152 results about "Phosphorylated Peptide" patented technology

The invention provides a synthetic method and application of a metal-organic framework (MOF) composite nanomaterial. The method comprises the following steps: dispersing ferriferrous oxide magnetic spheres which are synthesized through a traditional hydrothermal technology in a weakly alkaline solution of dopamine hydrochloride to carry out self-polymerization of dopamine on the surfaces of the magnetic spheres; and sequentially dispersing polydopamine coated magnetic spheres in a dimethylformamide solution of zirconium chloride and a dimethylformamide solution of 2-amino-terephthalic acid to obtain the MOF composite nanomaterial with the magnetic sphere surfaces coated with polydopamine and modified with an amino group and with zirconium as a center metal ion. The material has the advantages of large specific surface area, good hydrophilicity and suitable pore structure, can be applied to further researches of the proteomics, and can specifically enrich Which can specifically enrich phosphorylated peptide segments and glycopeptides; the synthetic method is simple and quick; and the synthesized material has good hydrophilicity and biocompatibility, and can be used for selectively enriching endogenous phosphorylation peptide segments and glycopeptide in complex biological samples.

There are provided a novel compound which captures a multisite phosphorylated peptide or protein specifically to a phosphorylation site and a method for detecting a multisite phosphorylated peptide or protein using the same. In particular, there are provided a compound which specifically detects an excessively phosphorylated tau protein observed in the brain affected by Alzheimer's disease and a method for diagnosing Alzheimer's disease in vitro or in vivo using the compound. By bringing a metal complex compound having two dipicolylamine (Dpa) moieties and a spacer including a chromogenic or luminescent functional group or atom group into contact with a multisite phosphorylated peptide or protein, the compound recognizes the distance between phosphate groups and specifically binds to the peptide or the protein, and a multisite phosphorylated peptide or protein or a kinase activity is optically detected by measuring the change, or a multisite phosphorylated peptide or protein or kinase activity is imaged by an optical imaging method applying the change in the luminescence.

The invention relates to polydopamine modified magnetic balls, a synthetic method of nanomaterial by fixing Ti<4+> on the surfaces and an application thereof. The synthetic method comprises the following steps: dispersing magnetic balls in a dopamine-contained water solution, quickly adding a trismetyl aminomethane buffer solution in the dopamine-contained water solution in which the magnetic balls are uniformly dispersed under the mechanical stirring condition, mechanically stirring for 10 to 16 hours at a room temperature, and washing and carrying out the magnetic separation to obtain nanomaterial which is good in dispersivity in the water and provided with the polydopamine modified magnetic balls on the surfaces, and dispersing the material in a 100 mmol/L Ti(SO4)2 solution, mechanically stirring for 2 to 3 hours at the room temperature, and washing and carrying out the magnetic separation to obtain polydopamine modified magnetic balls, and nanomaterial by fixing Ti<4+> on the surfaces of the magnetic balls.. The method for fixing Ti<4+> is very simple, a layer of polydopamine is modified on the surface, so that the material has excellent environmental stability and biocompatibility, and is good in dispersivity in the water, and the material can fix a large amount of Ti<4+>, and takes the magnetic balls as a core to cause the material to be simple to operate and carry out magnetic separation, the synthetic method is simple, and low in cost, and can enrich phosphorylated peptide by a high-flux MALDI-TOFMS.

The present invention is a method, and a kit for carrying out the method, of separating phosphorylated peptides from a mixture of phosphorylated and unphosphorylated peptides. The method involves reacting a collection of peptides, in which some of the peptides have one or more phosphate groups, with a first resin. The peptides are then reacted with a capture ligand which reacts with the phosphate group of the phosphorylated peptides to form a bond. The capture ligand is used to separate the phosphorylated peptides from the unphosphorylated peptides.

The invention relates to a rapid treatment method for a phosphoproteome sample and develops a novel sample treatment method integrating cell splitting decomposition and protein enzymolysis by using the advantage that protein rapid enzymolysis can be promoted through high-concentration trypsin, and application of the novel sample treatment method in analysis of phosphoproteomics. Benefiting from the high-concentration trypsin, a deep mechanism of the cell splitting decomposition and the protein enzymolysis is promoted; a cell sample can finish rapid conversion of a peptide fragment mixture in one step; and in a phosphoeptide enriching process, a non-phosphorylated peptide fragment and other substances with incompatible mass spectrums can be removed. The rapid treatment method can realize the rapid conversion from cells to the peptide fragment by 25 minutes; in a contrast normal group, the simplest method also needs at least 16 hours; and a relatively good mass spectrum identification coverage rate of the contrast group is obtained through analyzing a HeLa actual cell sample, and the accuracy and the high efficiency of the sample pre-treatment method are proved.

The invention relates to a preparation method of a hydrophilic metal ion immobilization affinity magnetic bead, which comprises the following steps: the surface of an amino magnetic bead is subjected to amino activation through glutaral; the activated magnetic bead is connected with an adenosine disodium triphosphate molecule through a surface modification reaction; the surface of the activated magnetic bead is bonded with a hydrophilic functional molecule containing a plurality of phosphate and hydroxyl radicals, so as to be beneficial for subsequent metal ion immobilization; at last, the immobilization of a metal ion is realized in a solution containing the metal ion; and the preparation of the hydrophilic metal ion immobilization affinity magnetic bead is accomplished. The hydrophilic metal ion immobilization affinity magnetic bead has the advantages that the preparation method is simple; compared with other metal ion immobilization affinity chromatographic columns or magnetic beads, the hydrophilic metal ion immobilization affinity magnetic bead prepared by the method is high in hydrophily, good in selectivity, good in preparation repeatability and stable in performance, and has a good specific concentration ability to phosphorylated peptides; the operation is simple; the complicated centrifugation operation can be avoided; and the hydrophilic metal ion immobilization affinity magnetic bead has wide application prospects in proteomics, especially in phosphorylated proteomics.

The present invention provides methods and materials for isolating, purifying, and/or enriching the concentration of compounds having one or more phosphate groups and/or derivatives thereof, including but not limited to phosphorylated peptides and/or phosphorylated proteins. In some aspects, the present invention provides nanostructured enrichment materials, such as metal oxide mesoporous materials, that selectively and reversibly bind with phosphorylated compounds with high specificity and are capable of controlled release of phosphorylated compounds bound to their active surfaces. Mesoporous materials of the present invention also provide enrichment materials having large active surface areas that provide for higher loading capacities for phosphorylated peptides and proteins relative to conventional affinity based methods. Nanostructured metal oxide mesoporous enrichment materials of the present invention are also compatible with implementation via a variety of separation platforms including flow through separation systems, elution based separation systems, column chromatography and affinity chromatography.

The invention relates to the enrichment and purification of phosphopeptide in phosphoproteome, in particular to the application of ZrO2 in the enrichment and purification of phosphopeptide. ZrO2 nano material shows high specificity, selectivity, binding capacity and sensitivity at a phosphopeptide section; meanwhile, the affinity of the ZrO2 nano material to the specificity of phosphopeptide ensures that the material can be used in phosphoproteome during protein post-translational modification. Due to the specificity combination of ZrO2 with the phosphopeptide section in complex proteolysis sample, large-scale identification of phosphorylated protein and determination of phosphorylation sites can be realized through combining with MALDI-TOF MS and nano-LC MS/MS.

The invention provides a peptide identification method by using a mesoporous silica composite combined with mass spectrum. A composite with perfluoroalkyl group modified mesoporous silica covering on the surface of Fe3O4 magnetic microsphere is used in matrix-assisted laser desorption ionization-time of flight mass spectrometric analysis of perfluoroalkyl group derived peptide. The material shows extremely strong affinity with the fluorous phase derived phosphorylated peptides, while maintaining good dispersion in aqueous solution. The Fe3O4 magnetic core in the material simplifies the process of enrichment and separation. A solid phase microextraction method based on the composite with perfluoroalkyl group modified mesoporous silica covering on the surface of Fe3O4 magnetic microsphere achieves selective enrichment of non-phosphorylated peptides, and can be successfully applied to the enrichment of endogenous phosphopeptides in human serum.

A method for the separation and assay of phosphorylated peptides or proteins from complex mixtures of proteins and peptides is described. The method uses a diazo moiety linked by an organic group to a substrate. Kits for performing the assay are also described. The method and kits are particularly useful for detecting changes in phosphorylates produced in living cells.

The invention relates to preparation of an immobilized metal ion affinity chromatographic material, and particularly relates to a method for preparing an immobilized metal affinity ion chromatographic monolithic column. In the method, a monolithic column material is prepared by using an organic-inorganic hybrid silica gel monolithic material of which the surface carries aldehyde groups as a vector; a phosphate buffer solution containing aminomethylphosphonic acid and sodium cyanotrihydroborate NaCNBH3 flows through the monolithic column material continuously to prepare a monolithic material vector of which the surface carries aldehyde chelation groups; and an aqueous solution of transition metal ions flows through the material vector to prepare the immobilized metal ion affinity chromatographic monolithic column. High-selectivity enrichment of phosphorylated peptide in a hybrid peptide section can be realized by utilizing the immobilized metal ion affinity chromatographic monolithic column. The invention has the advantages that the monolithic column has good selectivity to the phosphorylated peptide, can be recycled, and has the characteristics of large enrichment capability and high recovery rate; and the preparation method has the characteristics of simplicity, convenience, good permeability and small back pressure, and the prepared immobilized metal ion affinity chromatographic monolithic column has good mechanical strength and strong acid and alkali resistance.

According to the present invention, phosphorylated peptides and/or phosphorylated proteins are specifically separated. A sample containing a phosphorylated peptide and/or a phosphorylated protein is supplied to a separation unit filled with a metal oxide in the presence of an aliphatic hydroxycarboxylic acid. Upon separation of a phosphorylated peptide and/or a phosphorylated peptide with the use of a separation unit filled with a metal oxide, adsorption of carboxylic acid to an acidic peptide can be prevented in the presence of aliphatic hydroxycarboxylic acid. In addition, aliphatic hydroxycarboxylic acid does not inhibit adsorption of a phosphorylated peptide and a phosphoric acid group in the phosphorylated peptide to a metal oxide.

