Use Of Isotope Labeling Research Articles

Mass spectrometric detection for capillary isoelectric focusing separations of complex protein mixtures.

Capillary isoelectric focusing (CIEF) can provide high-resolution separations of complex protein mixtures, but until recently it has primarily been used with conventional UV detection. This technique would be greatly enhanced by much more information-rich detection methods that can aid in protein characterization. We describe progress in the development of the combination of CIEF with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry and its application to proteome characterization. Studies have revealed 400-1000 putative proteins in the mass range of 2-100 kDa from total injections of approximately 300 ng protein in single CIEF-FTICR analyses of cell lysates for both Escherichia coli (E. coli) and Deinococcus radiodurans (D. radiodurans). We also demonstrate the use of isotope labeling of the cell growth media to improve mass measurement accuracy and provide a means for quantitative proteome-wide measurements of protein expression. The ability to make such comprehensive and precise measurements of differences in protein expression in response to cellular perturbations should provide new insights into complex cellular processes.

An advantage for use of isotope labeling and NMR chemical shifts to analyze the structure of four homologous IgG-binding domains of staphylococcal protein A

Because of the complexity arising from the large molecular size and the amino acid sequence homologies of IgG-binding domains of Staphylococcal Protein A (SpA), we have introduced a combination of stable isotope labeling and both qualitative and quantitative investigations of the structural dependence of the NMR chemical shifts for its structure analysis. In order to enable selective isotope labeling with high efficiency, a mutated low molecular weight Protein A (LPA; MWt=27 kDa) which consists of E, D, A, B and 13 residues of the C-domain was used in this study. Amide proton chemical shifts, measured using uniformly 15N-labeled LPA and LPA labeled selectively with 15N-alanine, show that the turn between helices 1 and 2, and its tertiary interactions with helix 3, are very similar in all domains. This contradicts previous results obtained using independent structure calculations on isolated domains. The close similarity in NH and 15N chemical shifts of alanine residues in the interdomain linker suggests that the linker maintains a similar structure both in isolated domains and in the intact protein. We show that the high-field shifted methyl signal of Ala 48 is affected by the ring-current effect arising from Phe 30, and has a very similar helical environment in all four domains. Thus, helix 3 is present in all domains, as we previously reported [Kikuchi et al., J Biochem Biophys Method, 1999:38:203–208], even though it is not observed in the crystal structure [Deisenhofer J. Biochemistry 1981;20:2361–2370].

Hydrogen vs. electron transfer mechanisms in the chain decomposition of phenacyl bromides. Use of isotopic labeling as a mechanistic probe

Ring-substituted α-bromoacetophenones react with alcohols in a chain reaction leading to the corresponding acetophenone, HBr, and the carbonyl compound from oxidation of the alcohol. Two different mechanisms, involving hydrogen or electron transfer by ketyl radicals, have been proposed in order to accommodate the unusual selectivities of these reactions. By studying the efficiency of isotope incorporation from deuterated alcohols, it has been possible to determine the relative contributions from both mechanisms. For example, electron transfer dominates in the case of 2-propanol, while hydrogen transfer is more important for methanol. The results demonstrate that ring substitution in the starting ketone is not a main contributing factor in the discrimination between the two mechanisms. The only parameter that seems to be playing a major role is the nature (reducing strength) of the ketyl radicals. Key words: dehydrobromination, charge transfer, isotope effect, ketyl radicals.

Use of isotopic labeling in establishing reaction mechanisms: CD +3 + C 2H 6

The reaction of CD + 3 with C 2H 6 has been reinvestigated by the Fourier-transform ion cyclotron resonance technique. It proceeds, in addition to the previously observed “hydride transfer” pathway resulting in the C 2H + 5 ion, by at least two other channels. “Methylene transfer” results in the isotopic C 2H 2D + 3 ion, and “eliminative condensation” accompanied by molecular hydrogen ejection results in several C 3 ionic products. The reactions are investigated as a function of the collision energy, and their possible mechanisms are discussed.

Use of isotopic labelling in a SIMS study of the hydroxylation of TiO 2(100) surfaces

The interaction of TiO 2(100) surfaces with hydrogen and water vapour has been studied using mainly secondary ion mass spectrometry (SIMS). The influence of the stoichiometry of the surface on its reactivity has been checked. The existence of two kinds of hydroxyl groups is pointed out. The behaviour of these hydroxyl populations with the temperature was studied. Moreover the ability of SIMS to distinguish different isotopes of the same element was used: exposure of TiO 2(100) surfaces to isotope labelled molecules, D 2 and H 2 18O, was performed in order to specify the nature of these two hydroxyl groups.

