共查询到20条相似文献,搜索用时 15 毫秒
1.
Yu. P. Kozmin A. V. Manoilov M. V. Serebryakova O. A. Mirgorodskaya 《Russian Journal of Bioorganic Chemistry》2011,37(6):719-731
A method for direct introduction of 18O isotopes into carboxyl groups of peptides and proteins via the exchange with H2
18O in the presence of TFA is described. The isotope label is sufficiently stable in a wide pH range. Since the compounds labeled
by this method retain their physicochemical characteristics, they can be used as an internal standard in quantitative assay
of authentic compounds in the analyzed objects by means of mass spectrometry. This method is applicable to quantitative analysis
of peptides and proteins in biological environments, as well as for quantitative kinetic studies of metabolism and enzyme
activity. The quantitative analysis of polypeptides and proteins is combined with trypsinolysis. When necessary, the isotope
label can be simultaneously introduced into all peptides and proteins in a control biosample, making it applicable as a standard
for comparative analysis of experimental biosamples. 相似文献
2.
Shiwei Zhang Qiding Zhong Daobing Wang Zhanbin Huang Guohui Li 《Biotechnology letters》2017,39(12):1853-1857
Objectives
To determine the origin of 15N-labeled phenylalanine in microbial metabolic flux analysis using 15N as a tracer, a method for measuring phenylalanine δ15N using HPLC coupled with elemental analysis-isotope ratio mass spectrometry (EA-IRMS) was developed.Results
The original source of the 15N-labeled phenylalanine was determined using this new method that consists of three steps: optimization of the HPLC conditions, evaluation of the isotope fractionation effects, and evaluation of the effect of pre-processing on the phenylalanine nitrogen stable isotope. In addition, the use of a 15N-labeled inorganic nitrogen source, rather than 15N-labeled amino acids, was explored using this method.Conclusions
The method described here can also be applied to the analysis of metabolic flux.3.
Agnieszka?Sitarska Lukasz?Skora Julia?Klopp Susan?Roest César?Fernández Binesh?Shrestha Alvar?D.?Gossert
For a wide range of proteins of high interest, the major obstacle for NMR studies is the lack of an affordable eukaryotic expression system for isotope labeling. Here, a simple and affordable protocol is presented to produce uniform labeled proteins in the most prevalent eukaryotic expression system for structural biology, namely Spodoptera frugiperda insect cells. Incorporation levels of 80 % can be achieved for 15N and 13C with yields comparable to expression in full media. For 2H,15N and 2H,13C,15N labeling, incorporation is only slightly lower with 75 and 73 %, respectively, and yields are typically twofold reduced. The media were optimized for isotope incorporation, reproducibility, simplicity and cost. High isotope incorporation levels for all labeling patterns are achieved by using labeled algal amino acid extracts and exploiting well-known biochemical pathways. The final formulation consists of just five commercially available components, at costs 12-fold lower than labeling media from vendors. The approach was applied to several cytosolic and secreted target proteins. 相似文献
4.
A new method for synthesizing the labeled l-tryptophan is described in this work. l-Tryptophan, labeled with 98% 15N at position 1 was synthesized from the labeled anthranilic acid using Candida utilis mutants. The conversion ratio of 15N of 50% was achieved. The labeled anthranilic acid was synthesized by [15N] phthalimide that was prepared by 99.34% [15N] urea and phthalic anhydride in ortho-xylene medium at 140°C and under atmospheric pressure. 相似文献
5.
Heavy water (H218O) has been used to label DNA of soil microorganisms in stable isotope probing experiments, yet no measurements have been
reported for the 18O content of DNA from soil incubated with heavy water. Here we present the first measurements of atom% 18O for DNA extracted from soil incubated with the addition of H218O. Four experiments were conducted to test how the atom% 18O of DNA, extracted from Ponderosa Pine forest soil incubated with heavy water, was affected by the following variables: (1)
time, (2) nutrients, (3) soil moisture, and (4) atom% 18O of added H2O. In the time series experiment, the atom% 18O of DNA increased linearly (R
2 = 0.994, p < 0.01) over the first 72 h of incubation. In the nutrient addition experiment, there was a positive correlation (R
2 = 0.991, p = 0.006) between the log10 of the amount of tryptic soy broth, a complex nutrient broth, added to soil and the log10 of the atom% 18O of DNA. For the experiment where soil moisture was manipulated, the atom% 18O of DNA increased with higher soil moisture until soil moisture reached 30%, above which 18O enrichment of DNA declined as soils became more saturated. When the atom% 18O for H2O added was varied, there was a positive linear relationship between the atom% 18O of the added water and the atom% 18O of the DNA. Results indicate that quantification of 18O incorporated into DNA from H218O has potential to be used as a proxy for microbial growth in soil. 相似文献
6.
