Protein characterization, methods

Davis, M.T. Stahl, D.C. Swiderek, K.M. Lee, T.D. Capillary Liquid Chro-matography/Mass Spectrometry for Peptide and Protein Characterization, Methods A Companion to Methods in Enzymology 6,304-314 (1994). 

In this section, the development of protein characterization methods for samples in typical pharmaceutical laboratories will be examined. All of the method development procedures discussed in this chapter are based on the use of the whole-column detection cIEF instrument, since the IFF process can be observed and the easily associated with optimizing the focusing time. However, the basic conditions described in this chapter are also applicable to conventional cIEF method. 

Polyacrylamide gel electrophoresis is one of the most commonly used electrophoretic methods. AnalyMcal 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 nonreduced systems as weU as in combination with discontinuous and ief systems (39). 

Mann, M., and Wilm, M., 1995. Electro.spray ma.ss. spectrometry for protein characterization. Trends in Biochemical Sciences 20 219-224. A review of die ba.sic application of ma.ss. spectrometric methods to the analysis of protein. sequence and. structure. 

Current proteomics studies rely almost exclusively on 2D gel electrophoresis to resolve proteins before MALDI-TOF or ESI-MS/MS approaches. A drawback of the 2D gel approach is that it is relatively slow and work intensive. In addition, the in-gel proteolytic digestion of spots followed by mass spectrometry is a one-at-a-time method that is not well suited for high throughput studies. Therefore, considerable effort is being directed towards alternate methods for higher throughput protein characterization. 

Number of Proteins and Residues in Databases of Intrinsically Disordered Protein Characterized by Various Methods... 

Rigaut G, Shevchenko A, Rutz B, et al. A generic protein purification method for protein complex characterization and proteome exploration. Nat. Biotechnol. 1999 17 1030-1032. 

These efforts are characterized by a specific protein purification method. The aim of the investigation is to find the molecules involved... 

The identification of the fold is, however, only a minor part of protein characterization. Function is a loosely defined term, but must be viewed within a particular context, e.g., protein function can only take place with an interaction partner or within cellular cascades and networks. Fold predictions and homology searches can only give partial answers to such higher order functions. Thus, independent functional features have to be collected and put into context. Such features include not only molecular properties, but also cellular roles, expression patterns, dysfunctions, pathway context, and subcellular localization. The latter can be predicted by exploiting a variety of methods and localization sites. Kenta Nakai reviews many such sites and their implementation... 

Californium plasma desorption ( Cf-PD) dates back to 1973 [4-6,22,154-156] and was the first method to yield quasimolecular ions of bovine insulin. [157] Practically, Cf-PD served for protein characterization, a field of application which is now almost fully transferred to MALDI or ESI (Chaps. 10,11). [158]... 

The successful application of CE technology has resulted in dramatic growth of CE as an essential tool for protein characterization, R D, and QC of therapeutic biomolecules. CE methods have clearly been shown to be superior over traditional slab-gel methods. Many biopharmaceutical companies have adopted CE techniques in QC environments for determination of product purity, identity and consistency needed for the release of protein products. The success of validation per ICH guidelines has moved CE technology to a position of greater prominence and ensures the quality release of therapeutic proteins and antibodies. 

Martiny-Baron G, Haasen D, D Dorazio D et al (2011) Characterization of kinase inhibitors using reverse phase protein arrays. Methods Mol Biol 785 79-107... 

We have discovered a novel protein immobilization method, i.e., a Three-Dimensional Nanostructured Protein Hydrogel (3-D NPH), which is composed of protein-reactive polymer hybrid nanoparticles to detect protein-protein interactions. The 3-D NPH can be easily prepared by spotting a protein/reactive polymer mixture on a substrate. The resulting 3-D NPH is characterized by large amounts of immobilized proteins and a novel porous structure. 

In the literature Raman spectroscopy has been used to characterize protein secondary structure using reference intensity profile method (Alix et al. 1985). A set of 17 proteins was studied with this method and results of characterization of secondary structures were compared to the results obtained by x-ray crystallography methods. Deconvolution of the Raman Amide I band, 1630-1700 cm-1, was made to quantitatively analyze structures of proteins. This method was used on a reference set of 17 proteins, and the results show fairly good correlations between the two methods (Alix et al. 1985). 

Figure 12.12b illustrates the application of gel electrophoresis to protein characterization. In this illustration a cross-linked polyacrylamide gel is the site of the electrophoretic migration of proteins that have been treated with sodium dodecyl sulfate. The surfactant dissociates the protein molecules into their constituent polypeptide chains. The results shown in Figure 12.12b were determined with well-characterized polypeptide standards and serve as a calibration curve in terms of which the mobility of an unknown may be interpreted to yield the molecular weight of the protein. As with any experiment that relies on prior calibration, the successful application of this method requires that the unknown and the standard be treated in the same way. This includes such considerations as the degree of cross-linking in the gel, the pH of the medium, and the sodium dodecyl sulfate concentration. The last two factors affect the charge of the protein molecules by dissociation and adsorption, respectively. Example 12.5 considers a similar application of electrophoresis. 

Functional Diversity of Proteins 101 Methods for Characterization and Purification of Proteins 118... 

I n the previous three chapters we described the structures of amino acids and proteins, and in two cases we examined how these structures relate to their function. Some of the methods for structure determination were also discussed (e.g., sequence analysis in chapter 3 and x-ray diffraction in chapter 4). To analyze the structure of a protein we must isolate it from the complex mixture in which it exists in whole cells. The primary object of this chapter is to acquaint you with techniques used for protein purification. Because these procedures are often used for protein characterization as well, they will add to your repertoire of methods for protein characterization. 

Most of the recently developed methods for the detection, characterization, and quantitation of proteins are immunoassays based on the fact that proteins are antigens, compounds that can be recognized by an antibody. It is also true that by combining small molecules (haptens) with a larger carrier molecule such as a protein, these methods can be extended to small molecules of interest since antibodies can be produced that recognize epitopes (specific sites on the antigen recognized by the antibody) that include the hapten. 

It is very important to perform at least a limited protein characterization study before starting the NMR screening. Here it is assumed that the identity and purity of the target protein produced has been checked by methods such as MALDI-MS and N-terminal sequencing. 

MALDI-TOF provides limited capabilities for mixture analysis, LC/MS methods are used to provide more detailed interrogation of protein expression and peptide sequence. The use of LC/MS approaches for protein identification in conjunction with 2-DGE offers distinct advantages such as the ability to handle low picomole (miniaturized) level samples, enhanced separation, detection, the amenability to N-terminally blocked proteins, and fast analysis. The LC/MS methods for protein characterization focus on four distinct goals (1) confirmation of putative sequence, (2) identification of amino acid modifications, (3) identification of known proteins, and (4) sequence determination of unknown proteins. 

IEF is used as a purity test and for protein characterization, comparing it with the relative position of an authentic band of the target protein. It can also be used as a method to evaluate the stability of a biological product. Small changes such as the deamination of an amino acid will cause a change in the pi and a modification of the band pattern. 

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