Isotopes 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... 

Floey, M. R., Geieein, T. J., Martin, D., Aebersold, R. (2002). Advances in quantitative proteomics using stable isotope tags. Trends Biotechnd. 20, 23-29. 

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. 

Incorporation of a stable isotopic tag into proteins/peptides in metabolically active cells was first described to quantify protein abundance in yeast (43). Wild-type and mutant cell populations were grown in media containing the naturally abundant isotopes of nitrogen and enriched in 15N, respectively, followed by trypsin digestion and LC-ESI-MS/MS analysis to identify and quantify relative phosphopeptide levels in both populations (43). 

In another variant for quantitative proteome analysis by 2D PAGE, one could exploit the technique of stable isotope tagging. In this approach, the labeling strategy involves light/heavy forms of the same tagging molecule. An example of such an approach... 

Ehrenkranz, R.A., Gettner, P.A., NeDi, C.M., Sherwonit, E.A., Williams, J.E., Ting, B.T., and Janghorbani, M. (1991) Selenium absorption and retention by very-low-birth-weight infants studies with the extrinsic stable isotope tag Se. J. Pediatr. Gastroenterol. Nutr, 13, 125-133. 

The stable-isotope tags can be introduced into proteins or peptides by using different methods. These methods can be divided into the following categories ... 

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). 

By coupling GC with MS as the detection system (either ion trap or quadrupole mass spectrometer), structural information can be obtained regarding unknown or unexpected components. Another advantage of using mass spectrometric detection is that it can distinguish analytes tagged with stable isotopes, offering the possibility to use them as... 

Figure 16.1 The general design of an ICAT reagent consists of a biotinylation compound with a spacer arm containing stable isotope substitutions. The reactive group is used to label proteins or peptides at particular functional groups and the biotin affinity tag is used to isolate labeled molecules using immobilized (strept)avidin.

Figure 16.6 The solid phase ICAT reagent provides a thiol-reactive iodoacetyl group to capture cysteine peptides, a spacer containing stable isotopic labels, and a photo-cleavable group that can release the captured peptides for mass spec analysis. The VICAT mass tag is a solution phase labeling agent that also has a photo-cleavable site to release isolated peptides from a (strept)avidin affinity resin. This compound adds a fluorescent group to better detect labeled peptides as they are being isolated from a sample.

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... 

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. ... 

Kohno, T., Kusunoki, H., Sato, K., and Wakamatsu, K. (1998) A new general method for the biosynthesis of stable isotope-enriched peptides using a decahis-tidine-tagged ubiquitin fusion system an application to the production of masto-paran-X uniformly enriched with 15N and 15N/13C. J. Biomol. NMR 12,109-121. 

Figure 6.4 The structure of the ICAT reagent that consists of three elements an affinity tag (biotin), which is used to isolate ICAT-labeled peptides a linker that can incorporate stable isotopes and a reactive group with specificity toward thiol groups (cysteines). The reagent exists in two forms heavy (contains eight deuteriums) and light (contains no deuteriums). (Reprinted with permission from Gygi et al., 1999. Copyright 1999 Nature Publishing Group.)...

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