Use of isotopic labelling in a SIMS study of the hydroxylation of TiO2(100) surfaces

Use of isotopic labelling in a SIMS study of the hydroxylation of TiO2(100) surfaces

Application of 13C(ω 1)-half-filtered [ 1H, 1H]-NOESY for studies of a complex formed between DNA and a 13C-labeled minor-groove-binding drug

The complex formed between the anticancer drug 4-[ p-[ p-(4-quinolylamino)benzamido]anilino]pyridine (SN 6999) and the decadeoxyribonucleoside nonaphosphate d-(GCATTAATGC) 2 was investigated using two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) with a 13C(ω 1)-half-filter. The two quaternary methyl groups in SN 6999 had been labeled with 13C for these experiments. The simplified subspectra of [ 1H, 1H]-NOESY obtained with this procedure greatly facilitate the identification and assignment of intermolecular NOEs. Quite generally, the combined use of isotope labeling and heteronuclear filters in [ 1H, 1H]-NOESY provides an improved experimental basis for structural studies of drug/ DNA complexes.

Gas pyrolsis in combustion single-pulse shock tube studies of N- and O-atom-containing heterocyclics

Studies on the pyrolysis of heterocyclic compounds behind reflected shocks using the single-pulse shock tube technique are reviewed. Special attention is paid to oxygen- and nitrogen-containing heterocyclics. Compounds that can be considered as prototypes of fuel constituents and can simulate larger, more complex molecules are chosen. The reactions that take place during the pyrolysis of these compounds are very similar to the reactions that occur under combustion conditions. Distributions of reaction products in the pyrolysis of these compounds, measured over a wide temperature range, are shown for selected molecules and Arrhenius parameters for the rates of their production is given. The experimental techniques and analytical procedures for the identification and the quantitative determination of the reaction products are reviewed. Methods for determining unimolecular initiations versus free radical reactions, such as the use of isotope labeling and the introduction of free radical scavengers, are discussed. General pyrolysis mechanisms for several heterocyclic compounds are suggested.

The complete oxidation of acetonitrile on Ag(110): catalytic oxidation of -CH 2CN

The oxidation of -CH 2CN intermediates on Ag(110) results in the formation of CO 2, NO and H 2O produced apparently via surface formate and nitrile groups. With the use of isotopic labeling and temperature-programmed reaction spectroscopy, it has been shown that the formate is formed via attack of the methylene carbon of -CH 2CN by adsorbed atomic oxygen. For this transformation a metallacyclic intermediate, CHOCN, is proposed. Several possible routes to this intermediate are suggested, involving either OCH 2CN (a) or =CHCN, either of which is subsequently attacked by oxygen at the methylene carbon. The conversion of -CH 2CN to the formate occurs by reaction with adsorbed atomic oxygen at least 130 K below the temperature at which -CH 2CN itself reacts on the otherwise clean surface. The results obtained in ultra-high vacuum for the stability of -CH 2CN in the presence of coadsorbed oxygen atoms successfully predict the stability of -CH 2CN in a high pressure oxygen environment.

Carbon-carbon bond activation in the 1,2-ethanedioxy heterometallacycle by atomic oxygen on Ag(110)

The effects of coadsorbed oxygen atoms on the decomposition of the 1,2-ethanedioxy surface species (OCH 2CH 2O (a)) formed during the partial oxidation of ethylene glycol (CH 2OH) 2 on the Ag(110) surface has been studied using temperature programmed reaction spectroscopy (TPRS), isotope labelling and high resolution electron energy loss vibrational spectroscopy (EELS). Surface oxygen atoms react with OCH 2CH 2O (a) at 300 K to form gaseous water and formaldehyde, and surface formate (HCOO (a)), which decomposes at 415 K to yield CO 2 and H 2. Through the use of isotopic labelling it was found that the oxygen in the formaldehyde originates solely from the 1,2-ethanedioxy surface species, while in the surface formate one comes from OCH 2CH 2O (a) and the other from coadsorbed oxygen atoms. The kinetic isotope effect for the reaction of adsorbed d 4-1,2-ethanedioxy with surface oxygen indicates that the rate limiting step is the abstraction of a proton from a methylene carbon in OCH 2CH 2O (a) to form OH a and OCHCH 2O (a). Nucleophilic attack by surface oxygen at the carbonyl carbon of OCHCH 2O (a), followed by C-C bond cleavage then releases H 2CO and forms HCOO (a). This study underscores the importance of looking at a wide range of concentrations of the pertinent surface species in order to probe a wider variety of reaction channels.

