains essentially unchanged provided that the cells are grown under argon atmosphere to avoid nitrogen fixation. NMR analyses of [13C,15N]nisin A using H[13C]MQC, H[13C]MBC, 2D INADEQUATE, and H[15N]MQC techniques confirmed 1H spectral assignments previously reported for unlabeled material and readily provided carbon and nitrogen assignments. The results show that universal but not uniform 13C labeling occurs unless the nutrient source is completely isotopically enriched at high level (> or = 98%) because of differential levels of de novo amino acid synthesis. Application of NMR techniques such as TOCSY, DQF-COSY, NOESY, and H[13C]MQC to unlabeled and [13C]leucocin A afforded the complete 1H and 13C assignment. Leucocin A does not possess clearly defined conformational structure in DMSO or aqueous solutions.

Amino Acid Sequence↗

PubMed1979

Isotopically labelled compounds in the study of extracellular fluid space in dog bone.

The extracellular fluid space in dog bone has been examined using a series of isotopically labelled compounds. Sodium-77 bromide and indium-113m ethylenediaminetetracetic acid were used as extracellular fluid space markers, radioactive water as a total fluid space marker, and potassium-43 chloride to examine the existence of a bone membrane. The clearance of each tracer from bone was monitored for a period of 2h post-injection. Graphical analysis of the clearance curves shows that the number of exponential functions vary depending on the type of tracer used. The fact that a sum of three exponential terms can completely describe each curve indicates that a simple model consisting of three compartments is sufficient to approximate the clearance of these tracers from bone and its associated fluid space. It is concluded that bone consists of an extracellular fluid space, and that this space may well play an important part in the transference of solutes and the mechanisms involved in their localization on the hydroxyapatite crystals of bone.

Animals↗

PubMed2002

Stable isotope labelling in vivo as an aid to protein identification in peptide mass fingerprinting.

Peptide mass fingerprinting (PMF) is a powerful technique for identification of proteins derived from in-gel digests by virtue of their matrix-assisted laser desorption/ionization-time of flight mass spectra. However, there are circumstances where the under-representation of peptides in the mass spectrum and the complexity of the source proteome mean that PMF is inadequate as an identification tool. In this paper, we show that identification is substantially enhanced by inclusion of composition data for a single amino acid. Labelling in vivo with a stable isotope labelled amino acid (in this paper, decadeuterated leucine) identifies the number of such amino acids in each digest fragment, and show a considerable gain in the ability of PMF to identify the parent protein. The method is tolerant to the extent of labelling, and as such, may be applicable to a range of single cell systems.

Deuterium↗

PubMed2000

Expression, refolding, and isotopic labeling of human serum albumin domains for NMR spectroscopy.

Many different compounds bind to human serum albumin (HSA), which can be a significant problem in the drug discovery process. To aid in the design of drug molecules that do not bind to HSA, the structures of HSA/ligand complexes would be very useful. However, little information has been reported on the structures of small molecules complexed to HSA. In this paper, we describe a procedure for preparing isotopically labeled domains of HSA for nuclear magnetic resonance (NMR) studies. The procedure involves the expression in Escherichia coli, refolding, and a multistep purification. Domains I and III are capable of folding into stable structural units and producing well resolved (15)N/(1)H correlation spectra, whereas domain II forms significant aggregates at sub-millimolar concentration. Using our protocols, isotopically labeled and properly folded domains I and III can be effectively produced in large quantities for NMR-based structural studies and NMR-based screening. This provides a valuable tool for obtaining structural information on HSA/ligand complexes by NMR which will be useful in drug discovery.

Cloning, Molecular↗