Immobilized phosphorylated peptides

This amide, readily formed from an amine and the anhydride or enzymatically using penicillin amidase, is readily cleaved by penicillin acylase (pH 8.1, A -methylpyrrolidone, 65-95% yield). This deprotection procedure works on peptides, phosphorylated peptides, and oligonucleotides, as well as on nonpeptide substrates. The deprotection of racemic phenylacetamides with penicillin acylase can result in enantiomer enrichment of the cleaved amine and the remaining amide. An immobilized form of penicillin G acylase has been developed. ... 

Ferric ion was immobilized on a Chelating Sepharose Fast Flow column preparatory to the separation of seven enkephalin-related phosphopep-tides.17 Non-phosphorylated peptides flowed through the column, and the bound fraction contained the product. The capacity of the column was found to be 23 pmol/mL by frontal elution analysis. Cupric ion was immobilized on Chelating Superose for the isolation of bovine serum albumin.18 Cupric ion was immobilized on a Pharmacia HiTrap column for the separation of Protein C from prothrombin, a separation that was used to model the subsequent apparently successful separation of Factor IX from prothrombin Factor IX activity of the eluate was, however, not checked.19 Imidazole was used as the displacement agent to recover p-galactosidase from unclarified homogenates injected onto a nickel-loaded IMAC column.20 Pretreatment with nucleases and cleaning in place between injections were required procedures. A sixfold purification factor was observed. 

IMAP (immobilized metal ion affinity-based fluorescence polarization) Molecular Devices Trivalent metal ion containing beads bind to phosphorylated peptide producing FP signal change... 

Since the introduction of metal-ion affinity sorbents for the fractionation of proteins [1], the method became popular for the purification of a wide variety of biomolecules. Metal-ion affinity sorbents are also widely used for the immobilization of enzymes. At present, IMAC is a powerful method for separation of phosphorylated macromolecules, particularly proteins and peptides. The significance of techniques for separation and characterization of phosphorylated biomolecules is now increasing, because phosphorylation modulates enzyme activities and mediates cell membrane permeability, molecular transport, and secretion. Phosphorylated peptides can be separated from a peptide mixture on IDA-Sepharose with Fe " ions (Fig. 2). The majority of peptides pass freely through an IMAC column, whereas acidic peptides, including phosphorylated ones, are retained and can be released by a pH gradient. 

More recently, a number of reports have appeared demonstrating that phosphorylation could be measured in a microarray format using a known kinase-substrate pair. Two different detection approaches have generally been used, the first makes use of [y- / P]-ATP to label the immobilized substrate with a radioactive phosphate, which can be detected by autoradiography, phosphor imager, or silver staining [9,13,75-79]. The second detection method relies on phosphospecific antibodies, which are fluorescently labeled [13,51,58,76,80]. While the fluorescent detection may be preferable as it avoids working with radioactive ATP, it has been shown that only monoclonal anti-phosphotyrosine antibodies showed reliable results [76]. Alternatively, fluorescently labeled phosphor-chelators have been used to detect the phosphorylated peptide in an array [81]. Preliminary results have also been reported for mass spectrometry detection and surface plasmon detection (vide infra). 

Inamori, K, Kyo, M., Matsukawa, K, Inoue, Y., Sonoda, T., Tatematsu, K, Tanizawa, K., Mori, T. and Katayama, Y. (2008) Optimal surface chemistry for peptide immobilization in on-chip phosphorylation analysis. Anal. Chem. 80, 643-650. 

Phosphorylation on serine, threonine, and tyrosine residues is an extremely important modulator of protein function. Phosphorylation can be analyzed by mass spectrometry with enrichment of compounds of interest using immobilized metal affinity chromatography and chemical tagging techniques, detection of phosphopep-tides using mass mapping and precursor ion scans, localization of phosphorylation sites by peptide sequencing, and quantitation of phosphorylation by the introduction of mass tags (McLachlin and Chait 2001). 

Detection of phosphorylated proteins and peptides can be enhanced by selectively isolating these species. Online immobilized metal affinity chromatography (IMAC)-CE-ESI-MS is such a powerful analytical tool. The IMAC resin retains and preconcentrates phosphorylated proteins and peptides, CE separates the phosphorylated species and MS/MS identifies the components and their phosphorylation sites. Cao and Stults applied this method to the analysis of phosphorylated angiotensin II and tryptic digests of a- and /3-casein (CE conditions buffer, 0.1% acetic acid/10% methanol uncoated capillary). Beta-casein is a well-characterized protein with five phosphorylation sites and is widely used as a standard for protein phosphorylation studies. 

Fig. 7.4 Examples of solid-phase ECL assay formats, a DNA hyinidization assay based on an immobilized ssDNA hybridizes with a labeled target ssDNA. b Sandwieh-type DNA biosensor, c Assay used for integrase aetivity test with immobilized and Iree-labeled dsDNA. d Sandwich-type immunoassay, e Direct immunoassay, f Competitive assay in which analyte competes with labeled analyte for antibody-binding sites on immobilized antibody, g Protease activity assay in which cleavage of the immobilized peptide results in the decrease in ECL emission due to the removal of the ECL label, h Kinase activity assay using a labeled antibody to recognize the phosphorylated product. Adapted from Ref. [23]. Copyright 2008 American Chemical Society...

Muszyhska, G. Doborrowolska, G. Medin, A. Ekman, P. Porath, J.O. Model studies on iron(III) ion affinity chromatography. II. Interaction of immobilized iron(III) ions with phosphorylated amino acids, peptides and proteins. 

Immobilized metal-affinity chromatography (IMAC) is also known as metal-chelate affinity chromatography (MCAC). This method was first proposed by Porath et al. in 1975 [63] and is based on the specific interactions between immobilized metal ions and amino acid residues, such as histidine, fiyptophan, and cysteine in proteins or peptides [63]. IMAC has become an important tool for the detection and purification of metalloproteins, histidine-tagged proteins, and phosphorylated proteins. Areas in which this method is now used include proteomics [64—66], work with recombinant proteins [67—69], and disease diagnosis [70,71]. 

Jensen and co-workers showed that isobaric tags can be used in combination with 2D-PAGE and affinity purification using Fe(III)-IMAC (immobilized metal ion affinity chromatography) to quantify differences in phosphorylation states. They were able to quantify the relative amounts of a peptide with one phosphate at different amino acid positions by HPLC, because the three isoforms have different retention times. 

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