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A functionalized temperature-sensitive polymer, a preparing method thereof and applications of the polymer

A temperature-sensitive polymer technology, applied in the field of analytical chemistry, can solve the problems of reducing the enrichment reaction rate and efficiency, and achieve the reduction of solid-liquid interface mass transfer resistance and steric hindrance, high enrichment efficiency and selectivity, Reactive Bioorthogonal Effects

Active Publication Date: 2017-06-30
ACAD OF MILITARY SCI ACAD OF MILITARY MEDICAL SCI BEIJING INST OF LIFEOMICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, the existing enrichment carriers for O-GlcNAc glycosylated proteins / peptides are all solid-phase materials, which form a heterogeneous system with the target molecular protein in the solution during enrichment, and the mass transfer resistance and Factors such as steric hindrance between the solid phase material and the target protein will reduce the enrichment reaction rate and efficiency

Method used

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  • A functionalized temperature-sensitive polymer, a preparing method thereof and applications of the polymer
  • A functionalized temperature-sensitive polymer, a preparing method thereof and applications of the polymer
  • A functionalized temperature-sensitive polymer, a preparing method thereof and applications of the polymer

Examples

Experimental program
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Effect test

Embodiment 1

[0061] Embodiment 1, preparation and performance test of functionalized temperature-sensitive polymer

[0062] Synthetic routes for functionalized temperature-sensitive polymers such as figure 1 a) as shown.

[0063] 1) Synthesis of temperature-sensitive polymers: free-radical copolymerization of temperature-sensitive monomers and functional monomers under initiator conditions.

[0064] The details are as follows: add 50mL of 50% methanol to a 100mL round-bottomed flask, stir magnetically and pass N 2 to remove O from the round bottom flask 2 After 1h, weigh 800mg of N-isopropylacrylhydrazide and quickly add it to the above flask, and continue to pass N 2 30min at N 2 Slowly add 0.15mL methyl acrylate under atmosphere, pass N 2 And after stirring for 30min, slowly drop in 80mg of potassium persulfate which has been dissolved in 200μL of deoxygenated water, pass N 2 30min to ensure that the next free radical polymerization reaction is at N 2 Under the atmosphere, remov...

Embodiment 2

[0089] Example 2. Using the functionalized temperature-sensitive polymer obtained in Example 1 to enrich azide-labeled O-GlcNAc modified proteins

[0090] 1) Weigh an appropriate amount of α-crystallin standard protein and dissolve it in 20mM Hepes buffer solution (pH 7.9) to prepare a concentration of 3.5μgμL -1 standard protein solution. Use the in vitro enzyme catalysis kit to add various reagents according to the instructions to carry out azide labeling on α-crystallin protein overnight at 4°C. After α-crystallin standard protein azido-labeled, methanol, chloroform and deionized water were added in order to precipitate the protein, so as to remove excess UDP-GalNAz, and the excess methanol was blown dry with nitrogen. Finally, the α-crystallin protein was dissolved in 20mM Hepes buffer solution (pH 7.9), and stored at -80°C for later use;

[0091] 2) Weigh 4 parts of functionalized temperature-sensitive polymers, 0.4mg, 0.6mg, 0.8mg and 1.0mg respectively, dissolve in 20...

Embodiment 3

[0094] Example 3. Using the functional temperature-sensitive polymer obtained in Example 1 to enrich O-GlcNAc modified proteins in mixed samples

[0095] 1) Weigh an appropriate amount of α-crystallin standard protein and dissolve it in 20mM Hepes buffer solution (pH 7.9) to prepare a concentration of 3.5μgμL -1 standard protein solution. Use the in vitro enzyme catalysis kit to add various reagents according to the instructions to carry out azide labeling on α-crystallin protein overnight at 4°C. After α-crystallin standard protein azide labeling, add methanol, chloroform, and deionized water in sequence to precipitate the protein, thereby removing excess UDP-GalNAz, and blow dry excess methanol with nitrogen. Finally, the α-crystallin protein was dissolved in 20mM Hepes buffer solution (pH 7.9), and stored at -80°C for later use;

[0096] 2) The mixture of azide-labeled α-crystallin and bovine serum albumin (mass ratio 1:100) was dissolved in 20mM Hepes buffer solution (pH...

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Abstract

A functionalized temperature-sensitive polymer, a preparing method thereof and applications of the polymer is disclosed. The structure formula of the polymer is shown as a formula I. The preparing method includes subjecting a temperature-sensitive monomer and a functional monomer to free radical polymerization under the existence of an initiator to obtain a temperature-sensitive polymer; subjecting the temperature-sensitive polymer and hydrazine hydrate to an acylhydrazine conversion reaction to obtain a hydrazide-modified temperature-sensitive polymer; and reacting the hydrazide-modified temperature-sensitive polymer with 2-(diphenylphosphino) terephthalic acid 1-methyl 4-pentafluorophenyl diester. An azide-marked O-G1cNAc modified protein enriching mechanism of the functionalized temperature-sensitive polymer is based on a Staudinger ligation reaction between azide and triphenylphosphine, and the reaction is high in biological orthogonality, high in yield in an aqueous solution, specific and stable and does not need a catalyst, and therefore the reaction has good compatibility with subsequent mass spectrum identification. The method greatly increases the amino acid sequence coverage rate of low-abundance O-G1cNAc modified protein mass spectrum identification in a mixed sample.

Description

technical field [0001] The invention relates to a functionalized temperature-sensitive polymer and its preparation method and application, belonging to the field of analytical chemistry. Background technique [0002] O-linked β-N-acetylglucosamine (O-GlcNAc) is a post-translational modification ubiquitous in eukaryotic cells. Different from the classic O-glycosylation modification, O-GlcNAc glycosylation modification only exists in nuclear and cytoplasmic proteins, has only one monosaccharide structure, and is a highly dynamic, inducible and reversible modification method . Although the abundance of this modification on proteins is extremely low, O-GlcNAc modified proteins play an important role in organisms, from gene transcription to signal transduction and cell cycle regulation, there are O-GlcNAc glycosylated modified proteins participate. Studies have shown that abnormal levels of O-GlcNAc glycosylation modification on proteins are closely related to many human disea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08F220/14C08F220/54C08F8/30C08F8/40C07K1/32C07K14/47
CPCC07K1/32C07K14/47C08F8/40C08F220/14C08F220/54C08F8/30
Inventor 钱小红秦伟捷张万军刘彤
Owner ACAD OF MILITARY SCI ACAD OF MILITARY MEDICAL SCI BEIJING INST OF LIFEOMICS
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