Isotopic labelling approaches

Using this isotope labeling approach, metabolites in human plasma have been quantified (38) and altered metabolites have been detected in urine that are due to metabolic disorders such as tyrosinemia type II, argininosuccinic aciduria, homocystinuria, and phenylketonuria (20). Most recently, a smart isotope tag, 15N-cholamine, has been developed for effective detection of the same metabolites using both NMR and MS methods. This approach maximizes the combined strengths of two powerful analytical techniques for a variety of metabolomics applications. 15N-cholamine possesses dual characteristics an NMR-sensitive heteronuclear isotope with good chemical shift dispersion and a permanent charge that improves MS sensitivity (48). 

In the following section, both label-free and isotopic label approaches are described in detail. In both approaches, the point of the experiment is to compare the relative levels of peptides in two or more distinct groups ... 

Isotopic labelling approaches to studying soil organic phosphorus dynamics... 

For the majority of stable-isotope labeling approaches, including those described above, quantitative analysis is achieved in a mass scan. Limitations are encountered. 

Label-free quantitation is a rapid and fairly accurate approach for the survey of change in protein expression levels between two biological samples and is a promising alternative to stable isotope labeling approaches. It is a highly reproducible method that enables relative quantitation of the proteins across samples [14,15]. In a label-free quantitative approach, each sample has to be analyzed individually and sequentially by MS (unlike... 

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). 

One of the possibilities is to study experimentally the coupled system as a whole, at a time when all the reactions concerned are taking place. On the basis of the data obtained it is possible to solve the system of differential equations (1) simultaneously and to determine numerical values of all the parameters unknown (constants). This approach can be refined in that the equations for the stoichiometrically simple reactions can be specified in view of the presumed mechanism and the elementary steps so that one obtains a very complex set of different reaction paths with many unidentifiable intermediates. A number of procedures have been suggested to solve such complicated systems. Some of them start from the assumption of steady-state rates of the individual steps and they were worked out also for stoichiometrically not simple reactions [see, e.g. (8, 9, 5a)]. A concise treatment of the properties of the systems of consecutive processes has been written by Noyes (10). The simplification of the treatment of some complex systems can be achieved by using isotopically labeled compounds (8, 11, 12, 12a, 12b). Even very complicated systems which involve non-... 

The most straightforward experimental approach is isotopic labeling of certain atoms in the reactants. The detection method must distinguish between the possible isotopologs of the products. For example, in the reaction... 

By a combination of synthetic approaches, isotopic labeling, using tocopherols with 13C-labeling at C-5a and C-7a, EPR spectroscopy, and high-level DFT computations, it was shown that there is no radical formation at either C-5a or C-7a and that chromanol methide radical 10 does not occur in tocopherol.11 EPR failed to detect... 

A simple NMR technique, and arguably the most widely used and effective for hit validation, is the chemical shift perturbation method. In this approach, a reference spectrum of isotopically labeled target is recorded in absence and presence of a given test ligand (or a mixture of test ligands). Commonly, differences in chemical shift between free and bound protein target are observed in 2D [15N, 1H and/or 2D [13C, H] correlation spectra of a protein (or nucleic acid) upon titration of a ligand... 

The isotope dilution method can be used for the measurement of molecules or elemental species (about 60 elements have stable isotopes). This approach allows ultratrace analysis because, contrary to radioactive labelling where the measurement relies on detecting atoms that decay during the period of measurement, all of the labelled atoms are measured. 

Isotope labeling Phage display Protein arrays Computational approaches... 

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]-... 

Ong, S.-E., Blagoev, B., Kratchmarova, I., Kristensen, D.B., Steen, H., Pandey, A., and Mann, M. (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics 1, 376-386. 

Abstract To understand how membrane-active peptides (MAPs) function in vivo, it is essential to obtain structural information about them in their membrane-bound state. Most biophysical approaches rely on the use of bilayers prepared from synthetic phospholipids, i.e. artificial model membranes. A particularly successful structural method is solid-state NMR, which makes use of macroscopically oriented lipid bilayers to study selectively isotope-labelled peptides. Native biomembranes, however, have a far more complex lipid composition and a significant non-lipidic content (protein and carbohydrate). Model membranes, therefore, are not really adequate to address questions concerning for example the selectivity of these membranolytic peptides against prokaryotic vs eukaryotic cells, their varying activities against different bacterial strains, or other related biological issues. 

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