14] Isotopic labeling with hydrogen-2 and carbon-13 to compare conformations of proteins and mutants generated by site-directed mutagenesis, II

This chapter discusses the use of isotopic labeling to assign the aromatic partners of cross-peaks of both wild-type and mutant proteins through the use of deuteration of both aliphatic and aromatic amino acid side chains. Comparison of the data for isotopically labeled wild-type and mutant proteins allow independent assignment of the cross-peaks in these proteins. This contribution will conclude with a description of how the side chain assignments can be extended to site-specific assignments. For a protein the size of Snase, 149 amino acids plus a hexapeptide tail in the form one study, methods that depend on resolved scalar couplings or on sufficient resolution of the two-dimensional data will typically not be entirely adequate. Although isotopic labeling is both expensive and labor intensive, it does yield unambiguous information about both wild-type and mutant proteins. In favorable cases assignments can be made by comparing the NMR data of wild-type and mutant proteins. However, comparative methods are limited to instances of conformationally silent amino acids, or when there is a localized probe of the protein structure.

Peptide backbone conformation by solid-state nuclear magnetic resonance spectroscopy

Solid-state 14N NMR spectroscopy is used to determine the backbone structure of the peptides N-acetyl-L-valyl-L-leucine and L-alanylglycyl-glycine in single crystals. These samples provide examples of both glycyl and non-glycyl residues in peptide linkages. There is good agreement between the structures determined by solid-state NMR spectroscopy and X-ray diffraction, demonstrating that solid-state NMR spectroscopy can describe peptide backbone structures without the use of isotopic labels.

A mechanistic study of carbon monoxide hydrogenation over rhodium catalysts using isotopic tracers

The medium-pressure (1.01 MPa) hydrogenation of carbon monoxide has been investigated over rhodium catalysts in the steady state (at typically 523 K) by use of isotopic labeling ([ 13C]CO, [ 18O]CO, and [ 2H]H 2). When labeled carbon monoxide was introduced, neither methane nor ethanol nor methanol showed any incorporation; however, the labels were rapidly incorporated into the aldehydic function of ethanal. Labeled water, produced from the hydrogenation of [ 18O]CO, took more than 0.3 h to desorb. The results suggest that (i) ethanol and ethanal are produced independently with no common intermediate, (ii) the formation of alcohols is slow (taking over 0.5 h), and (iii) carbon monoxide is not hydrogenated directly to methane but goes through the hydrocarbonaceous residue present on the surface. The carbonaceous residue was found to play a central role in the mechanism, supplying in effect both hydrogen and CH 2 units to other reactive Surface Species.

The mechanism of acetate oxidation on Ag(110)

The effects of coadsorbed oxygen atoms and gaseous O 2 at pressures up to 0.7 Torr on the stability of the surface acetate intermediate on Ag(110) were investigated. Under an O 2 pressure of 0.7 Torr acetate decomposes at temperatures as low as 400 K, which is 200 K lower than the decomposition temperature of acetate in vacuum. This destabilization is due to population of the surface with atomic oxygen when 0.7 Torr O 2 is present. Acetate reacts with coadsorbed oxygen atoms via an intermediate which decomposes at 340 K to CO 2, water and formate. The formate subsequently decomposes at 400 K to give CO 2 and H 2. Through the use of isotopic labelling, it was found that the carbon and hydrogen atoms in the formate originate from the methyl group of the acetate, while the two oxygen atoms come from surface atomic oxygen. A mechanism for the reaction of acetate with atomic oxygen is presented in which a surface oxygen atom abstracts a proton from the acetate, and the resulting CH 2COO (a) species reacts further with another oxygen atom to form a glycolate (O 2CCH 2O (a)) intermediate. Nucleophilic attack at the methylene carbon by an oxygen atom, followed by C-C bond scission releases CO 2 and forms H 2CO 2(a), which subsequently loses a hydrogen atom to make formate. The results presented here show that acetate will decompose under commercial ethylene oxidation conditions, and thus cannot be ruled out as an intermediate in the total oxidation of ethylene. Isomerization of ethylene oxide to acetaldehyde, followed by oxidation of the acetaldehyde is a plausible route to acetate formation during ethylene oxidation. In addition, this work demonstrates that phenomena which occur at high pressures can be observed in ultra high vacuum provided the relevant surface species can be formed in vacuum and remain on the surface up to the temperatures at which the high pressure phenomena occur.

