Chemical labeling isotopic tags

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... 

For proteins of low abundance as well as membrane proteins, 2D PAGE suffers from a lack of resolving power. Another possibility, besides chromatography, to circumvent the limitations of 2D PAGE is to use chemically labeled tags, such as two different isotopes (ICAT, isotope-coded affinity tag) (Adam, 2002). A suitable... 

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). 

The ideal chemical isotopic tag is one that will react with every protein or peptide present in the sample and be reactive enough that the protein/peptide becomes fully labeled. However, the intrinsic reactivity of the compound should not be so high that it degrades upon storage under proper conditions. Also, the ideal tag will be stable upon mass spectrometry and not readily break apart under standard conditions. The ideal chemical tag will either be commercially available or relatively easy to synthesize, and the price should be reasonable. At a minimum, the tag needs to be available in a heavy and light form, but ideally would be available in a range of masses so that multiple replicates can be combined. It is also important that peptides labeled with the heavy and light forms of the tag coelute from HPLC otherwise, the quantitation is less accurate when... 

The trimethylammonium butyrate (TMAB) isotopic tags, which are synthesized as amine-reactive AMiydroxysuccinimide derivatives, were first described by Regnier and coworkers (72). The chemical name of the TMAB-A-hydroxysuccinimide reagent (TMAB-NHS) is [3-(2,5-dioxopyrroli-din-l-yloxycarbonyl)propyl] trimethylammonium chloride. The label incorporates a quaternary amine so that the labeled peptide retains a positive charge in place of the free amine (unlike acetylation and related labels described in the next section). 

The field of application for the isotope dilution method with radioactive tags, extends to measurements using stable isotope. Mass spectrometry or nuclear magnetic resonance are used to determine the variations in the isotopic concentrations. Chemical labelling using externally introduced tags consists of the addition to a sample of the same analyte but containing a stable isotope (e.g. H, C, N) as an internal standard. This method is as much used for molecular species as for atoms (around 60 have stable isotopes). 

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). 

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. ... 

TRACER. A chemical entity (almost invariably radioactive and usually an isotope) added to the reacting elements or compounds in a chemical process, which can be traced through the process by appropriate detection methods, e.g.. Geiger counter. Compounds containing tracers are often said to be tagged" or labeled." Carbon-14 is a commonly used... 

---