WO2022083799A1 - Garnissage chromatographique ayant une affinité pour les ions métalliques immobilisés, colonne chromatographique, et son procédé de préparation - Google Patents

Garnissage chromatographique ayant une affinité pour les ions métalliques immobilisés, colonne chromatographique, et son procédé de préparation Download PDF

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WO2022083799A1
WO2022083799A1 PCT/CN2021/141556 CN2021141556W WO2022083799A1 WO 2022083799 A1 WO2022083799 A1 WO 2022083799A1 CN 2021141556 W CN2021141556 W CN 2021141556W WO 2022083799 A1 WO2022083799 A1 WO 2022083799A1
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metal ion
immobilized metal
preparation
ion affinity
chromatographic column
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PCT/CN2021/141556
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English (en)
Chinese (zh)
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张文洋
晏石娟
黄文洁
吴绍文
殷志斌
孔谦
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广东省农业科学院农业生物基因研究中心
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Publication of WO2022083799A1 publication Critical patent/WO2022083799A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/282—Porous sorbents
    • B01J20/285—Porous sorbents based on polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3804—Affinity chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/282—Porous sorbents
    • B01J20/283—Porous sorbents based on silica

Definitions

  • the invention belongs to the field of chromatography, and in particular relates to an immobilized metal ion affinity chromatography filler, a chromatography column and a preparation method thereof.
  • Immobilized Metal Ion Affinity Chromatography is used for affinity purification of proteins, enzymes, polypeptides and amino acids that have the ability to bind metal ions.
  • the principle is to use the electrostatic interaction between the metal ions on the solid support and some amino acids or chemical modification groups exposed by the protein/polypeptide to enrich and purify the specific protein/peptide. After 30 years of development , has gradually become one of the most effective technologies for the separation and purification of biomolecules such as proteins.
  • IMAC immobilized metal ion affinity chromatography
  • the solid phase matrix of commercial IMAC products is usually resin microspheres or magnetic particles, so as to carry out the process of loading, washing and elution by centrifugation or magnetic adsorption, iminodiacetic acid (IDA), aminotriacetic acid (NTA) ) and ethylenediamine (TED) are representative chelating ligands for immobilizing metal ions, their carboxyl and amine groups can bind Al 3+ , Fe 3+ , Ga 3+ , Ni 2+ and other metal ions Chelated on the surface of the solid matrix, but these combinations have poor chelation performance and low selectivity.
  • IMAC materials whether commercial or reported in the literature, are usually in the form of microspheres or magnetic materials.
  • the enrichment of target proteins/peptides is completed in a centrifuge tube, and the processes of sample loading, washing and elution need to be manually It is time-consuming and labor-intensive to carry out.
  • the current commercial IMAC materials are monopolized by foreign companies, and the price is relatively expensive (such as: Thermo Fisher No. A32992, 4629 yuan for 30 times).
  • the first object of the present invention is to provide a kind of immobilized metal ion affinity with loose pores, high specific surface area, low back pressure, good physical and chemical stability, good mechanical stability, high selectivity, high sensitivity and high enrichment factor. and column packing.
  • the immobilized metal ion affinity chromatography column packing of the present invention is prepared by the following method:
  • Potassium water glass, ⁇ -glycidyl ether oxypropyl trimethoxysilane and water-dissolved adenosine triphosphate disodium are mixed and stirred, and then water-dissolved formamide is added and stirred to obtain a reaction solution, which is solidified by reaction to obtain a solid material, and then washed to obtain a fixed Metal ion affinity chromatography column packing.
  • the modulus of the potassium water glass is in the range of 2-4, and the Baumé degree is in the range of 20-50. More preferably, the modulus of potassium water glass is 3.3, and the Baumé degree is 40.
  • the curing is at a temperature of 100° C. for 10 hours.
  • the washing is successively washed with 1M ammonium nitrate, 0.1M nitric acid and water.
  • the amount-to-mass ratio of the potassium water glass, ⁇ -glycidyl ether oxypropyltrimethoxysilane, adenosine triphosphate disodium salt and formamide is 500-2000:1-10:2-50:20-130. More preferably, it is 1000:6:7.5:68.
  • the second object of the present invention is to provide a preparation method of an immobilized metal ion affinity chromatography column, which is prepared by the following method:
  • the chromatographic column is inserted into the above reaction solution, the reaction solution is filled with the chromatographic column, and then the reaction solution of the filled chromatographic column is reacted and solidified, and then washed to obtain an ATP-modified immobilized metal ion affinity chromatographic column.
  • the chromatographic column is a capillary, such as an elastic quartz capillary (outer diameter 360 microns, inner diameter 150 microns, length 15 cm), and an ATP-modified immobilized metal ion affinity capillary monolithic column is prepared.
  • the immobilized metal ion affinity chromatography column packing has the advantages of loose and porous, high specific surface area, low back pressure, good physical and chemical stability, good mechanical stability, high selectivity, high sensitivity and high enrichment multiple.
  • the ATP-modified immobilized metal ion affinity capillary monolithic column prepared by the one-step reaction method has the advantages of simple and rapid preparation steps, high repeatability and yield, and low preparation cost. It can be obtained by only mixing the raw materials uniformly and going through a baking reaction, and has good physical and chemical stability and good reproducibility after repeated use. Compared with the enrichment materials in the form of microspheres or magnetic materials commonly used in the art, the ATP-modified immobilized metal ion affinity capillary monolithic column of the present invention has the potential to be combined with a nanoliter liquid phase system, combined with an automatic sampling system.
  • the site coverage, detection limit, and selectivity of phosphorylated peptides in the present invention also reach the first-class level in the industry.
  • Figure 1 is a cross-sectional electron microscope image of the ATP-modified immobilized metal ion affinity capillary monolithic column, with dimensions of 200 ⁇ m and 5 ⁇ m, respectively.
  • Figure 2 is a first-order mass spectrogram of phosphorylated peptides enriched by ATP-modified immobilized metal ion affinity capillary monolithic column for ⁇ -casein cleavage products.
  • Figure 3 is the primary mass spectrogram of phosphorylated peptides enriched in 0.2ng and 2ng of ⁇ -casein digestion products by ATP-modified immobilized metal ion affinity capillary monolithic column.
  • Figure 4 is the first-order mass spectrogram of phosphorylated peptides enriched by ATP-modified immobilized metal ion affinity capillary monolith column enriched with 2ng ⁇ -casein and 2 ⁇ g BSA digestion products.
  • the ATP-modified immobilized metal ion affinity capillary monolithic column prepared by the one-step reaction method specifically comprises the following steps:
  • step S2 Weigh 7.5 mg of adenosine triphosphate disodium, dissolve it in 160 microliters of deionized water, slowly add it to the reaction solution in step S1, and continue to fully stir at room temperature for 30 minutes.
  • step S3 Take 60 microliters (about 68 mg) of formamide, mix with 40 microliters of deionized water, slowly add it to the reaction solution in step S2, and continue stirring for 1 minute at room temperature to obtain a reaction solution.
  • the cross-section of the ATP-modified immobilized metal ion affinity capillary monolithic column is shown in the electron microscope image in Figure 1. Under the field of view of 5 microns, it can be observed that the material is loose and porous, with an average pore size of about 1 micron, which proves that The material has a large specific surface area, which is the basis for its high enrichment efficiency. At the same time, this porous structure can avoid high back pressure during the loading process, and can complete the loading at a higher flow rate.
  • Example 2 Comprehensively investigate the enrichment ability of the ATP-modified immobilized metal ion affinity capillary monolithic column prepared in Example 1 of the present invention for phosphorylated peptides.
  • the ATP-modified immobilized metal ion affinity capillary monolithic column prepared in Example 1 of the present invention was used to enrich phosphorylated peptides obtained by trypsin treatment of standard phosphorylated protein ⁇ -casein or BSA.
  • the enrichment performance was investigated from the aspects of site coverage, detection limit and selectivity.
  • Dissolve 2 mg/ml BSA in 50 mM ammonium bicarbonate solution add DTT to 10 mM and heat at 56°C for 30 min, add iodoacetamide to 40 mM and incubate for 40 min in the dark to break the disulfide bond.
  • Add 1 mg of standard phosphorylated protein ⁇ -casein and 1 mg of BSA with broken disulfide bonds dissolve them in 1 ml of 50 mM ammonium bicarbonate, and treat with 20 ⁇ g of trypsin for 8 hours to obtain two 1 mg/mL protease cleavage solutions.
  • the standard phosphorylated protein ⁇ -casein contains two phosphorylated proteins, ⁇ -S1-casein and ⁇ -S2-casein, with 9 and 10 phosphorylation sites reported, respectively.
  • ⁇ -S1-casein contains two phosphorylated proteins, ⁇ -S1-casein and ⁇ -S2-casein, with 9 and 10 phosphorylation sites reported, respectively.
  • BSA bovine serum albumin
  • monolithic column Use a micro syringe pump to push 100 microliters of 80% acetonitrile, 1% TFA solution to flow through the ATP-modified immobilized metal ion affinity capillary monolithic column (hereinafter referred to as monolithic column) to complete the cleaning.
  • the experimental results show that the ATP-modified immobilized metal ion affinity capillary monolithic column prepared by the present invention has the enrichment effect on mono- and poly-phosphorylated peptides (Fig. 2).
  • the two ⁇ -casein proteins were included in the standard, and the site coverage was as high as 100% and 90%, respectively (Table 1), and the detection limit for phosphorylated peptide enrichment reached 10 fmol (Figure 3).
  • the selectivity of the peptides reached 1:1000 ( ⁇ -casein:BSA) (1ug ⁇ -casein mixed with 1 mg BSA) ( Figure 4), all of which are among the best in the industry.

