Protein Fractions, Analysis

Canals, J.M., Arola, L. and Zamora, F. (1998) Protein fraction analysis of white wine by FPFC, Am. J. Enol. Vitic., 49, 383-388. 

Further indications for an additional subunit were provided by a crosslinking analysis of C Eg solubilized H,K-ATPase, which exhibited ATPase and phosphatase activities, and ligand affinities comparable to the native enzyme [70]. Glutar-aldehyde treatment of soluble protein fractions resolved on a linear glycerol gradient revealed no active fraction enriched in monomeric (A/p = 94 kDa) H,K-ATPase. Instead, K -ATPase activity was only obtained in fractions enriched in particles of Mr = 175 kDa. This size also suggested that the functional H,K-ATPase unit is a heterodimer of a catalytic subunit and an additional subunit, since the apparent molecular mass of 175 kDa is probably too small to be a homodimer of the catalytic subunit. 

Table 5.1 lists several heart-cut and comprehensive techniques. Heart-cut 2DLC is very common and has great application for the increased resolution of one or several components from the first dimension (Augenstein and Stickler, 1990 Majors, 1980 Pasch et al., 1992 and Dixon et al., 2006). Heart-cut 2DLC for the analysis of polymers is often referred to as cross-fractionation (Balke and Patel, 1980). Protein digest analysis with MS/MS identification has been called multidimensional protein identification technology or MUDPIT. This is described in detail in Chapter 11. 

FIGURE 13.4 Total ion chromatograms from the ID LC/MS analysis of a yeast ribosomal protein fraction separated using 0.1% TFA (Panel a) and 0.1% formic acid (Panel b) as mobile phase modifiers. TFA produced narrower, more concentrated, peaks for mass analysis that did not overcome the significant electrospray ionization suppression associated with using this modifier for LC/MS studies, resulting in an overall reduction in component intensities. 

The ongoing development of top-down protein MS/MS capabilities (MS/MS) should prove quite valuable to researchers looking to identify and characterize proteins fractionated by 2DLC separations. Such methods are currently limited by restrictions on the maximum size of proteins analyzed, as well as analysis time-requirements that limit coupling of these methods with online LC analysis. Investigators from labs, such as Kelleher, McLafferty, Hunt (Coon et al., 2005 Meng et al., 2005 Han et al., 2006), and others are rapidly addressing these issues, and their methods will likely be adopted by many other researchers over the next few years. 

In the version we used, electrophoresis of proteins makes it possible to separate proteins and evaluate molecular weight and relative amount of each fraction. Analysis of this data, therefore, has permitted us to detect the following types of modification of proteins resulting from photodynamic treatment ... 

Chaplin used methanolysis for the analysis of carbohydrates in glycoproteins. His method was a variation of the foregoing procedures, with an improvement of using tert-hvAyX alcohol to remove hydrogen chloride by coevaporation, instead of prolonged trituration with silver carbonate. His method is useful for samples containing uronic acids and lipids. Mononen studied methanolysis, followed by deamination and reduction with borohydride, for determination of the monosaccharide constituents of glycoconjugates. This method was applied to a lipid-free, protein fraction of rat brain. 

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. 

Ldnnerdal, B., Stanislowski, A. G., Hurley, L. S. J. Inorg. Biochem. 12, 71 (1980) Woittierz, J. R. W. Elemental Analysis of Human Serum and Human Serum Protein Fractions by Thermal Neutron Activation, Netherlands Energy Research Foundation Report, ECN 147, January 1984... 

In order to investigate whether tomatinases from F. oxysporum and F. solani share similar molecular characteristics, F. solani tomatinase was partially purified. Comparative SDS-PAGE analysis of the protein fractions with and without tomatinase activity showed the presence of a 32.5 kDa band in all positive fractions, while this band was absent in fractions without tomatinase activity The apparent molecular mass of tomatinase of F. solani differs from that of F. oxysporum (50 kDa), S. lycopersici (110 kDa) [33], and Botrytis cinerea (70 kDa)[36]. The F. solani tomatinase presents a very low activity compared with F. oxysporum enzyme [35, 89]. Western blot analysis showed that the two enzymes also differ in their immunological characteristics since the polyclonal antibody against tomatinase of F. oxysporum f. sp. lycopersici did not recognize the tomatinase from F. solani. These results suggest that the enzyme from F. solani is a novel tomatinase species. 

It is unlikely that the protein fractions from this experiment contain a single type of protein. How many different proteins are present What is the relative abundance of each protein Is a-lactalbumin the predominant protein in the isolated fractions What are the approximate molecular weights of a-lactalbumin and other proteins These questions may be answered by analysis of the isolated fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (see Chapter 4). The technique of SDS-PAGE will be introduced and applied to the column-purified fractions, crude whey fraction, and standard a-lactalbumin. 

Medina and Phillips (36) analyzed the peptide pattern obtained by the enzymatic hydrolysis of proteins isolated from beef, pork, chicken, and soybean sequentially, using TLC and HPLC to identify the proteins. The analysis was performed using RP-HPLC on a /zBondapak C8 column under isocratic conditions using triethylamine phosphate buffer (0.0833 M, pH 3) as solvent. Figure 5 depicts the peptide pattern of fraction IV isolated from beef, soybean, chicken, and pork by TLC. Identification of the different foods was accomplished by applying discriminant analysis to the peptide pattern. 

Data from electrophoresis is normally recorded photographically. Densitometry may also be performed on the stained gel or bands may be excised/eluted for further analysis (Mayer et al., 1998). The bands may also be isolated from the gels by blotting methods such as electroblotting (McSweeney et al., 1994 O Malley et al., 2000) or immunoblotting (Addeo et al., 1995 Moio et al., 1992) for further characterization and identification. All of the electrophoretic methods, to a certain extent, provide good quality data. But due to the difficulty in quantitative analysis, very few examples are available on the quantification of protein fractions using... 

Alternatively, one strip (or, better, half of a strip cut lengthwise) is stained and used as a test for a second strip which is cut into appropriate lengths and eluted. There is, however, no certainty as to the limits of the zones of the protein fractions, for the factors involved in application and migration of protein samples are capable of destroying the parallelism of the two strips and of thus falsifying the results. Nevertheless, this method may be used and is even indispensable for the analysis of lipo-and glycoproteins with reference to the proteins themselves. 

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