WO2022083799A1 - Immobilized metal ion affinity chromatographic packing, chromatographic column, and preparation method therefor - Google Patents

Immobilized metal ion affinity chromatographic packing, chromatographic column, and preparation method therefor 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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French (fr)
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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    • 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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Abstract

An immobilized metal ion affinity chromatographic packing, a chromatographic column, and a preparation method therefor. Potassium sodium silicate, gamma-glycidoxypropyltrimethoxysilane and water-dissolved adenosine triphosphate disodium are mixed and stirred, then formamide dissolved with water is added and stirred to obtain a reaction solution, reaction and curing is performed to obtain a solid material, and then the material is washed to obtain an immobilized metal ion affinity chromatographic column packing. The ATP-modified immobilized metal ion affinity capillary monolithic column prepared by means of a one-step reaction method of the present invention features simple and rapid preparation steps, high repeatability and yield, and low preparation costs. The raw materials only need to be uniformly mixed and subjected to a one-time baking reaction to obtain the column, which has good physical and chemical stability, and good repeatability after multiple uses.

Description

一种固定化金属离子亲和色谱填料、色谱柱及其制备方法A kind of immobilized metal ion affinity chromatography packing, chromatography column and preparation method thereof 技术领域technical field
本发明属于色谱领域，具体涉及一种固定化金属离子亲和色谱填料、色谱柱及其制备方法。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.
背景技术Background technique
固定化金属离子亲和层析(IMAC)被用于亲和纯化与金属离子具有结合能力的蛋白质、酶、多肽和氨基酸等。其原理是利用固相载体上的金属离子与蛋白质/多肽暴露出的一些氨基酸或化学修饰基团之间的静电相互作用，对特定的蛋白质/多肽进行富集、纯化，经过三十年来的发展，已逐渐成为分离纯化蛋白质等生物分子最有效的技术之一。Immobilized Metal Ion Affinity Chromatography (IMAC) 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.
固定化金属离子亲和层析(IMAC)的核心在于固相基质、配体和金属离子的选择和制备工艺，上样、洗涤和洗脱条件也会影响富集的效果，其性能参数主要包括选择性、灵敏度、重现性、上样量、使用寿命和应用范围等。The core of immobilized metal ion affinity chromatography (IMAC) lies in the selection and preparation process of solid phase matrix, ligands and metal ions. The loading, washing and elution conditions will also affect the enrichment effect. Its performance parameters mainly include: Selectivity, Sensitivity, Reproducibility, Loading Volume, Lifetime and Application Range, etc.
目前商品化IMAC产品的固相基质通常为树脂微球或者磁性颗粒，以便通过离心或者磁吸附的方式进行上样、洗涤、洗脱的流程，亚氨基二乙酸(IDA)、氨基三乙酸(NTA)和乙二胺(TED)是具代表性的用于固定金属离子的螯合配体，它们的羧基和胺基能将Al 3+、Fe 3+、Ga 3+、Ni 2+等金属离子螯合在固相基质表面，但是这些组合的螯合性能较差，选择性不高。目前无论是商品化还是文献报道的IMAC材料，通常都是微球或者磁性材料的形式，在离心管中完成对目标蛋白质\多肽的富集，其上样、洗涤、洗脱的流程均需手动进行，较为耗时耗力，另外，目前商品化IMAC材料受国外公司垄断，价格较为昂贵(如：赛默飞货号A32992，4629元30次)。 At present, 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. At present, 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. In addition, 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).
发明内容SUMMARY OF THE INVENTION
本发明的第一个目的是提供一种具有疏松多孔、比表面积高、背压低、物理化学稳定性好、机械稳定性好、选择性高、灵敏度高、富集倍数高的固定化金属离子亲和色谱柱填料。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.
本发明的固定化金属离子亲和色谱柱填料，其是通过以下方法制备的：The immobilized metal ion affinity chromatography column packing of the present invention is prepared by the following method:
将钾水玻璃、γ-缩水甘油醚氧丙基三甲氧基硅烷和水溶解的三磷酸腺苷二钠混合搅拌，然后加入用水溶解的甲酰胺搅拌获得反应液，反应固化获得固体材料，然后洗涤，获得固定化金属离子亲和色谱柱填料。Potassium water glass, γ-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.
