Protein quantitation stable isotope label

The Tools of Proteomics A variety of methods and techniques including two-dimensional gel electrophoresis (2DE), capillary liquid chromatography, stable isotope labeling, and mass spectrometry has been developed for qualitative and quantitative protein... 

Guerrero et al. (2006) used this technique along with the quantitative mass spec strategy called SILAC (stable isotope labeling of amino acids in cell culture Ong et al., 2002) to identify the yeast proteins that interact with the 26 S proteasome. 

The proteomics research of a number of scientists was described in a C E News report of the 2001 Pittcon meeting.10 One group, that of Catherine Fenselau at the University of Maryland, has studied a new method for proteolytic stable isotope labeling to provide quantitative and concurrent comparisons between individual proteins from two entire proteome pools.11 Two lsO atoms are incorporated into the... 

Figure 4.10. Proteomic analysis by SILAC. Proteomic analysis by SILAC or stable isotope labeling of amino acids in cell culture utilize de novo metabolic incorporation of stable-isotope-labeled amino acids during protein synthesis. Cells can be cultured with various combinations of stable-isotope-labeled amino acids such as lysine or arginine. Tyrosine has been used in phosphoprotein studies of tyrosine residues. About five or six cell divisions are needed for complete labeling of proteins in cell cultures prior to experimentation. Labeled cells from control and treatment(s) lysates are combined and digested. Quantitation and identification are performed by LC-MS/MS.

In quantitative proteomics, two alternative strategies have been developed. The first one is based on 2-D PAGE combined with mass spectrometry for protein identification (Haynes and Yates, 2000). This method and current advances in the differential display of proteins by 2-D PAGE are discussed at length in another chapter of this book. The second approach exploits mass spectrometry in combination with stable isotope labeling for gaining accurate quantitative information on proteins. Quantitative MS via stable isotope labeling of proteins... 

The SI LAC approach has also been used to investigate metastatic prostate cancer development at the protein level (Everley et al., 2004). The fact that proteins showed altered concentration ratios by quantitative MS was confirmed by western blotting. In addition, proteomic approaches for quantitation of protein phosphorylation via SILAC combined with MS analysis have been described (Gruhler et al., 2005 Ibarrola et al., 2003, 2004). A recent study reports on identification as well as relative quantitation of in vivo mefhylation sites of proteins in HeLa cells by stable isotope labeling wifh C Hj-methionine (Ong et al., 2004). 

D. Bonenfant,T. Schmelze, E. Jacinto, J. L. Crespo,T. Mini, M.N. Hall, and P. Jenoe, Quantitation of changes in protein phosphorylation a simple method based on stable isotope labeling and mass spectrometry, Proc. Natl. Acad. Sci. USA 100... 

Blonder, J., Yu, L.R., Radeva, G., Chan, K.C., Lucas, D.A., Waybright, T.J., Issaq, H.J., Sharom, F.J. and Veenstra, T.D. (2006) Combined chemical and enzymatic stable isotope labeling for quantitative profiling of detergent-insoluble membrane proteins isolated using Triton X-100 and Brij-96. J. Proteome Res. 5, 349-360. 

Relative protein quantitation is the basis of all types of differential proteome analyses. In the 2D-gel approach protein staining with either visible or fluorescent dyes provides a reliable and sensitive method to detect changes in protein expression or isoform abundance. In the multidimensional LC approach quantitation relies mostly on stable isotope labeling and ratios between light and heavy isotopomers are determined by MS or MS/MS at the peptide level. Labeling can be performed on the protein level by... 

As far as protein-related biomarkers are considered, an important tool in biomarker discoveiy is quantitative protein-expression profiling (Ch. 18.4). Mixtures of nonlabelled and stable-isotopically-labelled peptides derived from two different states, e.g., a healthy and disease state, or with and without treatment, are analysed to search for up- or down-regulated protein concentrations. One may question... 

The other approach to quantitative protein profiling is based on stable isotope labelling of proteins and peptides and automated tandem mass spectrometiy (MS/MS) (Gygi et al., 1999b). This method, termed isotope-coded affinity tag labelling or ICAT, has been shown to be robust, reproducible and amenable to high throughput automation (Ideker et al.,... 

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