Mass spectrometry stable isotope tagging

Tao WA, Aebersold R (2003) Advances in quantitative proteomics via stable isotope tagging and mass spectrometry. Curr Opin Biotechnol 14 110-118... 

Che FY, Biswas R, Fricker LD. Relative quantitation of peptides in wild-type and Cpe(fat/fat) mouse pituitary using stable isotopic tags and mass spectrometry. J. Mass Spectrom. 2005 40 227-237. Decaillot FM, Che FY, Fricker LD, Devi LA. Peptidomics of Cpefat/fat mouse hypothalamus and striatum effect of chronic morphine administration. J. Mol. Neurosci. 2006 28 277-284. Naggert JK, Fricker LK, Varlamov O, Nishina PM, Rouille Y, Steiner DF, Carroll RJ, Paigen BJ, Leiter EH. Hyperproinsuli-naemia in obese fat/fat mice associated with a carboxypepti-dase E mutation which reduces enzyme activity. Nat. Genet. 1995 10 135-142. 

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... 

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... 

ICAT has also been adapted for absolute quantification purposes. The metal-coded affinity tag method introduces different lanthanide ions instead of stable isotope into peptides, which enables their absolute quantification by inductively coupled plasma mass spectrometry (ICP-MS) (55,56). Appending a fluorophore to an ICAT reagent also facilitates absolute quantification by fluorescence detection. Fluorescence isotope-coded affinity tag, as this... 

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). 

Certain methodologies used in conjunction to protein profiling by mass spectrometry. There are several methods to label proteins that assist their profiling by mass spectrometry. These methods involve labeling of proteins in vitro or in vivo with an isotope. Some of these techniques include Isotope Coded Affinity Tag (ICAT) and Stable Isotope Labeling with amino acids in Cell culture (SILAC). These are described below. 

Mass Spectrometric Identification of Interacting Proteins. The preparation of proteins obtained after TAP is introduced into a spectrometer, and the different proteins are identified. In such an analysis, a sample of protein affinity purified using the epitope tag and the one without the epitope tag is labeled with stable isotopes and then compared for the relative abundance of different peaks in the two samples to determine the interacting proteins in a particular preparation. Thus, this is essentially a quantitative ICAT mass spectrometry described in Chapter 4. In case the proteins are purified over a DNA column without the use of an epitope tag, it is compared with a sample that cannot bind with DNA in the matrix. The samples are labeled with stable isotopes. The interacting proteins are identified by their relative abundance. 

Bowman MJ, Zaia J. Tags for the stable isotopic labeling of carbohydrates and quantitative analysis by mass spectrometry. Anal Chem 2007 79 5777-5784. 

In most cases, standards are also needed for analysis, and these should be added to the sample as soon as possible, preferably at the time of sampling. The standard is often an isotope-labeled compound. A big advantage of mass spectrometry-based methods is that they can detect stable isotope labels and are not radioactive therefore, they do not pose a health hazard and can be freely used. Stable isotope labeling is also called isotope labeled affinity tags, especially in the field of proteomics. 

More conventionally, mass spectrometry is used to separate isotopes of a given element or isotopically tagged compounds. Four stable isotopic pairs have found application in the vast majority of molecular labelling experiments examined by mass spectrometry - >H/2H, 12C/13C, 14N/15N, and >60/i 0. In each of these cases, the heavier isotope is the rarer, with natural abundances of 0.15% ( H), 1.1% C C), 0.37% (>5N), and 0.20% C 0). The survey below will confine itself to molecular labelling experiments that utilize hydrogen, carbon, nitrogen and oxygen isotopes. 

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