Analytical Methods for Proteins

Shafer, Arch. Btochem. Biophys., 1979, 193, 284. 

Rubinstein, S. Chen-Kiang, S. Stein, and S. Udenfriend, Anal. Biochem., 1979, 95, 117. 

Two-dimensional gel electrophoresis (isoelectric focusing in one dimension followed by sodium dodecyl sulphate-gel electrophoresis or add-urea-gel electrophoresis in the other) is widely used in the analysis of complex mixtures of proteins. Bordier et aV showed that the polypeptide mapping gel system can be used in two dimensions to peptide map several proteins simultaneously. Proteins are first separated on a molecular weight basis in a sodium dodecyl sulphate gel. The gel is then placed on top of a slab gel and overlaid with a proteinase solution. The proteins 

Oakley, O. R. Kirsch, andN. R. Mortis, Anal. Biochem 1980, 105,361. 

A novel physical method has been developed by Kempner et al. for determining the molecular weight of the functional unit in multimeric enzymes. The lyophylized protein is exposed to gamma rays and the dose bringing about a 0.37 loss in activity is measured. The molecular weight of the functional unit is then apparently equal to 6.4 X 10 divided by the dose in Mrad. 

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Electrophoresis makes use of differences in the electrophoretic mobility of electrically charged particles (biomolecules, micro-organisms etc.) as a means to separate them. For this purpose, a homogeneous, rectified electrical field is used. Thanks to the excellent resolution and mild operating conditions, this is currently the best analytical method for protein separation, purification and characterization. It is also used as a preparative separation method which allows a few grams per hour to be purified. 

Salt, H. B., Micro analytical methods for proteins in blood plasma. Analyst 78, 4-14 (1953). 

Analytical Methods for Measuring Protein Content of Baker s Yeast (SCP)... 

Electrospray ionization mass spectrometry (ESI-MS) is an analytical method for mass determination of ionized molecules. It is a commonly used method for soft ionization of peptides and proteins in quadmpole, ion-trap, or time-of-flight mass spectrometers. The ionization is performed by application of a high voltage to a stream of liquid emitted from a capillaty. The highly charged droplets are shrunk and the resulting peptide or protein ions are sampled and separated by the mass spectrometer. 

In order to define this variety of food matrices, chemical composition differences that primarily influence chemical analytical measurements have to be considered. Major food components determining basic chemical make-up are the proximate composition of fat, protein, carbohydrate, ash, and moisture. Variations in ash content in general have a minor influence on analytical methods for other constituents and impact of moisture content can be controlled. Thus the major components influencing analytical performance are the relative levels of fat, protein, and carbohydrate. 

Zeheb, R., Chang, V., and Orr, G.A. (1983) An analytical method for the selective retrieval of iminobi-otin-derivatized plasma membrane proteins. Anal. Biochem. 129, 156-161. 

Antibodies provide a very convenient and specific analytical tool for protein determinations, a role considerably enhanced by the availability of a wide range of monoclonal antibodies. Many antibodies are capable of precipitating the target protein and the resulting turbidity can be measured by either tur-bidometric or nephelometric methods. The various types of alternative immunoassay offer increased sensitivity over the turbidometric methods. 

The objective of this book is to provide both an overview and practical uses of the techniques available to analytical scientists involved in the development and application of methods for protein-based biopharmaceutical drugs. The emphasis is on considering the analytical method in terms of the stage of the development process and its appropriateness for the intended application. The availability of techniques will reveal whether or not the analytical problem has a potential solution. Then will come the question of whether or not the technique is a truly appropriate solution. The theoretical considerations behind choosing the technique may be solid. However, the practicality of the method may not hold up to inspection. 

Stacey, K. A. The use of light-scattering for the measurement of the molecidar weight and size of proteins. In A laboratory manual of analytical methods of protein chemistry j Vol. 3, 245—275. Ed. R. Alexander u. R. J. Bloch, Pergamon Press 1961. 

One of the aldehyde groups of the gossypol is believed to react with the primary amine of the lysine units of the cottonseed proteins to form the Schiff base or imine type compound as shown at the top of Figure 12 (16, 17). An analytical method for determination of bound gossypol, developed by Pons et al. 

J. E. Eastoe and A. Courts, Practical Analytical Methods for Connective Tissue Proteins, Spon, London, 1963, Chapt. 6. 

Two articles have described analytical methods for the determination of sorafenib plasma concentrations, involving a similar protein precipitation step with acetonitrile [115, 116], There is at present only one published method for the quantification of lapatinib in human plasma after off-line SPE onto C18 cartridge, followed by evaporation and reconstitution in ACN/5.0 mM ammonium formate pH 3/formic acid (1,000 50 1, v/v/v) [117],... 

In the past decade, we have benefited from major improvements in the sensitivity and efficiency of both chromatography and electrophoresis. High-performance liquid chromatography (HPLC) has become firmly ensconced as a powerful method for protein analysis. Ironically, reversed-phase chromatography, which employs denaturing conditions, has found a particularly wide application, at least when only analytical information is sought. This is the case in most routine analytical work required for many biotechnological applications. 

Much time and effort were spent in compiling the analytical and preparative procedures for studying the amino acid composition of proteins of animal and plant origin, which resulted in his first book, The Determination of the Amino Acids, followed by five more dealing with the amino acid composition of proteins and foods, paper chromatographic and electrophoretic methods, and analytical methods of protein chemistry. 

The most common implementation of proteomic analysis involves protein separation two-dimensional gel electrophoresis (2DGE), quantification of proteins with analytical methods for their identification in mass spectrometer (MS), and at the very least data integration and analysis using bioinformatic tools. 

TABLE 4 Analytical Methods for Characterization and Quality Control of Pharmaceutical Peptides and Proteins... 

Gilg, D., Riedl, B., Zier, A., and Zimmermann, M. F. (1996), Analytical methods for the characterization and quality control of pharmaceutical peptides and proteins, Pharm. Acta Helv., 71, 383-394. 

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