本发明的固定化金属离子亲和色谱柱填料的制备原理示意图如下所示：The schematic diagram of the preparation principle of the immobilized metal ion affinity chromatography column packing of the present invention is as follows:
1、钾水玻璃的制备1. Preparation of potassium water glass
mKOH+nSiO 2→mK 2O·(n-m)SiO 2+mH 2O mKOH+nSiO 2 →mK 2 O·(nm)SiO 2 +mH 2 O
2、钾水玻璃在甲酰胺、加热条件下水解2. Hydrolysis of potassium water glass under formamide and heating conditions
Figure PCTCN2021141556-appb-000001
Figure PCTCN2021141556-appb-000001
3、与步骤2同时进行的硅偶联剂与ATPNa 2的反应: 3. The reaction of silicon coupling agent and ATPNa 2 carried out simultaneously with step 2:
Figure PCTCN2021141556-appb-000002
Figure PCTCN2021141556-appb-000002
4、硅胶基底上的ATP修饰4. ATP modification on silica substrate
Figure PCTCN2021141556-appb-000003
Figure PCTCN2021141556-appb-000003
5、填料发挥富集功能5. Filler plays an enrichment function
Figure PCTCN2021141556-appb-000004
Figure PCTCN2021141556-appb-000004
优选，Preferably,
S1、将钾水玻璃与γ-缩水甘油醚氧丙基三甲氧基硅烷混合均匀得到S1的混合液；S1, the potassium water glass and γ-glycidyl ether oxypropyl trimethoxysilane are evenly mixed to obtain the mixed solution of S1;
S2、将三磷酸腺苷二钠用水溶解后加入到S1的混合液中，得到S2的混合液；S2, dissolving adenosine triphosphate disodium with water and adding it to the mixed solution of S1 to obtain the mixed solution of S2;
S3、将甲酰胺用水溶解后加入到S2的混合液中，搅拌获得反应液，反应获得固体材料，然后洗涤，获得固定化金属离子亲和色谱柱填料。S3, dissolve the formamide in water and add it to the mixed solution of S2, stir to obtain a reaction solution, react to obtain a solid material, and then wash to obtain an immobilized metal ion affinity chromatography column packing.
优选，所述的钾水玻璃的模数范围2-4，波美度范围为20-50。进一步优选是钾水玻璃的模数3.3，波美度为40。Preferably, 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.
优选，所述的固化是在温度100℃下固化10小时。Preferably, the curing is at a temperature of 100° C. for 10 hours.
优选，所述的洗涤是先后用1M硝酸铵、0.1M硝酸和水洗涤。Preferably, the washing is successively washed with 1M ammonium nitrate, 0.1M nitric acid and water.
优选，所述的钾水玻璃、γ-缩水甘油醚氧丙基三甲氧基硅烷、三磷酸腺苷二钠盐和甲酰胺的用量质量比是500-2000:1-10:2-50:20-130。进一步优选为1000:6:7.5:68。Preferably, the amount-to-mass ratio of the potassium water glass, γ-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.
本发明的第二个目的是提供一种固定化金属离子亲和色谱柱的制备方法，其是通过以下方法制备的：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:
是将色谱柱插入上述反应液中，使反应液注满色谱柱，然后注满的色谱柱的反应液反应固化，再经洗涤，获得ATP修饰的固定化金属离子亲和色谱柱。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.
所述的色谱柱是毛细管，如弹性石英毛细管(外径360微米，内径150微米，长度15厘米)，制得ATP修饰的固定化金属离子亲和毛细管整体柱。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.
本发明的固定化金属离子亲和色谱柱填料，其具有疏松多孔、比表面积高、背压低、物理化学稳定性好、机械稳定性好、选择性高、灵敏度高、富集倍数高的优点。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.
