Protein spectral analysis

Field, H.I., Fenyo, D., Beavis, R.C. (2002). RADARS, a bioinformatics solution that automates proteome mass spectral analysis, optimises protein identification, and archives data in a relational database. Proteomics 2, 36 17. 

Chong BE, Lubman DM, Miller FR, et al. Rapid screening of protein profiles of human breast cancer cell lines using non-porous reversed-phase high performance liquid chromatography separation with matrix-assisted laser desorption/ionization time-of-flight mass spectral analysis. Rapid Commun. Mass Spectrom. 1999 13 1808-1812. 

H. I. Field, D. Fenyo, and R. C. Beavis. RADARS, a Bioinformatics Solution that Automates Proteome Mass Spectral Analysis, Optimises Protein Identification, and Archives Data in a Relational Database. Proteomics, 2, no. 1 (2002) 36-47. 

It has been recognized that many of the time-consuming interactive tasks carried out by an expert during the process of spectral analysis could be done more efficiently by automated computational systems [6]. Over the past few years, this potential has been realized to some degree. Today automated methods for NMR structure determination are playing a more and more prominent role and can be expected to largely supersede the conventional manual approaches to solving three-dimensional protein structures in solution. 

Equations (1-3) are widely used for protein dynamics analysis from relaxation measurements. The primary goals here are (A) to measure the spectral densities J(co) and, most important, (B) to translate them into an adequate picture of protein dynamics. The latter goal requires adequate theoretical models of motion that could be obtained from comparison with molecular dynamics simulations (see for example Ref. [23]). However, accurate analysis of experimental data is an essential prerequisite for such a comparison. 

The characteristics that discourage the use of RPLC for preparative isolation of bioactive proteins favor its use as an analytical tool for studying protein conformation. Chromatographic profiles can provide information on conformational stability of a protein and the kinetics of folding and unfolding processes. Information about solvent exposure of certain amino acid residues (e.g., tryptophan) as a function of the folding state can be obtained by on-line spectral analysis using diode array UV-vis detection or fluorescence detection. 

Unfortunately, the utility of this method for many of the more interesting enzymes is restricted by the complexity of the protein. If more than one type of cluster is present, the multiple component analysis on the extruded mixture may lead to ambiguous conclusions. In addition non-metallochromophores may interfere. Holm and co-workers (Wong et ai, 1979) circumvented some of these problems for Fe S proteins by their choice of spectral analysis and exogenous thiolate ligand. Namely, they used F NMR spectroscopy to analyze the products of thiolate extrusion with /)-trifluoromethylbenzenethiol. Contact shifts for the fluorine resonances are considerably different for 2Fe and 4Fe clusters. Important restrictions on the use of the F NMR detection are the quantity of protein needed, the synthesis of the ligand, and access to the spectrometer. 

Sun, T., and R.E. Lovins, Quantitative protein sequencing using mass spectrometry use of low ionizing voltages in mass spectral analysis of methyl- and phenylthiohydantoin amino acid derivatives. Anal Biochem, 1972. 45(1) 176-91. 

Several modifications of protoheme are indicated in Fig. 16-5. To determine which type of heme exists in a particular protein, it is customary to split off the heme by treatment with acetone and hydrochloric acid and to convert it by addition of pyridine to the pyridine hemochrome for spectral analysis. By this means, protoheme was shown to occur in hemoglobin, myoglobin, cytochromes of the b and P450 types, and catalases and many peroxidases. Cytochromes a and a3 contain heme a, while one of the terminal oxidase... 

Howell, N.K., Arteaga, G.E., Nakai, S., and Li-Chan, E.C.Y. 1999. Raman spectral analysis in the C-H stretching region of proteins and amino acids for investigation of hydrophobic interactions. J. Agric. Food Chem. 47 924-933. 

Chromophores, in CD spectral analysis of proteins, 219-221 Cl. see Chemical ionization... 

Soluble proteins, purification of extracellular, 275 recombinant, 276-277 Solutions, clarification for CD spectral analysis, 228-229... 

Structure, of proteins CD spectral analysis, 219-243 for classification, 274 fluorescence spectroscopy, 245-265... 

Fluorescence microspectrophotometry typically provides chemical information in three modes spectral characterization, constituent mapping in specimens, and kinetic measurements of enzyme systems or photobleaching. All three approaches assist in defining chemical composition and properties in situ and one or all may be incorporated into modem instruments. Software control of monochrometers allows precise analysis of absoiption and/or fluorescence emission characteristics in foods, and routine detailed spectral analysis of large numbers of food elements (e.g., cells, fibers, fat droplets, protein bodies, crystals, etc.) is accomplished easily. The limit to the number of applications is really only that which is imposed by the imagination - there are quite incredible numbers of reagents which are capable of selective fluorescence tagging of food components, and their application is as diverse as the variety of problems in the research laboratory. 

Fig. 18.3. Raman spectral analysis of foetal osteoblast (FOB) differentiation. Unsupervised PCA of FOB cells cultured for 3 days in bioactive glass (BG) conditioned media (triangle) or control media (circle) (a). BG-treated cells formed a distinct cluster separate from control cells after 3 days culture. Least square (LS) analysis (which decomposes the cell spectra into the linear combination of Raman spectra obtained from the pure chemical constituents of the cell, e.g. nucleic acid, proteins, lipids, phospholipids and carbohydrates) of the relative RNA concentration of FOBs cultured for 1, 3 and 14 days in culture media (black) or BG condition media (grey), revealed a significantly reduced relative RNA concentration in FOBs culture in BG-conditioned media (b). FOBs cultured in BG-conditioned media appeared to accelerate FOB differentiation into mature adult osteoblast phenotypes (parallel gene and protein expression experiments confirmed this). Significant difference to control (p <0.05) [38]...

Molybdopterin synthase is a 27 kDa protein (in its inactive resting state) that is comprised of 16 and 10 kDa subunits [67], The protein has a tendency to dissociate and the exact stoichiometry of the active synthase remains uncertain. Activation of the protein requires that the smaller subunit undergo a single sulfur-for-oxygen exchange, a finding that has been confirmed by mass spectral analysis of both active and inactive 10 kDa subunits. Since conversion of the precursor to MPT requires two sulfur atoms, it has been postulated that the active form of the synthase contains two 10 kDa subunits [67], The synthase not only provides sulfur atoms for MPT precursor conversion but also transports MPT to the apo-molybdenum (or presumably apotungsten) enzyme. 

The metabolism of triazine herbicides in plants has been diligently studied since their introduction. Much of what is currently known about the metabolic pathways was obtained only after newer and more advanced methods of chromatography and spectral analysis were discovered. The study of triazine herbicides has resulted in improved methodologies and understanding of plant proteins, biochemistry, and metabolic pathways. These pathways will serve as a reference point for future researchers in their quest for a complete understanding of plant metabolic chemistry. 

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