Quantitation of proteins

The Biuret method depends on the reaction of compounds containing several peptide bonds with Cu2+ under alkaline conditions to form a violet-coloured species. 

The reagent required is commercially available, but it can also be prepared by dissolving 1.5 g CuS04 5H20, 6 g Na tartrate in 500 ml H20, adding 300 ml 10% (w/v) 

Biuret Cu2+ reaction with peptide bonds 1-20 mg NH4+, Tris, Good s buffers Rapid insensitive 

Lowry Cu2+ reaction with peptide bonds reduction with phosphomolybdate and phosphotungstate with Tyr und Trp 2—100 pg NH/, Mercapto compounds Slow, depends on amino acid composition unstable colour 

BCA Cu2+ reaction with peptide bonds Cu+ reaction with bicinchonate 0.2-50 pg NH4+. EDTA Suitable for solutions containing detergent 

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The use of the in vivo labeling methods described above is limited by the fact that the sample must be grown in the presence of the labeling isotopes. In many cases, it is not feasible to perform in vivo metabolic labeling. For example, for human clinical samples it is not possible to perform in vivo labeling and yet it is highly desirable to obtain accurate quantitative information on protein expression levels within these samples. Therefore, robust methods are needed for quantitation of protein levels in the absence of in vivo labeling with isotopes. 

Oda, Y., Huang, K., Cross, F. R., Cowbum, D., and Chait, B. T. (1999). Accurate quantitation of protein expression and site-specific phosphorylation. Proc. Natl. Acad. Sci. USA 96, 6591-6596. 

Nirmalan NJ, Harnden P, Selby PJ, et al. Development and validation of a novel protein extraction methodology for quantitation of protein expression in formalin-fixed paraffin-embedded tissues using western blotting. J. Pathol. 2009 217 497-506. 

Quantitation and qualification of large molecules follow similar principles as procedures for small molecules but involve different foci and approaches. The quantitation of proteins and peptides includes relative and absolute amount evaluations. Most proteomic applications in drug discovery are concerned with relative abundances of proteins. 

Kirby R, Cho EJ, Gehrke B, Bayer T, Park YS, Neikirk DP, McDevitt JT, Ellington AD (2004) Aptamer-based sensor arrays for the detection and quantitation of proteins. Anal Chem 76 4066-4075... 

The word protein describes only one type of polymer involving mainly a-amino acids and yet it includes many thousands of different molecules. It is possible to measure the total protein content of a sample despite the fact that relatively simple preparative techniques may be capable of demonstrating the presence of different proteins. However, if interest lies in only one of these proteins, then a measure of the total protein content would be completely inappropriate. Methods for the quantitation of proteins are either suitable for all proteins or designed to measure individual proteins. Such specific methods may depend on either a preparative stage in the analysis or the use of a specific characteristic of the protein in question. 

Procedure 11.1 Quantitation of protein using the Biuret reaction ... 

Procedure 11.3 Quantitation of protein using bidnciionlnic acid... 

Observations that the presence of protein affects the colour change of some indicators used in acid-base titrations led to the development of methods for the quantitation of proteins based on these altered absorption characteristics of such dyes. As the presence of protein alters the colour produced by these indicators when measuring pH, so in the quantitation of proteins using dye-binding methods the control of pH is vital. 

Procedure 11.4 Quantitation of protein using Coomassie brilliant blue dye... 

Zhu MM, Hambley D, Gross ML Quantitation of protein-ligand interactions in solution by H/D exchange (PLIMSTEX), chapter 11. 

Powell, K.D. Ghaemmaghami, S. Wang, M.Z. Ma, L Oas, T.G. Fitzgerald, M.C. A general mass spectrometry-based assay for the quantitation of protein—ligand binding interactions in solution. 

Proteomics includes a variety of technologies that include differential protein display on gels, protein chips, quantitation of protein amoimts, analysis of post-translational modifications, characterization of protein complexes and networks and bioinformatics. All this information in combination with genome and phenotype studies will ultimately yield a comprehensive picture of a cellular or tissue proteome (Wasinger and Corthals 2002). 

Affinity capture-release electrospray ionization mass spectrometry (ACESIMS) and isotope-coded affinity tags (ICAT) are two recently introduced techniques for the quantitation of protein activity and content with applications to clinical enzymology... 