本发明通过一步反应法制备的ATP修饰的固定化金属离子亲和毛细管整体柱，制备步骤简单快速、重复性和良品率高、制备成本低。仅需将原料混合均匀后经历一次烘烤反应便可得到，其物理化学稳定性好，多次使用后具有较好的重现性。与本领域常用的微球或者磁性材料形式的富集材料相比，本发明的ATP修饰的固定化金属离子亲和毛细管整体柱具备与纳升级液相系统联用的潜力，结合自动进样系统，可使劳动密集的上金属离子、上样、洗涤、洗脱等操作自动化进行，同时本发明对磷酸化肽的位点覆盖率、检出限、选择 性也达到业界一流的水平。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. , which can automate labor-intensive operations such as metal ion loading, sample loading, washing, and elution, and at the same time, the site coverage, detection limit, and selectivity of phosphorylated peptides in the present invention also reach the first-class level in the industry.
附图说明Description of drawings
图1是ATP修饰的固定化金属离子亲和毛细管整体柱的横切面电镜图，分别为200μm及5μm尺度。Figure 1 is a cross-sectional electron microscope image of the ATP-modified immobilized metal ion affinity capillary monolithic column, with dimensions of 200 μm and 5 μm, respectively.
图2是ATP修饰的固定化金属离子亲和毛细管整体柱富集α-casein酶切产物中磷酸化肽的一级质谱图。Figure 2 is a first-order mass spectrogram of phosphorylated peptides enriched by ATP-modified immobilized metal ion affinity capillary monolithic column for α-casein cleavage products.
图3是ATP修饰的固定化金属离子亲和毛细管整体柱富集0.2ng和2ngα-casein酶切产物中磷酸化肽的一级质谱图。Figure 3 is the primary mass spectrogram of phosphorylated peptides enriched in 0.2ng and 2ng of α-casein digestion products by ATP-modified immobilized metal ion affinity capillary monolithic column.
图4是ATP修饰的固定化金属离子亲和毛细管整体柱富集2ngα-casein及2μg BSA酶切产物中磷酸化肽的一级质谱图。Figure 4 is the first-order mass spectrogram of phosphorylated peptides enriched by ATP-modified immobilized metal ion affinity capillary monolith column enriched with 2ngα-casein and 2μg BSA digestion products.
具体实施方式Detailed ways
下面将结合具体实施例来进一步说明本发明,应该说明的是，下述实施例仅是为了解释本发明，并不对其内容进行任何形式的限定。本发明的设计思想或同类物质的简单替代，均应包含在本发明的保护范围之内。除非特别说明，本发明采用的试剂、材料、方法和设备均为本技术领域现有常规的试剂、材料、方法和设备。The present invention will be further described below in conjunction with specific embodiments. It should be noted that the following embodiments are only for explaining the present invention, and do not limit its content in any form. The design idea of the present invention or the simple substitution of similar substances shall be included within the protection scope of the present invention. Unless otherwise specified, the reagents, materials, methods and equipment used in the present invention are conventional reagents, materials, methods and equipment in the art.
实施例1 ATP修饰的固定化金属离子亲和毛细管整体柱的制备Example 1 Preparation of ATP-modified immobilized metal ion affinity capillary monolithic column
本发明通过一步反应法制备的ATP修饰的固定化金属离子亲和毛细管整体柱，具体包括以下步骤：The ATP-modified immobilized metal ion affinity capillary monolithic column prepared by the one-step reaction method specifically comprises the following steps:
S1.在微型反应瓶中加入740微升(约1000mg)钾水玻璃(模数3.3，波美度40)，在室温搅拌条件下缓慢加入6微升(约6mg)γ-缩水甘油醚氧丙基三甲氧基硅烷(GLYMO， Cas号：2530-83-8)，持续在室温条件下充分搅拌30分钟。S1. Add 740 microliters (about 1000 mg) of potassium water glass (modulus 3.3, Baumé degree 40) to a micro reaction flask, and slowly add 6 microliters (about 6 mg) of γ-glycidyl ether oxypropion under stirring at room temperature trimethoxysilane (GLYMO, Cas number: 2530-83-8), and continued stirring well at room temperature for 30 minutes.
S2.称取7.5mg三磷酸腺苷二钠，用160微升去离子水溶解后，缓慢加入S1步骤中的反应液中，继续持续在室温条件下充分搅拌30分钟。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.