The Bakerian lecture, 1984. Biosynthesis of the pigments of life.

Many vitally important functions in living systems are carried out by metal ions held as complexes within organic ligands, the organic part of the molecule being a tetrapyrrolic macrocycle. Chlorophyll, haemoglobin, the cytochromes and vitamin B12 all fall into this family of 'pigments of life', a list that emphasizes their central importance in living systems. Research on the biosynthesis of these pigments has involved the synergistic combination of synthesis, structure determination, carbon nuclear magnetic resonance and isotopic labelling with radioactive and stable isotopes in conjunction with enzymology and kinetics. The lecture describes the logical series of experiments based on these approaches which have led to a step-by-step knowledge of the biosynthesis of the parent macrocycle (uroporphyrinogen-III) from which the other pigments are derived. One main pathway from the parent macrocycle involves oxidative transformations and leads eventually to protohaem required inter alia for haemoglobin and myoglobin. The second important pathway makes use of C-methylation to convert the parent macrocycle through many stages finally into vitamin B12. The biosynthetic studies on vitamin B12 are outlined with particular emphasis on the use of isotopic labelling with both radioactive and stable isotopes of carbon and hydrogen. Roughly two-thirds of the entire biosynthetic pathway to vitamin B12 has now been elucidated. The scarcity of several of the known intermediates on the pathway severely hampers future researches and progress towards the total synthesis of these key materials is reviewed. Finally, the lecture brings out the evolutionary interest of what has been discovered about the biosynthesis of the pigments of life.

Kinetic studies in mass spectrometry—VI: The [M  CH3] reaction in substituted ethylbenzenes

AbstractThe [M] → [M  CH3] reaction in a series of m‐ and p‐X substituted ethylbenzenes has been studied by wide range electron energy kinetics and metastable ion characteristics techniques. By this approach, qualitative measures of activation evergy differences between [XC6H4CH2]+ ions derived from m‐ and p‐X isomer substrates have been secured, for both their formation and further decomposition. These evergy differences are consistent with (but do not prove) ion structures that have been suggested by previous work in this area, involving the use of isotope labeling, and ionization and appearance potential methods.

Fluorescein-conjugated bovine albumin; physical and biological properties.

Fluorescein-bovine albumin conjugates have been prepared and found not to differ appreciably in size, shape, and homogeneity from the precursor, bovine serum albumin. Fluorescein has also been conjugated to rat plasma proteins. Their disappearance rates from the circulation of rats correspond with those obtained from the use of isotope labeling. Their sites of localization in rat tissues were shown to be in the cytoplasm but not in the nuclei of Kupffer cells, fixed macrophages, granulocytes, and proximal renal tubules. Adsorption to endothelium was a characteristic finding. Extracellular localizations were predominantly in the lumina of blood vessels and proximal renal tubules (but never in the lumina of collecting tubules), and the interstitial fluid of skeletal and cardiac muscle (but not that of glandular organs such as the adrenals, liver, and spleen). BAC absorption from the skin of rabbits requires days whereas sodium fluorescein absorption is measured in hours, attesting to the persistence of the colloidal state of BAC in vivo. Fluorescein conjugates have been used to visualize the transcapillary passage of circulating proteins in the mesenteric circulation of frogs and rats by direct microscopic observation and found to diffuse slowly in the manner predicted for plasma proteins. The normal cutaneous vessels of the rat are impermeable in the gross to the labeled proteins; second degree burn promptly increases the permeability of these vessels rendering the presence of the label detectable in the gross in the skin. The process of labeling does not render guinea pig albumin antigenic, although slight antigenicity results from labeling whole plasma protein. It is believed that sufficient biological evidence is presented to support the conclusion that fluorescein-conjugated plasma proteins, particularly albumin, behave in vivo like their native precursors.