Ideally, for purified or partially purified protein, the protein standard should have an aromatic amino acid content similar to that of the sample protein. For the total protein of a crude lysate, bovine serum albumin (BSA) is a commonly used standard for spectrophotometric quantitation of protein concentration. A 3 mg/ml solution of BSA should have an A280 of 1.98, based on an A2S0 of 6.61 for a 1% (w/v) solution. 

Accordingly, the quantitation of proteins by peptide bond absorption at 205 nm (A2os) is more universally applicable among proteins. Furthermore, the absorptivity for a given protein at 205 nm is several-fold greater than that at 280 nm (Scopes, 1974 Stoscheck, 1990). Thus lower concentrations of protein can be quantitated with the A205 method. The disadvantage of this method is that some buffers and other components absorb at 205 nm (Stoscheck, 1990). 

Nucleic acids have substantial absorbance at 280 nm and can interfere with A2so quantitation of protein in crude samples. To resolve the protein concentration in such samples, measure the absorbance at 260 nm and 280 nm and calculate the protein concentration as follows (Warburg and Christian, 1942 Layne, 1957) protein concentration (mg/ml) = 1.55 x A2g0 -0.76 x A 260- This estimation of protein concentration is valid up to 20% (w/v) nucleic acid or an A2g )/A2(Jo ratio <0.6. 

Hensley, K., Maidt, M. L., Pye, Q. N., Stewart, C. A., Wack, M., Tabatabaie, T., and Floyd, R. A. (1997). Quantitation of protein-bound 3-nitrotyrosine and 3,4-dihydroxy-phenylalanine by high-performance liquid chromatography with electrochemical array detection. Anal. Biochem. 251 187-195. 

Most of the recently developed methods for the detection, characterization, and quantitation of proteins are immunoassays based on the fact that proteins are antigens, compounds that can be recognized by an antibody. It is also true that by combining small molecules (haptens) with a larger carrier molecule such as a protein, these methods can be extended to small molecules of interest since antibodies can be produced that recognize epitopes (specific sites on the antigen recognized by the antibody) that include the hapten. 

Warren EN, Elms PJ, Parker CE, et al. Development of a protein chip a MS-based method for quantitation of protein expression and modification levels using an immunoaffmity approach. Anal. Chem. (2004) 76 4082-4092. 

The use of isotope-coded affinity tag (ICAT) approaches for identification and quantitation of proteins contained in complex mixtures is a developing methodology that bypasses 2-DGE (Gygi et al., 1999). The ICAT approach is a derivative of an isotope dilution method and involves the introduction of a postgrowth biotin affinity tag onto cysteine residues via iodoacatamide chemistry (Figure 6.4). Proteome samples obtained from two cellular states are labeled one with the d0-ICAT and the other with d8-ICAT. The two... 

Environment, relative abundance, and interactions of fluorophore. Quantitation of proteins and fluorophoric compounds. 

H2. Haimovich, J., Hurwitz, E., Novik, N., and Sela, M., Use of protein-bacteriophage conjugates for detection and quantitation of proteins. Biochim. Biophys. Acta 207, 125-129 (1970). 

UV detection at 215 nm allows quantitation of protein content and thus... 

Figure 4.3. Fields of proteomic research. Proteomic research can be classified into six general research fields. Proteomic mapping and proteomic profiling constitute the first tier of proteomic analysis based upon identification and quantitation of proteins within a defined space of interest that can range from the entire organism to the protein level. The second tier of proteomic analyses is shown below involving global characterization of structure, function, posttranslational modifications, and association with other proteins (or other biochemical components).

In parallel with these advances in mRNA detection and assessment, significant advances in the detection, characterization, and quantitation of proteins have been made. This is not only a result of the rapidly expanding database of protein sequences but also of the extraordinary advances in sensitivity of mass spectrometry techniques. It is proposed, with some real sense of expected success, that protein characterization at the whole genome level will be forthcoming (Domon and Aebersold, 2006). 

Shigenaga, M. K., Quantitation of protein-bound 3-nitrotyrosine by high-performance liquid chromatography with electrochemical detection. Meth. Enzymol 301, 27-40 (1999). 

T7. Thomas, J. A., and Beidler, D., A thin-gel isoelectric focusing method for quantitation of protein S-thiolation. Anal. Biochem. 157, 32—38 (1986). 

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). 

Isobaric tags for relative and absolute quantitation of proteins in proteomic research Janus kinase c-Jun iV-terminal kinase... 

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