S3.取60微升(约68mg)甲酰胺，与40微升去离子水混匀后，缓慢加入S2步骤中的反应液中，室温下继续搅拌1分钟，得到反应液。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.
S4.切取一批外径360微米、内径150微米、长度15厘米的弹性石英毛细管，插入S3所得反应液中，利用毛细现象使反应液注满毛细管。S4. Cut out a batch of elastic quartz capillaries with an outer diameter of 360 microns, an inner diameter of 150 microns and a length of 15 cm, insert them into the reaction solution obtained in S3, and fill the capillaries with the reaction solution by capillary phenomenon.
S5.将注满的毛细管放入100℃烘箱中固化10小时，所得整体柱两端分别切去2厘米。S5. Put the filled capillary into an oven at 100° C. to cure for 10 hours, and cut off 2 cm from both ends of the obtained monolithic column.
S6.利用压力注射池，先后用1M硝酸铵、0.1M硝酸、去离子水各200微升洗涤上述整体柱，由此得到成品的ATP修饰的固定化金属离子亲和毛细管整体柱。S6. Wash the monolithic column with 200 μl each of 1M ammonium nitrate, 0.1M nitric acid and deionized water using a pressure injection cell, thereby obtaining a finished ATP-modified immobilized metal ion affinity capillary monolithic column.
ATP修饰的固定化金属离子亲和毛细管整体柱的截面如附图1的电镜图所示，在5微米的视野尺度下，可观察到该材料呈疏松多孔状，平均孔径约为1微米，证明材料具有较大的比表面积，这是其具有高富集效率的基础，同时这种多孔结构能避免上样过程中出现较高的背压，能够在较高的流速下完成上样。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.
实施例2综合考察本发明实施例1制得的ATP修饰的固定化金属离子亲和毛细管整体柱对磷酸化肽的富集能力。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.
将本发明实施例1制得的ATP修饰的固定化金属离子亲和毛细管整体柱用于对标准磷酸化蛋白α-casein或BSA经胰蛋白酶处理所得磷酸化肽的富集。从位点覆盖度、检测限、选择性等方面考察其富集性能。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 α-casein or BSA. The enrichment performance was investigated from the aspects of site coverage, detection limit and selectivity.
溶解于50mM碳酸氢铵溶液的2mg/ml BSA加入DTT至10mM于56℃加热30min，加入碘代乙酰胺至40mM于暗处孵育40min，以断开二硫键。取1mg标准磷酸化蛋白α-casein及1mg断开二硫键的BSA，溶解于1ml的50mM碳酸氢铵，分别用20μg胰蛋白酶处理8小时，获得两种1mg/mL的蛋白酶切液。(标准磷酸化蛋白α-casein含有α-S1-casein和α-S2-casein两种磷酸化蛋白，分别有9和10个磷酸化位点被报道，经胰蛋白酶处理后，能产生一系列的单磷酸化肽、多磷酸化肽和非磷酸化肽，未经特异性富集时在质谱中主要观察到非磷酸化肽的信号；牛血清白蛋白BSA是一种常见的蛋白，没有磷酸化位点，经胰蛋白酶处理后，产生多种非磷酸化肽)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. Take 1 mg of standard phosphorylated protein α-casein and 1 mg of BSA with broken disulfide bonds, dissolve them in 1 ml of 50 mM ammonium bicarbonate, and treat with 20 μg of trypsin for 8 hours to obtain two 1 mg/mL protease cleavage solutions. (The standard phosphorylated protein α-casein contains two phosphorylated proteins, α-S1-casein and α-S2-casein, with 9 and 10 phosphorylation sites reported, respectively. After trypsin treatment, a series of Monophosphorylated peptides, polyphosphorylated peptides, and non-phosphorylated peptides, the signals of non-phosphorylated peptides were mainly observed in mass spectrometry without specific enrichment; bovine serum albumin (BSA) is a common protein and is not phosphorylated site, after trypsin treatment, produces a variety of non-phosphorylated peptides)
具体步骤如下：Specific steps are as follows:
S1.利用微量注射泵推动100微升体积分数80％乙腈、1％TFA溶液流经ATP修饰的固定化金属离子亲和毛细管整体柱(下称整体柱)，完成清洗。S1. 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.