Juranić N Nemutlu E Zhang S Dzeja P Terzic A Macura S 《Journal of biomolecular NMR》2011,50(3):237-245
Intramolecular correlations among the 18O-labels of metabolic oligophosphates, mapped by J-decoupled 31P NMR 2D chemical shift correlation spectroscopy, impart stringent constraints to the 18O-isotope distributions over the whole oligophosphate moiety. The multiple deduced correlations of isotopic labels enable
determination of site-specific fractional isotope enrichments and unravel the isotopologue statistics. This approach ensures
accurate determination of 18O-labeling rates of phosphometabolites, critical in biochemical energy conversion and metabolic flux transmission. The biological
usefulness of the J-decoupled 31P NMR 2D chemical shift correlation maps was validated on adenosine tri-phosphate fractionally 18O labeled in perfused mammalian hearts. 相似文献
7.
Kota Kera Dennis D. Fine Daniel J. Wherritt Yoshiki Nagashima Norimoto Shimada Takeshi Ara Yoshiyuki Ogata Lloyd W. Sumner Hideyuki Suzuki 《Metabolomics : Official journal of the Metabolomic Society》2018,14(5):71
Introduction
Oxygen from carbon dioxide, water or molecular oxygen, depending on the responsible enzyme, can lead to a large variety of metabolites through chemical modification.Objectives
Pathway-specific labeling using isotopic molecular oxygen (18O2) makes it possible to determine the origin of oxygen atoms in metabolites and the presence of biosynthetic enzymes (e.g., oxygenases). In this study, we established the basis of 18O2-metabolome analysis.Methods
18O2 labeled whole Medicago truncatula seedlings were prepared using 18O2-air and an economical sealed-glass bottle system. Metabolites were analyzed using high-accuracy and high-resolution mass spectrometry. Identification of the metabolite was confirmed by NMR following UHPLC–solid-phase extraction (SPE).Results
A total of 511 peaks labeled by 18O2 from shoot and 343 peaks from root were annotated by untargeted metabolome analysis. Additionally, we identified a new flavonoid, apigenin 4′-O-[2′-O-coumaroyl-glucuronopyranosyl-(1–2)-O-glucuronopyranoside], that was labeled by 18O2. To the best of our knowledge, this is the first report of apigenin 4′-glucuronide in M. truncatula. Using MSn analysis, we estimated that 18O atoms were specifically incorporated in apigenin, the coumaroyl group, and glucuronic acid. For apigenin, an 18O atom was incorporated in the 4′-hydroxy group. Thus, non-specific incorporation of an 18O atom by recycling during one month of labeling is unlikely compared with the more specific oxygenase-catalyzing reaction.Conclusion
Our finding indicated that 18O2 labeling was effective not only for the mining of unknown metabolites which were biosynthesized by oxygenase-related pathway but also for the identification of metabolites whose oxygen atoms were derived from oxygenase activity.8.
The present study evaluates the variations of 137Ba abundance in pectoral fin spine of 1-month-old juvenile Persian sturgeon (Acipenser persicus) upon marking using the stable isotope approach. The marking of the fish was achieved by incorporation of 137Ba2+ in the calcified lattice of the pectoral fin spine through substitution with structural Ca2+. This process was carried out by rearing juveniles in treatment tanks containing elevated concentrations of 137Ba for 1, 3 and 5 days. The marked fish were then retained in natural abundance fresh and brackish waters, to evaluate the trend of exchange of 137Ba from the fin spines. The abundance of 137Ba in fin spines during marking and post-marking experiments were detected by inductively coupled plasma mass spectrometry (ICP-MS). The results showed that a significant isotope mark can be obtained with no mortality and 100% marking rate on the first day of exposure to the isotope. The marked juveniles maintained their isotopic signature for at least 25 days. Statistical analysis of the obtained 138Ba/137Ba ratios demonstrated that the successful incorporation of 137Ba2+ in pectoral fin spines provides an effective marking method for Persian sturgeon restocking programs. 相似文献
9.
