Phosphoproteins analysis

Brugge JS, Yonemoto W, Darrow D. 1983. Interaction between Rous Sarcoma virus transforming protein and two cellular phosphoproteins Analysis of the turnover and distribution of this complex. 

H. Zhang, X. Zha, Y. Tan, P.V. Hornbeck, A.J. Mastrangelo et al., Phosphoprotein analysis using antibodies broadly reactive against phosphorylated motifs, j. Biol. Chem. 2002, 277, 39379-39387. 

N. (2001) Characterization of phosphoproteins from electrophoretic gels by nanoscale Ee(lll) affinity chromatography with off-line mass spectrometry analysis. Proteomics,... 

Consensus docking sites for actin-based motihty, defined by the oligoproline modules in Listeria monocytogenes ActA surface protein and human platelet vasodilator-stimulated phosphoprotein (VASP). Analysis of... 

I Identification, structure analysis of phosphoproteins and metal-containing proteins by MALDI-FTICR-MS... 

Hydrolytic Decomposition of Phosphoprotein. The kinetic analysis of phosphate liberation revealed that phosphate is not steadily liberated but that an initial lag period occurs which is followed by a transient burst of phosphate liberation123, 177> 192 This burst has been interpreted as resulting from the accumulation of an acid-labile intermediate arising from an acid-stable precursor. The burst is fol-... 

A more conclusive kinetic analysis is not only made difficult because of the temperature-dependent occurrence of different phosphoprotein species, but also by the fact that the spontaneous decay of gradient-independent as well as of gradient-dependent phosphoprotein depends on the concentration of phosphate. This phosphate dependence is not taken into account by the tentative reaction scheme. 

VojteSek, B., Bartek, J., Midgley, C. A., and Lane, D. P. 1992. An immunohistochemical analysis of human nuclear phosphoprotein p53. New monoclonal antibodies and epitope mapping using recombinant p53. J. Immunol. Methods 757 237-244. 

Grebb JA, Greengard P. 1990. An analysis of synapsin II, a neuronal phosphoprotein, in postmortem brain tissue from alcoholic and neuropsychiatrically ill adults and medically ill children and young adults. Arch Gen Psychol 47 1149-1156. 

Figure 4.10. Proteomic analysis by SILAC. Proteomic analysis by SILAC or stable isotope labeling of amino acids in cell culture utilize de novo metabolic incorporation of stable-isotope-labeled amino acids during protein synthesis. Cells can be cultured with various combinations of stable-isotope-labeled amino acids such as lysine or arginine. Tyrosine has been used in phosphoprotein studies of tyrosine residues. About five or six cell divisions are needed for complete labeling of proteins in cell cultures prior to experimentation. Labeled cells from control and treatment(s) lysates are combined and digested. Quantitation and identification are performed by LC-MS/MS.

A comprehensive analysis of protein phosphorylation includes the identification of the phosphoprotein or phosphopeptide, the localization of the modified amino acid, and if possible, the quantification of phosphorylation. 

Indeed, the analysis of phosphoproteins and the detection and localization of the phosphorylated residues are not straightforward for various reasons ... 

The following three sections show concrete examples of the analysis of phosphoproteins as potential drug targets and demonstrate the applicability and enormous power of proteomics in this field. 

Kamps, M.P., Sefton, B.M (1989) Acid and Base Hydrolysis of Phosphoproteins Bound to Immo-bilon Facilitates the Analysis of Phosphoamino Acids in Gel-Fractionated Proteins, Anal. Biochem. 176, 22-27. 

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... 

Comparison of triple-quadrapole and Q-TOF use in these type of studies was reported [22]. While similar sensitivity can be achieved, the selectivity of the Q-TOF is better due to its high resolving power, thereby minimizing isobaric interferences. Bateman et al. [23] reported a novel mode of operation of a (J-TOF instrument for precursor-ion analysis of phosphoproteins. The first-stage quadrapole is operated in radiofrequency-only mode, serving as a wide-band ion-transmission... 

J.E. Labdon, E. Nieves, U.K. Schubart, Analysis of phosphoprotein pl9 by LC-MS. Identification of two Pro-directed Ser phosphorylation sites and a blocked amino terminus, J. Biol. Chem., 267 (1992) 3506. 

The phosphoproteome is a specific subset of the proteome, and phosphoproteomics is the comprehensive analysis of the entire complement of phosphoproteins in biological systems, including detection of all phosphoproteins, location of the exact site of phosphorylation, and quantification of differentially expressed phosphoproteins. 

A general protocol for mass spectrometric analysis of phosphoproteins is illustrated in Figure 15 various steps of this protocol are cleavage of purified phosphoproteins, isolation and preferential enrichment of phosphopeptides, selective detection of phosphopeptides in the digest, identification of the phosphorylation sites using tandem mass spectrometry, and identification of phosphopeptides/proteins through a database search. 

Similar to the analysis of ordinary proteins, phosphoproteins also need to be cleaved into small manageable peptide fragments any one of the proteases listed in Table 1 can be used. The concept of chemical tagging through /3-elimination/Michael addition reactions has been adopted to facilitate the cleavage process. The [3-elimination reaction converts phosphoserine and phosphothreonine to dehydroalanine and... 

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... 

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