Protein characterization, analytical techniques

Biomolecule Separations. Advances in chemical separation techniques such as capillary zone electrophoresis (cze) and sedimentation field flow fractionation (sfff) allow for the isolation of nanogram quantities of amino acids and proteins, as weU as the characterization of large biomolecules (63—68) (see Biopolymers, analytical techniques). The two aforementioned techniques, as weU as chromatography and centrifugation, ate all based upon the differential migration of materials. Trends in the area of separations are toward the manipulation of smaller sample volumes, more rapid purification and analysis of materials, higher resolution of complex mixtures, milder conditions, and higher recovery (69). 

Centrifugation can be used either as a preparative technique for separating and purifying macromolecules and cellular components or as an analytical technique to characterize the hydrodynamic properties of macromolecules such as proteins and nucleic acids. 

HPLC analysis of food proteins and peptides can be performed for different purposes to characterize food, to detect frauds, to assess the severity of thermal treatments, etc. To detect and/or quantify protein and peptide components in foods, a number of different analytical techniques (chromatography, electrophoresis, mass spectrometry, immunology) have been used, either alone or in combination. The main advantages of HPLC analysis lie in its high resolution power and versatility. In a single chromatographic run, it is possible to obtain both the composition and the amount of the protein fraction and analysis can be automated. 

ITABLE 4.5. Some analytical techniques use in protein characterization and stability assessment... 

Polyacrylamide gel electrophoresis is one of the most commonly used electrophoretic methods. Analytical uses of this technique center around protein characterization, for example, purity, size, or molecular weight, and composition of a protein. Polyacrylamide gels can be used in both reduced and nonrcduced systems as well as in combination with discontinuous and isoelectric focusing (ief) systems. 

TABLE 6 Analytical Techniques for Physicochemical Characterization of Proteins... 

Application of a comprehensive array of analytical techniques to fully characterize the drug product at each stage of development. Table 6 provides examples of methods to probe virtually every property of the protein and develop a hngerprint of the molecule. 

In our laboratory, we have developed a novel MALDI-MS based assay in combination with separation techniques to rapidly identify and confirm the presence of viral proteins (immature or mature) and impurities in the rAd vector [138], The approach combines powerful multidimensional analytical techniques to fully characterize viral proteins, including RP-HPLC, SDS-PAGE, MALDI-MS, MS/MS, and database searching methods (Figure 19-24). This assay involves dissociation/separation of intact viruses by RP-HPLC, separation of the SDS-dissociated viruses by SDS-PAGE, and enzymatic digestion of the dissociated viral proteins from the RP-HPLC fractions and the gel bands, followed by MALDI-MS, MALDLpost source decay (PSD) studies, and database search. 

LC/MS analysis of proteins and peptides is an important part of drug discovery process, as illustrated in this chapter. The combination of various HPLC techniques and advanced MS methods provides unique analytical capabilities of structural identifications for therapeutic proteins and target proteins. The continuous evolution of proteomics research provides both an opportunity and a challenge for further developments in separation techniques and MS characterization methods. It is expected that these analytical techniques will continue to play important roles in drug discovery in the future. 

The common analytical techniques used in the characterization of proteins and the nature of the information they provide are listed in Table 3.4. The biotechnology literature is replete with examples of application, of these techniqnes to the characterization of rDNA-derived proteins some examples can be found in Heinrikson (1988), Hancock et al. (1988), and Rinas et al. (1990). 

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