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  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un garnissage chromatographique ayant une affinité pour les ions métalliques immobilisés, une colonne chromatographique, et un procédé de préparation associé. Du silicate de sodium de potassium, du gamma-glycidoxypropyltriméthoxysilane et de l'adénosine triphosphate disodique dissoute dans l'eau sont mélangés et agités, puis du formamide dissous avec de l'eau est ajouté et agité pour obtenir une solution de réaction, la réaction et le durcissement étant réalisés pour obtenir un matériau solide, puis le matériau est lavé pour obtenir un garnissage de colonne chromatographique ayant une affinité pour les ions métalliques immobilisés. La colonne monolithique capillaire ayant une affinité pour les ions métalliques immobilisés modifiée par ATP, préparée au moyen d'un procédé de réaction en une étape de la présente invention, comprend des étapes de préparation simples et rapides, une répétabilité élevée et un rendement élevé, et de faibles coûts de préparation. Les matières premières doivent uniquement être mélangées uniformément et soumises à une réaction de cuisson en une seule fois pour obtenir la colonne, qui présente une bonne stabilité physique et chimique, et une bonne répétabilité après de multiples utilisations.
PCT/CN2021/141556 2021-06-15 2021-12-27 Garnissage chromatographique ayant une affinité pour les ions métalliques immobilisés, colonne chromatographique, et son procédé de préparation WO2022083799A1 (fr)

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