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N-phosphopeptide and protein enrichment material and preparation and application thereof

A technology for phosphorylation of peptides and proteins, applied in peptide preparation methods, analytical materials, chemical instruments and methods, etc., can solve the problems of phosphorylation modification loss, etc., and achieve the advantages of reduced loss, good enrichment effect and fast mass transfer Effect

Active Publication Date: 2018-05-29
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, N-phosphorylation modification, because its P-N bond will be hydrolyzed rapidly when the pH is less than 3, resulting in the loss of phosphorylation modification, so the traditional method of enriching O-phosphorylated peptides or proteins cannot be applied to N-phosphorylation Enrichment of peptides or proteins

Method used

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  • N-phosphopeptide and protein enrichment material and preparation and application thereof
  • N-phosphopeptide and protein enrichment material and preparation and application thereof
  • N-phosphopeptide and protein enrichment material and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] 1. Preparation of sub-two-micron core-shell silica gel with vertical pores:

[0047] a) Mix the hydrolysate (6.7mL ammonia water, 5.1mL water and 70mL absolute ethanol) and 4mL ethyl orthosilicate, stir in a 22℃ water bath for 40min, and heat up to 55℃;

[0048] b) Add 0.64mL water and 4mL ethyl orthosilicate, react at 55°C for 40min;

[0049] c) Repeat step (2) 4 times to get Sead1;

[0050] d) Replace the Sead1 solution with an equal amount of newly prepared hydrolysate, and repeat step (2) 10 times to obtain Sead2.

[0051] e) Disperse Sead2 in 100mL water, add 1g cetyltrimethylammonium chloride, 5.8mL tridecane, 0.1-100mg ammonium fluoride and 0.1-10mL ammonia, react at 90°C for 24h to obtain NPs;

[0052] f) Sintering the dried NPs at 550°C for 6 hours to form the initial shell layer of core-shell microspheres.

[0053] g) Disperse NPs in 30 mL of 5N HCl solution, and reflux for 12 hours at 120°C.

[0054] 2. Synthesis of a phosphate recognition functional molecule:

[0055] a) ...

Embodiment 2

[0065] 1. Preparation of sub-two-micron core-shell silica gel with vertical pores:

[0066] a) Mix the hydrolysate (6.7mL ammonia water, 5.1mL water and 70mL absolute ethanol) and 4mL ethyl orthosilicate, stir in a 22℃ water bath for 40min, and heat up to 55℃;

[0067] b) Add 0.64mL water and 4mL ethyl orthosilicate, react at 55°C for 40min;

[0068] c) Repeat step (2) 4 times to get Sead1;

[0069] d) Replace the Sead1 solution with an equal amount of newly prepared hydrolysate, and repeat step (2) 10 times to obtain Sead2.

[0070] e) Disperse Sead2 in 100mL water, add 1g cetyltrimethylammonium bromide, 5.8mL tridecane, 0.1-100mg ammonium fluoride and 0.1-10mL ammonia, react at 90°C for 24h to obtain NPs;

[0071] f) Sintering the dried NPs at 550°C for 6 hours to form the initial shell layer of core-shell microspheres.

[0072] g) Disperse NPs in 30mL 5N HCl solution, and reflux for 12h at 120°C.

[0073] 2. Synthesis of a phosphate recognition functional molecule:

[0074] a) 3.37g N,N'...

Embodiment 3

[0085] 1. Preparation of sub-two-micron core-shell silica gel with vertical pores:

[0086] a) Mix the hydrolysate (6.7mL ammonia, 5.1mL water and 70mL absolute ethanol) and 4mL ethyl orthosilicate, stir in a water bath at 22°C for 40min, and heat up to 55°C;

[0087] b) Add 0.64mL water and 4mL ethyl orthosilicate, react at 55°C for 40min;

[0088] c) Repeat step (2) 4 times to get Sead1;

[0089] d) Replace the solution of Sead1 with an equal amount of newly prepared hydrolysate, and repeat step (2) 10 times to obtain Sead2.

[0090] e) Disperse Sead2 in 100mL water, add 1g cetyltrimethylammonium bromide, 5.8mL tridecane, 0.1-100mg ammonium fluoride and 0.1-10mL ammonia, react at 90°C for 24h to obtain NPs;

[0091] f) Sintering the dried NPs at 550°C for 6 hours to form the initial shell layer of core-shell microspheres.

[0092] g) Disperse NPs in 30mL 5N HCl solution, and reflux for 12h at 120°C.

[0093] 2. Synthesis of a phosphate recognition functional molecule:

[0094] a) 3.37g N,...

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Abstract

The present invention relates to a novel silica gel material capable of recognizing N-phosphopeptides and proteins under neutral conditions and application thereof in N-phosphopeptide and protein enrichment. Non-porous silica microspheres are prepared by a seed growth method, a core-shell microsphere initial layer is formed by a template-guided dissolution and re-deposition method, and finally sub-2-micron core-shell silica gel with vertical pores can be obtained by acid reflux. N-tert-butyloxycarbonyl-L-tyrosine and N, N'-dipicolylamine are reacted to form a support molecule, and a phosphaterecognition functional molecule is formed by complexation of a metal ion on the support molecule. Finally, the novel phosphoric acid recognition functional silica gel material is obtained by covalently bonding the phosphoric acid recognition functional molecule with the sub-2-micron core-shell silica gel having the vertical pores through an amide bond. The novel phosphoric acid recognition functional silica gel material can rapidly and specifically enrich the N-phosphopeptides and proteins in a neutral buffer system.

Description

Technical field [0001] The invention relates to a novel silica gel material capable of identifying N-phosphorylated peptides and proteins under neutral conditions and its application in N-phosphorylated peptides and protein enrichment. Background technique [0002] The phosphorylation modification of protein is one of the most common post-translational modifications in nature. It is involved in all life activities such as cell proliferation, apoptosis, development, differentiation, and signal transduction (Nat. Biotechnol, 2005, 23, 94-101). Phosphorylation modification is mainly divided into O-phosphorylation modification (occurring on serine, threonine or tyrosine) and N-phosphorylation modification (occurring on histidine, lysine or arginine) according to the different amino acids in which it occurs. Acid), S-phosphorylation modification (occurs on cysteine). Among them, O-phosphorylation modification is the most studied post-translational modification. Its PO bond has good s...

Claims

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Application Information

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IPC IPC(8): B01J20/22B01J20/30B01J20/28C07K1/14G01N1/40
CPCB01J20/103B01J20/22B01J20/28021B01J2220/4806B01J2220/4812C07K1/14C07K1/145G01N1/405
Inventor 张丽华胡晔晨江波翁叶靖高航杨开广张玉奎
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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