Analysis protein, cross-linking

These thermal analysis studies serve to establish a direct relationship between a heat-induced AR method and the reversal of formalin-induced intra- and intermolecular protein cross-links.10 2831 Further, while formalin-treatment provides thermal stability to RNase A, this stabilization is not sufficient to prevent thermally induced protein denaturation at temperatures (>100°C) typically used in heat-induced AR methods.32 34 The implications of this finding for the mechanism of AR will be discussed further in Section 15.6. 

Several attempts of FruA-catalyzed DHAP additions to simple aliphatic dialdehydes like glyoxal or glutaric dialdehyde have been reported in the literature, but in no case had a product been isolated and characterized [72,85]. Malonic dialdehyde cannot be used because it tends to enolize under protic conditions and engages in polycondensations. Our own extensive studies corroborate that enzymatic assays indicate a consumption of DHAP, but no defined products result according to t.Lc. or NMR analysis. Problematic also is the fact that aliphatic dialdehydes irreversibly destroy enzymatic activity by protein cross-linking [86,87]. 

Miller CAIII, Costa M. 1989. Analysis of proteins cross-linked to DNA after treatment of cells with formaldehyde, chromate and czy-diamminechloroplatimun(II). Mol Toxicol 2 11-26. 

Schlosser, P. M., Patrick, D. L., Conolly, R. B., Janszen, D. B., and Kimbell, J. S. (2003). Benchmark dose risk assessment for formaldehyde using airflow modeling and a single-compartment, DNA-protein cross-link dosimetry model to estimate human equivalent doses. Risk Analysis 23, 473-487. 

Seebacher, J. MaUick, P. Zhang, N. Eddes, J.S. Aebersold, R. Gelb, M.H. Protein cross-linking analysis using mass spectrometry, isotope-coded cross-linkers, and integrated computational data processing. J. Proteome Res. 2006, 5, 2270-2282. 

Thiede, B. and Wittmann-Liebold, B. (2000) Analysis of RNA-protein cross-link sites by matrix-assisted laser desorption/ionization mass spectrometry and N-terminal microsequencing. Methods Enzymol., 318, 438-446. 

Urlaub, H., Hartmuth, K., Kostka, S., Grelle, G., and Luhrmann, R. (2000) A general approach for identification of RNA-protein cross-linking sites within native human spliceosomal small nuclear ribonucleoproteins (snRNPs). Analysis of RNA-protein contads in native U1 and U4/U6.U5 snRNPs./. 

Lee, Y.J. (2008) Mass Spectrometric Analysis of Cross-linking Sites for the Structure of Proteins and Protein Complexes. Mol. BioSyst. 4 816-823. 

The side chains of proteins can undergo post-translational modification in the course of thermal processes. The reaction can also result in the formation of protein cross-links. A known reaction which mainly proceeds in the absence of carbohydrates, for example, is the formation of dehydroalanine from serine, cysteine or serine phosphate by the elimination of H2O, H2S or phosphate. The dehydroalanine can then lead to protein cross-links with the nucleophilic side chains of lysine or cysteine (cf. 1.4.4.11). In the presence of carbohydrates or their degradation products, especially the side chains of lysine and arginine are subject to modification, which is accompanied by a reduction in the nutritional value of the proteins. The structures of important lysine modifications are summarized in Formula 4.95. The best known compounds are the Amadori product -fructoselysine and furosine, which can be formed from the former compound via the intermediate 4-deoxyosone (Formula 4.96). To detect of the extent of heat treatment, e. g., in the case of heat treated milk products, furosine is released by acid hydrolysis of the proteins and quantitatively determined by amino acid analysis. In this process, all the intermediates which lead to furosine are degraded and an unknown portion of already existing furosine is destroyed. Therefore, the hydrolysis must occur under standardized conditions or preferably by using enzymes. Examples showing the concentrations of furosine in food are presented in Table 4.13. 

Fancy, D.A. and Kodadek, T., Chemistry for the analysis of protein-protein interactions Rapid and efficient cross-linking triggered by long wavelength light, Proc. Natl. Acad. Sci. USA, 96, 6020-6024, 1999. 

In the previously described electrophoretic methods, the capillary was filled with electrolytes only. Another mode of operation in capillary electrophoresis involves filling the capillary with gel or viscous polymer solutions. If desired, a column can be packed with particles and equipped with a frit.68 This mode of analysis has been favorably used for the size determination of biologically important polymers, such as DNA, proteins, and polysaccharides. The most frequently used polymers in capillary gel electrophoresis are cross-linked or linear polyacrylamide,69 cellulose derivatives,70-75 agarose,76 78 and polyethylene glycols. 

Formaldehyde fixes proteins in tissue by reacting with basic amino acids— such as lysine,5 7—to form methylol adducts. These adducts can form crosslinks through Schiff base formation. Both intra- and intermolecular cross-links are formed,8 which may destroy enzymatic activity and often immunoreactiv-ity. These formaldehyde-induced modifications reduce protein extraction efficiency and may also lead to the misidentification of proteins during proteomic analysis. 

Guerrero C, Tagwerker C, Kaiser P, et al. An integrated mass spectrometry-based proteomic approach quantitative analysis of tandem affinity-purified in vivo cross-linked protein complexes (QTAX) to decipher the 26 S proteasome-interacting network. Mol. Cell. Proteomics. 2006 5 366-378. 

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