The present invention relates to an N-phosphorylated peptide enrichment method. According to the method, O-phosphorylated peptide interference in a peptide sample is removed through beta-elimination, and the N-phosphorylated modified peptide in the sample is enriched by using a magnetic mesoporous titania composite nanometer material; the O-phosphorylated modification on serine and threonine is removed through a beta-elimination method under an alkaline condition so as to form stable dehydroalanine and beta-methyl dehydroalanine; a magnetic Fe3O4 nanometer material is synthesized through a solvent method, titanium dioxide is wrapped on the surface through a sol-gel method, and hole amplification is performed through a hydrothermal method to obtain the magnetic mesoporous titania composite nanometer material; and the magnetic mesoporous titania composite nanometer material can specifically enrich the N-phosphorylated peptide under a weak acid buffer system. According to the present invention, the beta-elimination and specific enrichment combined method is used for enriching the N-phosphorylated peptide in the sample.

The invention discloses a preparation method of a flexible metallic oxide nanofiber phosphorylated peptide enrichment material. The preparation method of the flexible metallic oxide nanofiber phosphorylated peptide enrichment material includes the steps that 1, metal salt is added into corresponding solvent, stirring is conducted to generate metal ions, then, a chelating agent is added, stirring is conducted again, and a precursor solution is obtained, wherein the ratio of the metal salt to the solvent is 1 g:(10-80) ml, and the molar ratio of the metal salt to the chelating agent is 1:(0.01-0.4); 2, the obtained precursor solution is subjected to electrostatic spinning to obtain a precursor nanofiber membrane; 3, the precursor nanofiber membrane is calcined in air, the calcining temperature gradually increases to 500-1200 DEG C from room temperature, calcining is kept for 30-120 min at the highest calcining temperature, and then the flexible metallic oxide nanofiber membrane is obtained. The preparation method of the flexible metallic oxide nanofiber phosphorylated peptide enrichment material is simple in process, and phosphorylated protein and phosphorylated peptide fragments canbe effectively enriched and purified efficiently and repeatedly.

The invention discloses a phosphorylated peptide adsorption enrichment method comprising (1) a non specific adsorption inhibitor and part of a to-be-tested sample are added a sample loading buffer, and an enrichment filter column is used for phosphorylated adsorption enrichment; (2) enrichment products are collected, the abundance of 20 kinds of amino acids in non phosphorylated peptide can be calculated as first abundance, the abundance of the non phosphorylated peptide in the 20 kinds of amino acids can be calculated as second abundance, at least one amino acid with higher ratio of first abundance to second abundance can be selected as an additive; (3) the sample loading buffer added with the additive is used for phosphorylated adsorption enrichment of the to-be-tested sample. By use of the competitive adsorption effects of amino acids and peptides, adsorption of the non phosphorylated peptide can be reduced, adsorption selectivity of the phosphorylated peptide enrichment can be increased, and the method lays the foundation for improving the accuracy and stability of phosphorylation detection.

The invention discloses a sequential separation and mass spectrum identification method of multi-site phosphorylation peptide. By using spinel and inverse-spinel type magnetic nano ferrite materials as a separation substrate, the method is used for realizing the fast separation from a complex sample background under the action of an external magnetic field by using the difference of the coordination property between metal ions on an octahedron binding site in the materials and different phosphorylation peptides and the inherent magnetism of the materials. The nano ferrite materials related by the invention can be used for separating a complex polypeptide mixture into non-phosphorylation peptide, mono-phosphorylation peptide and multi-site phosphorylation peptide, so that the signal inhibiting between the molecules of different samples is reduced or eliminated, in-vivo or in-source mono-phosphorylation peptide can be distinguished, and high signal-to-noise ratio and low interference are realized. The method is simple and can be used for effectively enriching low-abundance phosphorylation peptides without using a complex apparatus and realizing the sequential separation and the mass spectrum identification of the multi-site phosphorylation peptide; and the sample analysis and operation are simple without complex sample pre-treatment.

The invention relates to high-selectivity enrichment of phosphorylated peptide, in particular to a method for preparing polymer materials for enriching phosphorylated peptide fragments. The method adopts monomers provided with phosphate groups to perform polymerization, so as to directly form the polymer materials provided with the phosphate groups. The materials are acted by a solution containing zirconium ions to generate phosphate ester zirconium groups, so that the materials can specially enrich and purify the phosphorylated peptide from a complex proteolysis solution. The method adopts the polymerization method to directly synthesize the polymer materials provided with functional groups, does not need to additionally perform chemical modification, and has the characteristics of simplicity and convenience.

The invention belongs to the field of biochemical analysis and relates to an enrichment and identification method for lysine nitrogen-junctional phosphorylation post-translational modification. By the retention difference before and after loss of a phosphate group on the lysine nitrogen-junctional phosphorylated peptide through chromatographic separation, specific enrichment of lysine phosphorylated peptide is realized by chromatography, and then identification is achieved through mass spectrometry. Through the above method, large-scale enrichment and identification of lysine nitrogen-junctional phosphorylation post-translational modification sites are realized.