Proteins SILAC

Figure 1 The SILAC method for comparative proteomics. A real experiment requires collection of thousands of mass spectra corresponding to different peptides. In the case portrayed, the drug has lowered the abundance of the affected protein in the treated cells compared to the control cells. Multiple peptide fragments of the affected protein would show this effect, supporting the conclusion that the protein was affected.

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.

It has been demonstrated very recently that SILAC is applicable to multiplex experiments in order to measure dynamics of protein abundances in cells in response to stimuli such as growth factors or drugs (Molina et al, 2005). Henrietta Lacks (HeLa) cells were grown in normal medium or in media lacking normal... 

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). 

Amanchy, R., Kalume, D. E., Pandey, A. (2005). Stable isotope labeling with amino adds in cell culture (SILAC) for studying dynamics of protein abundance and posttranslational modifications. 

A protein microarray study by Jones et al. (33) was designed to test the affinity of all proteins that contain SH2 and PTB domains, which specify binding to the phosphorylated domains of all four ErbB family members. The study uncovered that some ErbB family members were more promiscuous than others, which has important implications for the ErbB signaling network in general and specifically in that the promiscuous ErbB family members are much more commonly overexpressed in several cancer cell types. Eurthermore, 116 new ErbB interaction partners were discovered. Another study by Schulze et al. (34) was also designed to identify all interaction partners for the phosphorylated ErbB family members but using a novel methodology that combines SILAC and LC-MS/MS. This study defined the specific ErbB sites where the interaction partners bind. 

Figure 7 Protocol for quantification of proteins by the SILAC method. Reproduced from C. Dass, Fundamentals of Contemporary Mass Spectrometry, Wiley-lnterscience Hoboken, NJ, 2007, with permission from Wiley-lnterscience, Copyright 2007.

Unlike mammalian cells, which cannot synthesize all amino acids, SILAC is not well suited to studying the protein/peptide levels of microorganisms, most of which are prototrophic for all amino acids. Application of SILAC in such cases is mostly restricted to auxotrophic strains, which renders the technique ineffective for the proteome analysis of many commercially important microbes. Native SILAC (nSILAC), a recently developed modification of... 

SILAC is difficult for studies on rodents due to the expense of isotopic label and the need for lengthy exposure times to ensure adequate incorporation of the label into the proteome. Despite the high cost, SILAC had been performed for some specific analyses of protein turnover in rodents (48,49). 

Since light and heavy amino acids are chemically identical, the labeling process will not affect the chemical properties of the peptides and therefore differentially labeled peptides will co-elute from the HPLC column. However, these peptides are isotopically distinct from each other the peaks from light and heavy labeled peptides can be accurately distinguished and quantified by using mass spectrometry. An example of a study using SILAC includes the quantitative proteomic analysis of 495 proteins in renal cells towards the exploration of molecular mechanisms of calcineurin-inhibitors induced nephrotoxicity [82], In a second study, a... 

SILAC-based proteomics approach was successfully used to identify a total of 63 differentially expressed proteins between a HepG2 human hepatoma cell line and an immortal hepatic cell line L02 [83]. Among these differentially expressed proteins, phosphoglyc-erate mutase was found highly upregulated and might play important role in hepatocarcinogenesis, which demonstrated the remarkable power of this SILAC platform in biomarker discovery. 

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. 

The other two methods introduce isotopes in the protein at sites other than the phosphorylation sites. PhlAT and ICAT are used to label the proteins in vitro, whereas SILAC is used to label proteins in vivo. SILAC is useful for in vivo labeling of the proteins in cell cultures grown under different conditions that may influence the extent of phosphorylatiom. The ICAT and SILAC methods are described in Chapter 3. 

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