Chemical labeling stable isotopic labels

Another widely used approach to the elucidation of metabolic sequences is to feed cells a substrate or metabolic intermediate labeled with a particular isotopic form of an element that can be traced. Two sorts of isotopes are useful in this regard radioactive isotopes, such as and stable heavy isotopes, such as or (Table 18.3). Because the chemical behavior of isotopically labeled compounds is rarely distinguishable from that of their unlabeled counterparts, isotopes provide reliable tags for observing metabolic changes. The metabolic fate of a radioactively labeled substance can be traced by determining the presence and position of the radioactive atoms in intermediates derived from the labeled compound (Figure 18.13). 

The approach recruited to chemical proteomics in Reference [17] is called SILAC (stable isotope labeling with amino acids in cell culture) and is important in comparative proteomics (Figure 1). SILAC works well with cultured mammalian cells, but prokaryotes defeat it by metabolizing the label (usually supplied in lysine and arginine) into other amino acids. For applications beyond cultured eukaryotic cells, the reductive methylation route to differential labeling [18] is among the alternatives [15]-... 

Although use of radio and stable isotope labels involving the trio of covalently-bonded nitrogenous functions in 3 and in 78, provided evidence that isocyano is the precursor of the isothiocyano and formamido groups [30, 81], it remains to be shown that a biosynthetic equivalent of the in vitro chemically-proven fusion process between isocyano and free sulfur (e.g., cf. Introduction) exists in the cells of sponges. In marine biota, various ionic forms of sulfur in a number of oxidation states, as well as organo-polysulfides are known. However, any association with the isonitrile group and a sulfated species has yet to be established. 

The isotope-coded affinity tag approach utilizes chemical labeling that allows quantitation when combined with mass spectrometry. ICAT is desirable because the chemical labeling takes advantage of the mass defects of monoisotopic stable isotopes. ICAT uses an ICAT reagent to differentially label protein samples on their cysteine residues. ICAT is advantageous because it permits the evaluation of low-abundance proteins and proteins at both extremes of molecular weights and isoelectric points.60... 

There are two basic aims of chemical derivatization simplification of the fragmentation pattern by either promoting or demoting formation of a chosen ion series, and stable-isotopic labelling for simple assignment of ions to proper ion series (Fig. 6.21). Both methods may provide good results and their advantages and limitations will be discussed. 

Probably, one of the most valuable advances in this field has dealt with the first chemoenzymatic synthesis of the stable isotope-enriched heparin from a uniformly double labelled 13C, 15N /V-acetylheparosan from E. coli K5. Heteronuclear, multidimensional nuclear magnetic resonance spectroscopy was employed to analyze the chemical composition and solution conformation of N-acety 1 hcparosan, the precursors, and heparin. Isotopic enrichment was found to provide well-resolved 13C spectra with the high sensitivity required for conformational studies of these biomolecules. Stable isotope-labelled heparin was indistinguishable from heparin derived from animal tissues and might be employed as a novel tool for studying the interaction of heparin with different receptors.30... 

In addition to the stable isotope labeling ( 0 versus 0) of proteins for quantifiable proteomic analyses as described above, chemical approaches to the protein-labeUng problem have developed in great variety. These so-called affinity tags can be used to label specific side chain groups such as sulfhydryl or amino groups, active sites for serine and cysteine hydrolases and many others. This active research area has been reviewed recently by A. Leitner and W. Lindner in a Proteomics article entitled Chemistry meets proteomics The use of chemical tagging reactions for MS-based proteomics. ... 

Fig. 3.9 ALIS-MS results for the titration of 5 pM HSA with warfarin in the presence of a 5 im concentration of its stable isotope-labeled congener warfarin-De. Increasing concentrations of warfarin reduce the response of warfarin-De due to isosteric binding competition. Reprinted from [39] with permission from the American Chemical Society.

Liang, H.R. Foltz, R.L. Meng, M. Bennett, P. Ionization enhancement in atmospheric pressure chemical ionization and suppression in electrospray ionization between target drugs and stable-isotope-labeled internal standards in quantitative liquid chromatography/tandem mass spectrometry. Rapid Commun. Mass Spectrom. 2003, 17, 2815—2821. 

The chemical reactions involving positron emitters have to be specially designed to take into account the short half life of the radionuclide, the limited number of labelled precursors and the sub-micromolar amounts of these precursors. Moreover, the reactions must be possible without any addition of the stable isotope (especially when ligands of receptors are synthesized). Several practical considerations that influence the design of positron-emitter radiotracers with a high specific radioactivity and their experimental handling have been reviewed [4,8,14-19]. 