For larger proteins, and proteins not amenable to expression in bacterial hosts, it is difficult to deduce structures using
NMR methods based on uniform 13C, 15N isotopic labeling and observation of just nuclear Overhauser effects (NOEs). In these cases, sparse labeling with selected
15N enriched amino acids and extraction of a wider variety of backbone-centered structural constraints is providing an alternate
approach. A limitation, however, is the absence of resonance assignment strategies that work without uniform 15N, 13C labeling or preparation of numerous samples labeled with pairs of isotopically labeled amino acids. In this paper an approach
applicable to a single sample prepared with sparse 15N labeling in selected amino acids is presented. It relies on correlation of amide proton exchange rates, measured from data
on the intact protein and on digested and sequenced peptides. Application is illustrated using the carbohydrate binding protein,
Galectin-3. Limitations and future applications are discussed. 相似文献
10.
Jin-Lin Zhang Anne M. Wetson Suo-Min Wang Ali R. Gurmani Ai-Ke Bao Chun-Mei Wang 《Biological trace element research》2011,139(1):108-117
Salinity is an increasing problem for agricultural production worldwide. The result of low-affinity Na+ uptake is toxic to the cytoplasm of most crop plants. Nevertheless, the pathways for this low-affinity Na+ uptake are still uncertain. In this work we used 22Na+ isotope tracing technology to investigate factors associated with determination of root 22Na+ influx in the salt accumulation halophyte Suaeda maritima. We found that a 2 min of exposure to the 22Na+ labeled uptake solution was optimal for determining 22Na+ influx into excised roots of S. maritima and that 7 min of blotting is suitable in 22Na+ influx experiments. 22Na+ influx did not increase linearly with the increasing external Na+ concentration, in the range tested, of 2 to 300 mM NaCl. But root 22Na+ influx and root Na+ concentration were well correlated. 22Na+ influx into excised roots of S. maritima was not, however, well correlated with the plant size. All the above results indicated further that this 22Na+ isotope influx procedure is a good method for quantify Na+ uptake rate by the roots of the salt accumulation halophyte. 相似文献
11.
This report describes a novel method for overexpression of 13C-labeled oligosaccharides using genetically engineered Saccharomyces cerevisiae cells, in which a homogeneous high-mannose-type oligosaccharide accumulates because of deletions of genes encoding three
enzymes involved in the processing pathway of asparagine-linked oligosaccharides in the Golgi complex. Using uniformly 13C-labeled glucose as the sole carbon source in the culture medium of these engineered yeast cells, high yields of the isotopically
labeled Man8GlcNAc2 oligosaccharide could be successfully harvested from glycoprotein extracts of the cells. Furthermore, 13C labeling at selected positions of the sugar residues in the oligosaccharide could be achieved using a site-specific 13C-enriched glucose as the metabolic precursor, facilitating NMR spectral assignments. The 13C-labeling method presented provides the technical basis for NMR analyses of structures, dynamics, and interactions of larger,
branched oligosaccharides. 相似文献
12.
Joon-Young Jung Seok Hun Yun Dae-Kyun Im Jinwon Lee Min-Kyu Oh 《Biotechnology and Bioprocess Engineering》2016,21(6):814-822
13C metabolite profiling to quantify the dynamic changes of central carbon metabolites was attempted using mass isotopomer distribution analysis in two yeast strains, Saccharomyces cerevisiae and Kluyveromyces marxianus. Mass and isotopomer balances of the intermediates were examined and calculated in both yeast species and central carbon metabolic fluxes were successfully determined. Metabolic fluxes of pentose phosphate pathway in K. marxianus were 1.66 times higher than S. cerevisiae. The flux difference was also supported by relatively high abundance of partially labeled fructose 6-phosphate and 3-phosphoglycerate as well as an increased concentration of labeled L-valine in K. marxianus. Metabolic flux analysis combined with dynamic metabolite profiling has provided better understanding in the central carbon metabolic pathways of two model organisms and can be applied as a method to analyze more complicated metabolic networks in other organisms. 相似文献
13.
Isotope labeling by residue type (LBRT) has long been an important tool for resonance assignments at the limit where other
approaches, such as triple-resonance experiments or NOESY methods do not succeed in yielding complete assignments. While LBRT
has become less important for small proteins it can be the method of last resort for completing assignments of the most challenging
protein systems. Here we present an approach where LBRT is achieved by adding protonated 14N amino acids that are 13C labeled at the carbonyl position to a medium for uniform deuteration and 15N labeling. This has three important benefits over conventional 15N LBRT in a deuterated back ground: (1) selective TROSY-HNCO cross peaks can be observed with high sensitivity for amino-acid
pairs connected by the labeling, and the amide proton of the residue following the 13C labeled amino acid is very sharp since its alpha position is deuterated, (2) the 13C label at the carbonyl position is less prone to scrambling than the 15N at the α-amino position, and (3) the peaks for the 1-13C labeled amino acids can be identified easily from the large intensity reduction in the 1H-15N TROSY-HSQC spectrum for some residues that do not significantly scramble nitrogens, such as alanine and tyrosine. This approach
is cost effective and has been successfully applied to proteins larger than 40 kDa.
Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
14.
This study reports the sequence specific chemical shifts assignments for 76 residues of the 94 residues containing monomeric unit of the photosynthetic light-harvesting 2 transmembrane protein complex from Rhodopseudomonas acidophila strain 10050, using Magic Angle Spinning (MAS) NMR in combination with extensive and selective biosynthetic isotope labeling methods. The sequence specific chemical shifts assignment is an essential step for structure determination by MAS NMR. Assignments have been performed on the basis of 2-dimensional proton-driven spin diffusion 13C–13C correlation experiments with mixing times of 20 and 500 ms and band selective 13C–15N correlation spectroscopy on a series of site-specific biosynthetically labeled samples. The decreased line width and the reduced number of correlation signals of the selectively labeled samples with respect to the uniformly labeled samples enable to resolve the narrowly distributed correlation signals of the backbone carbons and nitrogens involved in the long -helical transmembrane segments. Inter-space correlations between nearby residues and between residues and the labeled BChl a cofactors, provided by the 13C–13C correlation experiments using a 500 ms spin diffusion period, are used to arrive at sequence specific chemical shift assignments for many residues in the protein complex. In this way it is demonstrated that MAS NMR methods combined with site-specific biosynthetic isotope labeling can be used for sequence specific assignment of the NMR response of transmembrane proteins. 相似文献
15.
A novel type of trivalent BNg five-membered cational species B5Ngn3+(Ng = He~Rn, n = 1~5) has been found and investigated theoretically using the B3LYP and MP2 methods with the def2-QZVPPD and def2-TZVPPD basis sets. The geometry, harmonic vibrational frequencies, bond energies, charge distribution, bond nature, aromaticity, and energy decomposition analysis of these structures were reported. The calculated B?Ng bond energy is quite large (the averaged bond energy is in the range of 209.2~585.76 kJ mol-1) for heavy rare gases and increases with the Ng atomic number. The analyses of the molecular wavefunction show that in the BNg compounds of heavy Ng atoms Ar~Rn, the B?Ng bonds are of typical covalent character. Nuclear independent chemical shifts display that both B53+ and B5Ngn3+(n=1~5) have obvious aromaticity. Energy decomposition analysis shows that these BNg compounds are mainly stabilized by the σ-donation from the Ng valence p orbital to the B53+ LUMO. These findings offer valuable clues toward the design and synthesis of new stable Ng-containing compounds. 相似文献
16.
Boisbouvier J Bryce DL O'neil-Cabello E Nikonowicz EP Bax A 《Journal of biomolecular NMR》2004,30(3):287-301
New methods are described for accurate measurement of multiple residual dipolar couplings in nucleic acid bases. The methods use TROSY-type pulse sequences for optimizing resolution and sensitivity, and rely on the E.COSY principle to measure the relatively small two-bond 2DCH couplings at high precision. Measurements are demonstrated for a 24-nt stem-loop RNA sequence, uniformly enriched in 13C, and aligned in Pf1. The recently described pseudo-3D method is used to provide homonuclear 1H-1H decoupling, which minimizes cross-correlation effects and optimizes resolution. Up to seven 1H-13C and 13C-13C couplings are measured for pyrimidines (U and C), including 1DC5H5, 1DC6H6, 2DC5H6, 2DC6H5, 1DC5C4, 1DC5C6, and 2DC4H5. For adenine, four base couplings (1DC2H2, 1DC8H8, 1DC4C5, and 1DC5C6) are readily measured whereas for guanine only three couplings are accessible at high relative accuracy (1DC8H8, 1DC4C5, and 1DC5C6). Only three dipolar couplings are linearly independent in planar structures such as nucleic acid bases, permitting cross validation of the data and evaluation of their accuracies. For the vast majority of dipolar couplings, the error is found to be less than ±3% of their possible range, indicating that the measurement accuracy is not limiting when using these couplings as restraints in structure calculations. Reported isotropic values of the one- and two-bond J couplings cluster very tightly for each type of nucleotide. 相似文献
17.
