Proteins quantitation

Interacting centers Proteins Quantitative study Refs. 

Eftink, M.R. and Chiron, C.A. (1976) Exposure of tryptophanyl residues in proteins quantitative determination by fluorescence quenching studies. Biochemistry 15, 672-679. 

Fried, V.A., Ando, M.E., and Bell, A.J. (1985) Protein quantitation at the picomole level An o-phthaldial-dehyde pre-TSK column-derivatization assay. Anal. Biochem. 146, 271-276. 

Ross, P.L., Huang, Y.N., Marchese, J.N., Williamson, B., Parker, K., Hattan, S., Khainovski, N., Pillai, S., Dey, S., Daniels, S., Purkayastha, S., Juhasz, P., Martin, S., Bartlet-Jones, M., He, F., Jacobson, A., and Pappin, D.J. (2004) Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol. Cell. Proteomics 3, 1154-1169. 

Biochemical MT (protein) quantitation Bg, Dp, Pc Liver (Bg), digestive gland (Dp, Pc) Metal poisoning... 

In addition to the approaches covered in a recent review [20], the first comprehensive model covering receptor dimerization and internalization has been recently described [21], In this work, the authors use direct time-dependent measurement of the phosphorylation of the four ErbB receptor species, as well as protein quantitation to develop a comprehensive mechanistic model of receptor dimerization and internalization. Once this comprehensive, quantitative framework was developed, the authors were able to show that receptor dephosphorylation, a key step in the downregulation of ErbB-driven signaling, was restricted to intracellular compartments. 

What follows is not an exhaustive or up-to-the-minute survey of the methods available for protein quantitation, but a practical guide to selecting the appropriate assay for each stage of drug development. A case study further illustrates the application of the standard protein methods to the drug development process. The reader is referred to reviews on the topic for further details.12... 

Is there a component in your protein solution that will interfere with your chosen protein quantitation method Many methods will provide misleading results in the presence of standard biopharmaceutical reagents such as detergents, chao-tropes, bases, or reductants. It is imperative to determine if excipients present in the sample interfere with the chemistry of the protein quantitation method. 

Accurate total protein quantitation of fatty tissues was achieved by adding 0.1 ml 10% sodium deoxy-cholate, pH 8.0 and 2.9 ml biuret reagent to each protein pellet following an acetone/ether wash step. After sonication, each sample was heatedfor 30 sec in a boiling water bath to develop full color. 

Peterson, G.L. 1979. Review of the Folin phenol protein quantitation method of Lowry, Rose-brough, Farr and Randall. Anal. Biochem. 100 201-220. 

Rapid advances have been made in proteomic research, with growing success in protein quantitation utilizing MS technologies [74-76]. The quantitative tools from proteomics and other analytical innovations have great potential to be translated to macromolecule drug bioanalytical applications, and are discussed in the following sections. 

DE, two-dimensional gel electrophoresis WB, Western blot MS, mass spectrometry LC, liquid chromatography 2DE DICE, two-dimensional difference gel electrophoresis, SRM, selected-reaction monitoring MRM, multiple reaction monitoring AQUA, absolute protein quantitation SMIM, selected MS/MS ion monitoring. ... 

Carr SA, Anderson L (2008) Protein quantitation through targeted mass spectrometry the way out of biomarker purgatory Clin Chem 54 1749-1752... 

Aptamers placed on a matrix bind specific proteins from dilute solution in the context of many other proteins [22]. Thus, an array of different aptamers on a chip could be used to capture different proteins (from blood, for example) at specific locations (in X, Y space) for subsequent (protein) quantitative measurements and informatics (Figure 20.5) ... 

The QMS platform combines the identification of proteins with their quantitative detection in one procedure. While protein identification can be deduced from peptide mass fingerprinting (PMF) or MS/MS spectra (see section Mass Spectrometry (MS)), protein quantitation is based on analysing either peak areas or signal intensities, or a combination of both. Several computer programs, in most cases reagent specific ones, are available. For each peak, quantitation values are calculated before differentially expressed proteins are identified by the comparison of control and treated samples. 

Ross PL, Huang YN, Marchese JN, Williamson B, Parker K, Hattan S, Khainovski N, Pillai S, Dey S, Daniels S, Purayastha S, Juhasz P, Martin S, Bartlet-Jones M, He F, Jacobson A, Pappin DJ (2004) Multiplexed protein quantitation in Saccharmyces cerevisiae using amino-reactive isobaric tagging reagents. Mol Cell Proteomics 3.12 1154-1169... 

In quantitative proteomics, two alternative strategies have been developed. The first one is based on 2-D PAGE combined with mass spectrometry for protein identification (Haynes and Yates, 2000). This method and current advances in the differential display of proteins by 2-D PAGE are discussed at length in another chapter of this book. The second approach exploits mass spectrometry in combination with stable isotope labeling for gaining accurate quantitative information on proteins. Quantitative MS via stable isotope labeling of proteins... 

The total half-cystine content of pig heart lipoamide dehydrogenase is 10 per FAD 6S). The basis for the protein quantitation has been discussed in Section II,C. Older data suggested that there were 2 cystine residues and 6 cysteine residues 83), but more recent data 61, 63) give strong evidence that the active center cystine residue is the only cystine residue. Only 7 thiols react with DTNB under denaturing conditions (dS) however, recalculation of data of Brown and Perham 86), taking... 

Kruger, N.J. (1994) The Bradford Method for Protein Quantitation, Methods Mol. Biol. 32, 9-15. 

Walker, J.M. (1994b) The Bicinchonic (BCA) Assay for Protein Quantitation, Methods Md. Biol. 

Mass spectrometry is the method of choice for identification of proteins, quantitation of proteins expression at a given time but also for characterization of post-translational modifications. Indeed, allowing direct analysis of peptides and proteins, the MALDI and ES ionization modes have been revolutionary in the field of mass spectrometry applied to biology. 

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