An analytical chemical technique that utilizes radioactive (or stable) isotopes for the quantitative analysis of the amount of substance. In the absence of a kinetic isotope effect, isotopic isomers react identically with respect to their unlabeled counterparts. The method offers the advantage that specific activity (or gram-atom excess in the case of stable isotopes) is an intensive variable. Therefore, one only needs to recover sufficient labeled metabolite to determine amount of substance and disintegrations per minute (or, gram-atom excess) to reach an accurate determination of specific activity. The technique is feasible so long as one can accurately determine the initial and final specific activities. 

The precision of MS assays is in the range typical of most clinical assays (i.e., under 5-15%). The best choice of internal standard is the stable-isotope-labeled form (preferably 13C) of the compound of interest (e.g., P-hydroxy myristic acid or muramic acid). Specific trace detection of chemical markers in complex matrices requires appropriate negative controls. Procedures are often described that do not employ the mass spectrometer and false positives are often reported. The mere analysis of blank filters or water blanks is not satisfactory since chemical noise contributed by the sample is much greater and is not accounted for with this form of control. 

Perhaps no reagent is more important than the stable-isotope-labeled internal standard in any clinical assay utilizing mass spectrometry for quantification. Internal standards are important in many aspects of the analysis and are somewhat different than standards utilized in other clinical, non-mass-spectrometric assays. The ideal internal standard is an enriched isotopic version of the analyte being measure. For example, in the case of phenylalanine, a standard available may contain six 13C molecules rather than 12C in the aromatic ring. This has the net effect of shifting the mass of phenylalanine by six units while also maintaining nearly identical chemical... 

Value of Isotopic Labeling. The experiments described demonstrate the usefulness of stable-isotope additions in understanding trophic interactions and biogeochemical fluxes in whole ecosystems. Using stable isotopes as chemical tracers in natural, undisturbed systems is especially helpful in interpreting the results of perturbation experiments. 

Considerations in Stable-Isotope Experiments. Isotopic tracer additions should label the specific pool of interest quickly, before chemical or biological transformations of no interest distribute the tracer throughout the ecosystem. For calculation of transfers of material from one pool to another the isotope must be well mixed within the ecosystem in the compound it is supposed to trace, and the isotopic content between both pools should be given time to reach equilibrium. 

The quantification of metabolites in dried blood spots primarily ensures that the quality of the isotopes standards is excellent in terms of chemical and isotopic purity. When using MS/MS, it is essential that the fragments produced by the collision cell and the product ions detected ensure that both labeled and unlabeled metabolites are identical. Most importantly, the choice of the isotope label and the structural positions must be such that they are stable and do not exchange with other isotopes during sample preparation. Finally, it is imperative that the mass shift is sufficiently high (at least 3 Da) for small molecules less than 1000 Da and that the label occurs at a mass free from other compound interference. Figure 4 illustrates the concepts of quantification using stable isotope with Phe measurement in a dried blood spot as an example. 

Internal standards play critical roles in ensuring the accuracy of final reported concentrations in quantitative LC-MS bioanalysis through the correction of variations during sample preparation, LC-separation, and MS detection. The physical-chemical properties of an internal standard, particularly hydrophobicity and ionization properties should be as close as possible to those of the corresponding analyte to better track the variations the analyte experiences. For this reason, stable isotope labeled internal standards should be used whenever possible. However, efforts should still be made to obtain clean extracts, adequate chromatographic separation, and optimized ionization mode and conditions. 

LC-MS/MS assays typically rely on the use of an internal standard that mimics the performance of the analyte to improve the precision, reproducibility and reliability of the assay. An ideal internal standard candidate is a stable-isotope labeled ( stable labeled ) form of the drug. Because synthesizing stable labeled chemicals can be expensive and time-consuming, it is very common to use a chemically similar structural analog of the analyte(s) as the internal standard, especially during the early phases of drug development. 

The internal standard should show physical and chemical properties that are as close as possible to those of the molecule that has to be measured. It must be pure, absent from the sample and, of course, inert towards the compounds in the sample. The internal standards can be classified into three categories structural analogues that are labelled with stable isotopes, structural homologues and compounds from the same chemical family. These various types of internal standards are classified here in descending order according to their usefulness and their price. In fact, the starting material for labelled compounds is fairly... 

C-labelled propionates double-labelled with deuterium or fluorine have been needed to study the stereochemistry of the chain elongation steps in the biosynthesis of polyether antibodies75. NMR spectroscopy allows one to identify the stable isotope-labelled positions in these compounds, thus avoiding a chemical degradation76. 

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