Natural variability in stable isotope ratios and element concentrations in calcified structures of fish (e.g. scales and otoliths) has provided biogeochemical ‘tags’ for studying origins and movements of marine species, but has been little used in freshwater studies. We examine whether variability in scale δ15N and δ13C values of Salmo trutta L., could provide a tag of fish over small spatial scales in a small river catchment (River Dee, U.K.) and compared their performance as tags with that of scale/otolith element concentrations. Whole scale δ15N and δ13C values differed among six study sites and fish could be classified to their site of origin with a high degree of accuracy. Classifying fish to their site of capture was marginally superior using scale δ15N and δ13C values compared to that achieved using Sr, Mn, Ba and Mg in scale hydroxyapatite or otolith aragonite. Scale δ15N and δ13C values could therefore provide non-lethally collectable biogeochemical tags superior in performance to element concentrations in otoliths and scales. A comprehensive study of δ15N and δ13C values within freshwater systems would develop our understanding of factors influencing geographical variability in baseline δ15N and δ13C signatures. 相似文献
18.
A simple isotope labeling approach for selective 13C/15N backbone labeling of proteins is described. Using {1,2-13C2}-pyruvate as the sole carbon source in bacterial growth media, selective incorporation of 13Cα-13CO spin-pairs into the backbones of protein molecules with medium-to-high levels of 13C-enrichment is possible for a subset of 12 amino acids. The isotope labeling scheme has been tested on a pair of proteins—a
7-kDa immunoglobulin binding domain B1 of streptococcal protein G and an 82-kDa enzyme malate synthase G. A number of protein
NMR applications are expected to benefit from the {1,2-13C2}-pyruvate based protein production. 相似文献
19.
Summary Various plant secondary products have been implicated in the promotion of good health or the prevention of disease in humans,
but little is known about the way they are absorbed in the gut, or in which tissues they are deposited throughout the body.
While these issues could be studied if the phytochemicals were isotopically labeled, generating labeled molecules often is
problematic because many compounds of interest can be synthesized only in planta at present. In order to generale 14C-labeled phytochemicals of high radioactive enrichment, we developed an enclosed-chamber labeling system in which cell suspension
cultures can be safely and efficiently grown when supplied with 14C-enriched precursors. The system is designed to hold culture flasks within a clear, polyacrylic compartment that is affixed
to the top of a rotary shaker. The flow-through gas exchange nature of the system allows for O2 replenishment and complete capture of respired 14CO2 throughout the entire period of cell culture. Air is circulated internally with the aid of a small fan, and chamber air temperature
is monitored continuously with an internal temperature probe and data logger. Production runs of 12–14 d with Vaccinium pahalae (ohelo berry) and Vitis vinifera (grape) suspension cultures, using [14C]sucrose as the carbon source, demonstrated a 20–23% efficiency of 14C incorporation into the flavonoid-rich fractions. Further studies with ohelo cell cultures showed that flavonoids were produced
with either sucrose or glucose as the carbohydrate source, although flavonoid productivity (measured as anthocyanins) was
higher with sucrose. This comprehensive chamber system should have broad applicability with numerous cell types and can be
used to generate a wide array of labeled phytochemicals. 相似文献
20.
Ulrich Weininger 《Journal of biomolecular NMR》2017,67(3):191-200
NMR-spectroscopy enables unique experimental studies on protein dynamics at atomic resolution. In order to obtain a full atom view on protein dynamics, and to study specific local processes like ring-flips, proton-transfer, or tautomerization, one has to perform studies on amino-acid side chains. A key requirement for these studies is site-selective labeling with 13C and/or 1H, which is achieved in the most general way by using site-selectively 13C-enriched glucose (1- and 2-13C) as the carbon source in bacterial expression systems. Using this strategy, multiple sites in side chains, including aromatics, become site-selectively labeled and suitable for relaxation studies. Here we systematically investigate the use of site-selectively 13C-enriched erythrose (1-, 2-, 3- and 4-13C) as a suitable precursor for 13C labeled aromatic side chains. We quantify 13C incorporation in nearly all sites in all 20 amino acids and compare the results to glucose based labeling. In general the erythrose approach results in more selective labeling. While there is only a minor gain for phenylalanine and tyrosine side-chains, the 13C incorporation level for tryptophan is at least doubled. Additionally, the Phe ζ and Trp η2 positions become labeled. In the aliphatic side chains, labeling using erythrose yields isolated 13C labels for certain positions, like Ile β and His β, making these sites suitable for dynamics studies. Using erythrose instead of glucose as a source for site-selective 13C labeling enables unique or superior labeling for certain positions and is thereby expanding the toolbox for customized isotope labeling of amino-acid side-chains. 相似文献