Method for preparing immobilized metal affinity material by using 5-pyridoxal phosphate

A technology of affinity materials and metals, which is applied in the field of analytical chemistry, can solve the problems of complex synthesis process and high toxicity, and achieve the effect of simple modification method and increased practicability

Inactive Publication Date: 2017-07-07
WUHAN UNIV
View PDF3 Cites 7 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, in the following years, a series of phosphate-group-containing materials were prepared and used for the isolation and enrichment of phosphorylated polypeptides in complex biologica

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for preparing immobilized metal affinity material by using 5-pyridoxal phosphate
  • Method for preparing immobilized metal affinity material by using 5-pyridoxal phosphate
  • Method for preparing immobilized metal affinity material by using 5-pyridoxal phosphate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] First prepare amino-modified nanomaterials to obtain Y-NH 2 ; Then connect 5-pyridoxal phosphate to Y-NH 2 On, Y-NH with surface-modified phosphate2 -PLP; Finally, metal ions are immobilized on the phosphate to obtain Y-NH 2 -PLP-M n+ .

[0031] 1. Amino-modified nanomaterials (Y-NH 2 ) preparation, according to different nanomaterials, its preparation method is as follows:

[0032] ① SiO 2 -NH 2 Preparation: 0.5g nano-SiO 2 Disperse in 25mL of 1M HCl aqueous solution for activation for 30min; after washing with water and ethanol, disperse it in 50mL of ethanol; add 2mLAPTES to the above suspension, stir at room temperature for 3h to obtain amino-bonded silica gel nanoparticles.

[0033] ② Fe 3 o 4 @SiO 2 -NH 2 Preparation: 2.0g FeCl 2 4H 2 O and 5.4 g FeCl 3 ·6H 2 O was dissolved in 2M HCl aqueous solution, nitrogen gas was passed through to remove dissolved oxygen, then 30mL concentrated ammonia water (25wt%) was added and stirred for 30min to obtain Fe...

Embodiment 2

[0039] In order to investigate three different nanomaterials modified titanium ions (Y-NH 2 -PLP-Ti 4+ ) on the extraction effect of phosphorylated peptides, so as to select an optimal substrate material, we compared three kinds of Y-NH 2 -PLP-Ti 4+ (Y=SiO 2 , OCNT or Fe 3 o 4 @SiO 2 ) Extraction effect on phosphorylated polypeptide in β-casein and BSA enzymatic hydrolysis mixture:

[0040] Prepare β-casein with 100mM Tris-HCl (pH=8.5) buffer solution to a concentration of 1mg / mL; add pancreatic protease, and reacted at 37°C for 16h. The polypeptide product after enzymatic hydrolysis was drained by a vacuum centrifugal concentrator and stored in a -20°C refrigerator for later use.

[0041] Weigh 1 mg BSA and dissolve it in 100 μL of denaturing buffer (pH=8.5) containing 8M urea and 100 mM Tris-HCl, and then add 5 μL of 100 mM tris(2-formylethyl)phosphine hydrochloride solution (tricarboxymethylphosphoric acid, TCEP), and react at room temperature for 20 min to reduce ...

Embodiment 3

[0047] In order to investigate three commonly used metal ions (Ti 4+ , Ga 3+ , Fe 3+ ) on the extraction effect of phosphorylated peptides, so as to select an optimal metal ion to obtain the optimal material, we compared the Fe 3 o 4 @SiO 2 -NH 2 -PLP-M n+ (M=Ti 4+ , Ga 3+ or Fe 3+ ) Extraction effect on phosphorylated polypeptide in β-casein and BSA enzymatic hydrolysis mixture:

[0048] In order to compare three Fe bound to different metal ions 3 o 4 @SiO 2 -NH 2 -PLP-M n+ (M=Ti 4+ , Ga 3+ or Fe 3+ ) for the enrichment ability of phosphorylated peptides, the mixture of phosphorylated protein (β-casein) and non-phosphoprotein (BSA) hydrolyzate was used as the analyte (β-casein:BSA=1:500).

[0049] Weigh 10mg Fe respectively 3 o 4 @SiO 2 -NH 2 -PLP-M n+Disperse in 1mL of water, mix well, take 20μL of the dispersion, equilibrate with the sample solution (5% TFA-50% ACN (v / v)), then add it to 100μL of the sample solution containing the peptide mixture, vort...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a method for preparing an immobilized metal affinity material by using a chelating agent 5-pyridoxal phosphate, and an application of the immobilized metal affinity material in sample pretreatment. The method comprises the following steps: firstly selecting several common nano materials to carry out chemical modification so as to prepare amino modified nano materials to obtain Y-NH2; grafting the 5-pyridoxal phosphate on the Y-NH2 to obtain a surface modified phosphate radical Y-NH2-PLP; and finally immobilizing metal ions on the phosphate radical to obtain Y-NH2-PLP-M<n+>. The modification method of the invention is simple and time-saving, and does not destroy the morphological characteristics of the base material, and the modification is used for realizing the functionalization of the base material, thereby increasing the practicability of the base material. The Y-NH2-PLP-M<n+> can be directly used as extraction material for the sample pretreatment of complex biological samples, the material is separated from the sample in the extraction process through centrifugal or external magnetic field action, and the immobilized metal affinity material has been successfully applied to extraction of phosphorylated polypeptide of standard peptide mixed liquor, milk enzymatic hydrolysate, human serum samples and rat brain lysate.

Description

technical field [0001] The invention belongs to the technical field of analytical chemistry, and in particular relates to a method of using pyridoxal 5-phosphate for preparing immobilized metal-affinity materials and its application in sample pretreatment. Background technique [0002] Protein phosphorylation is a reversible post-transcriptional modification that plays an important role in various physiological functions. At present, mass spectrometry is widely used in the study of phosphorylated peptides due to its high sensitivity and high throughput. However, due to the low content of phosphorylated proteins in biological samples, and the ionization efficiency of phosphorylated peptides in mass spectrometry is lower than that of non-phosphorylated peptides, it is difficult for us to analyze them directly. Therefore, it is very important to enrich the phosphorylated peptides in the sample before mass spectrometry analysis. [0003] As a fast, easy-to-use and economical p...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): B01J20/281C07K7/08C07K14/47C07K1/14
CPCB01J20/281B01J2220/42B01J2220/52C07K7/08C07K14/47
Inventor 冯钰锜王倩
Owner WUHAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap