Stable isotope labeling with amino acids

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]-... 

Stable Isotope Labeling with Amino Acids... 

SILAC Stable-isotope labeling with amino acids in cell culture... 

Labeling proteins with heavy and light tags and screening the hit compound versus an inactive control, followed by mass spectrometric comparison of the two samples, is another approach that avoids many of the common pitfalls in affinity methods (3). Techniques such as stable isotope labeling with amino acids in cell culture and isotope-coded affinity tagging (ICAT) exemplify these techniques. 

SILAC stable isotope labelling with amino acids in cell cultmes... 

Everley PA, Krijgsveld J, Zetter BR, Gygi SP (2004) Quantitative cancer proteomics stable isotope labeling with amino acids in cell culture (SILAC) as a tool for prostate cancer research. Mol Cell Proteomics 3(7) 729-735... 

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. 

The sample proteins are usually prepared by a several methods. Among these methods the following are commonly used phosphoprotein isotope-coded affinity tag (PhlAT), isotope-coded affinity tag, (ICAT) and stable isotope labeling with amino acids in cell culture (SILAC). PhlAT introduces isotopes directly into phosphoserine and phosphothreonine residues of the 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. 

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.

Quantification of Proteins with Stable-Isotope Labeling by Amino Acids in Cell Culture 469... 

Later, Ong et al. introduced a method termed SILAC (stable isotope labeling by amino acids in cell). In SILAC, two cell-culture populations are grown under identical conditions, except that one is supplied with the labeled amino acids (e.g. arginine with six atoms) and the other is with the non-labeled. After five or six doublings, two kinds of amino acids have fully incorporated into proteins. Every peptide pair is separated by the mass shift by the labeled amino acid. This approach cannot be applied to tissues or body fluids, and is... 

Table 2.1.3 Multiple reaction monitoring of amino acids for their tandem mass spectrometry quantitation. In daily practise not all mentioned amino acids are measured in one run, but a set of ten dedicated evaluation programs has been developed, covering groups of amino acids associated with groups of disorders. Amino acids presented in italics indicate stable-isotope-labeled internal standards ... 

Reference values of this approach are not different from those for other amino acid analyses. An example of a mass chromatogram, representing the plasma of a PKU patient, is shown in Fig. 2.1.1. When evaluating the results of MS/MS amino acid analyses, one has to reahze that the hquid chromatographic separation is by far less efficient that the AAA separation. For this reason, any amino acid may (partly) coelute with other amino acid(s), which potentially interferes with its mass spectromet-ric behavior. This effect is known as quenching. In order to overcome this as much as possible, stable-isotope-labeled internal standards (as many as possible) should be used. However, this matrix effect of ion suppression is the major pitfall in the MS/MS analysis of amino acids. Consequently, the MS/MS analysis of amino acids cannot be regarded as a reference method, similar to all other amino acid analytical methods. 

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