Phosphoproteins, separation

Myers, J., et al. (1996). A Method for Enhancing the Sensitivity and Stability of Stains-all for Phosphoproteins Separated in Sodium Dodecyl Sulfate-polyacrylamide Gels, Anal. Biochem. 240 300 302. 

This system allows the separation of alkali-labile proteins (e.g., acylphosphate phosphoproteins) under denaturating conditions according to their molar mass. Despite the low acrylamide concentration (%T = 5.61, %C = 3.61), the separation force is remarkable. Because it is a SDS-containing system, the migration is from to + despite the low pH. 

To serve as an effective regulatory mechanism, phosphorylation must be reversible. In general, phos-phoryl groups are added and removed by different enzymes, and the processes can therefore be separately regulated. Cells contain a family of phosphoprotein phosphatases that hydrolyze specific -Ser, -Thr, and -Tyr esters, releasing Pj. The phosphoprotein phosphatases we know of thus far act only on a subset of phosphoproteins, but they show less substrate specificity than protein kinases. 

According to Bloomfield and Mead (1974), The ultimate goal of all workers on casein is to reconstitute micelles with native properties from the separated constituents of skim-milk. This assertion reflects the large number of studies in the literature on the precipitation and association properties of the caseins.There are, however, legitimate scientific goals in this kind of work other than the creation of artificial casein micelles, such as the elucidation of the mechanisms by which phosphoproteins profoundly influence the nucleation and growth of calcium phosphate phases. 

The earliest assays were based on the use of 32P as a label either on the ATP cofactor for kinases or on a peptide substrate for phosphatases. With kinases, the transfer of the 32P from the /position of ATP to a peptide or protein substrate resulted in a 32P-labeled peptide or protein that would be separated away from the ATP by capture on a filter and subsequent washing. The quantity of phosphoprotein could be quantified by scintillation counting. 

One of the most crucial considerations in proteomic analysis is sample preparation because this will ultimately dictate the number and type of proteins that can be processed. The first priority is to establish the precise protein system to be studied [e.g., will this be a comprehensive and exhaustive catalogue of every expressed protein within a tissue or cellular extract, or is only a small subset of a cellular proteome (e.g., only phosphoproteins or membrane-bound proteins) sufficient for analysis ]. Whether a full or partial proteome, or even a limited number of specific proteins is required for analysis, it is crucial that the extraction technique provide maximal protein recovery while preserving the integrity of the protein complex to be examined. Furthermore, the method of preparation must be totally compatible with the separation methods to be used. This is particularly important for separation technologies that are reliant on protein-protein interactions or drug/ligand/antibody, etc. interactions. Poor recovery of proteins is clearly... 

A comprehensive proteomics approach has been developed to identify the components of phosphoprotein complexes and includes the following experimental steps isolation of native phosphoproteins and associated proteins by affinity chromatography, ID PAGE separation of the affinity-isolated proteins, proteolysis of the protein spots, LC-ESI-MS/MS analysis of the cleaved peptides, and database searching for indentification of the proteins involved in the complex formation.178... 

The structures of F class and V class ion pumps are sIm liar to one another but unrelated to and more complicated than P-class pumps. F- and V-class pumps contain several different transmembrane and cytosolic subunits. All known V and F pumps transport only protons. In a process that does not Involve a phosphoprotein Intermediate. V-class pumps generally function to maintain the low pH of plant vacuoles and of lysosomes and other acidic vesicles In animal cells by pumping protons from the cytosolic to the exoplasmic face of the membrane against a proton electrochemical gradient. F-class pumps are found In bacterial plasma membranes and In mitochondria and chloroplasts. In contrast to V pumps, they generally function to power the synthesis of ATP from ADP and Pj by movement of protons from the exoplasmic to the cytosolic face of the membrane down the proton electrochemical gradient. Because of their Importance In ATP synthesis in chloroplasts and mitochondria, F-class proton pumps, commonly called ATP synthases, are treated separately In Chapter 8. 

Mass spectrometry of phosphopeptides has become a powerful tool for phosphorylation site identification. However, proteolytic digests examined by MS are often likely to fail to detect phosphopeptides because the ionization of phosphorylated peptides in positive ion mode MS is generally less efficient compared with the ionization of their nonphosphorylated counterparts resulting in ion suppression effects. A further problem is that phosphopeptides may not be retained by RP chromatography because they are too small and/or hydrophilic to bind to the CIS stationary phase. Therefore, capillary electrophoresis coupled to MS is a powerful method to enhance the detection of phosphoproteins and phosphopeptides due to their efficient separation by CE. 

Stupak, J., Liu, H. Z., Wang, Z. R, Brix, B. J., Fliegel, L., and Li, L., Nanohter sample handling combined with microspot MALDl-MS for detection of gel-separated phosphoproteins. Journal of Proteome Research, 4, 515-522, 2005. 

Casein is a naturally occurring macromolecule that accounts for approximately 80% of the protein content of cow s milk it is a phosphoprotein that can be separated into various electrophoretic fractions, such as aj-casein, /c-casein, fi-casein, and y-casein in which each constituent differs in primary, secondary, and tertiary structure, amino acid composition, and molecular weight (Ghosh et al., 2009 Audic et al., 2003 Barreto et ai., 2003]. It finds use in making adhesives and paper coatings. 

Milk normally has a pH of 6.0-6.3. If bacteria grow in it they release acids such as lactic acid which lower the pH and render the milk sour. When the isoelectronic point for casein is reached at about pH 4.7, this phosphoprotein is precipitated and the milk curdles, enabling it to be separated into the traditional curds and whey. The latter corresponds roughly to milk serum (Figure 12.8). 

Hydrogen bonding is frequently crucial in determining the structure and properties of phosphorus compounds. In some of these compounds, however, the P atom itself may not be closely involved and the more important hydrogen bonds are formed by distant parts of the molecule. Notable examples of this situation are found in the nucleic acids, phosphoproteins and other bio polymers (Chapter 10). This section will be mostly concerned with relatively simple compounds where the P atom is separated by not more than a single atom from the participating H atom. 

The various techniques involved in fluorescence spectrometry are of great importance in analytical studies of phosphoproteins, and in DNA technology. In the latter case they are employed to detect or estimate the fractions separated by gel electrophoresis and stained with fluorescent dyes. These techniques have become invaluable for DNA sequencing (Section 14.3). 

Acidic peptides are released in the pH range 5.5-6.2 and phoshorylated peptides are eluted in the pH range 6.9-7.5. Elution of retained peptides can also be performed with sodium phosphate. IMAC has been successfully used for the characterization of casein phosphopeptides in cheese extracts. Phosphoproteins can be separated under very similar conditions as phosphopeptides. IMA sorbents were already used for fractionation of proteins... 

Cell sap contains a specific phosphoprotein phosphatase (P) which dephosphoiylates protein 67, eIF-2 and several other dsRNA-dependent phosphorylated proteins (5). The protein phosphatase P is separated from PK-i on DEAE-cellulose and elutes at 25O1DM KCl. As shown in Figure 5> P stimulates mengo RNA translation in interferon-treated cell extracts (SlO-int) supplemented with dsRNA, but not in control cell extracts (SIO-cont). This suggests a regulatory mechanism which controls the level of PK-i dependent inhibition of protein synthesis. 

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