S2.利用微量注射泵推动100微升0.1M氯化锆溶液流经整体柱，完成对Zr 4+的固定。 S2. Use a micro syringe pump to push 100 microliters of 0.1M zirconium chloride solution to flow through the monolithic column to complete the fixation of Zr 4+ .
S3.取一定量的蛋白酶切物(位点覆盖率实验为2μgα-casein酶切物\100pmol，检测限实验为0.2ngα-casein酶切物\10fmol或2ngα-casein酶切物\100fmol，选择性实验为2ngα-casein酶切物与2μg BSA酶切物)溶于100微升体积分数80％乙腈、1％TFA溶液，利用微量注射泵推动该溶液流经整体柱，完成对磷酸化肽的富集。S3. Take a certain amount of protease digest (the site coverage experiment is 2μgα-casein digest\100pmol, and the detection limit experiment is 0.2ngα-casein digest\10fmol or 2ngα-casein digest\100fmol, selectivity In the experiment, 2ng of α-casein digest and 2μg of BSA digest) were dissolved in 100 microliters of 80% acetonitrile, 1% TFA solution, and the solution was pushed through the monolithic column by a micro syringe pump to complete the enrichment of phosphorylated peptides. set.
S4.利用微量注射泵推动100微升体积分数80％乙腈、1％TFA溶液流经整体柱，完成对非特异性结合多肽的清洗。S4. Use a micro syringe pump to push 100 microliters of 80% acetonitrile and 1% TFA solution to flow through the monolithic column to complete the washing of non-specifically bound polypeptides.
S5.将整体柱末端与弹性石英毛细管、nano喷针、nanoESI离子源及Orbitrap Fusion质谱组成连接，利用微量注射泵按照0.5微升/分钟的流速持续推动5％氨水，对被整体柱富集 的磷酸化肽进行洗脱，用质谱实时检测洗脱产物。S5. Connect the end of the monolithic column with the elastic quartz capillary, nano spray needle, nanoESI ion source and Orbitrap Fusion mass spectrometer, and use a micro syringe pump to continuously push 5% ammonia water at a flow rate of 0.5 μl/min. Phosphorylated peptides are eluted and the eluted products are detected in real time by mass spectrometry.
表1.ATP修饰的固定化金属离子亲和毛细管整体柱富集α-casein酶切物磷酸化肽的鉴定结果Table 1. Identification results of ATP-modified immobilized metal ion affinity capillary monolithic column enriched with α-casein digested phosphorylated peptides
Figure PCTCN2021141556-appb-000005
Figure PCTCN2021141556-appb-000005
Figure PCTCN2021141556-appb-000006
Figure PCTCN2021141556-appb-000006
实验结果表明本发明制备的ATP修饰的固定化金属离子亲和毛细管整体柱对单、多磷酸化肽均有富集效果(图2)，对α-casein-S1和α-casein-S2(这个标准品中就包含了这两种α-casein蛋白)的位点覆盖率分别高达100％和90％(表1)，对磷酸化肽富集的检测限达到10fmol(图3)，对磷酸化肽的选择性达到1:1000(α-casein:BSA)(1ugα-casein和1mg BSA混合)(图4)，均达到了业界一流的水平。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 α-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 (α-casein:BSA) (1ug α-casein mixed with 1 mg BSA) (Figure 4), all of which are among the best in the industry.

Claims (10)

  1. 一种固定化金属离子亲和色谱柱填料的制备方法，其特征在于，将钾水玻璃、γ-缩水甘油醚氧丙基三甲氧基硅烷和水溶解的三磷酸腺苷二钠混合搅拌，然后加入用水溶解的甲酰胺搅拌获得反应液，反应固化获得固体材料，然后洗涤，获得固定化金属离子亲和色谱柱填料。A method for preparing an immobilized metal ion affinity chromatography column filler, characterized in that potassium water glass, γ-glycidyl ether oxypropyltrimethoxysilane and water-dissolved disodium adenosine triphosphate are mixed and stirred, and then added with water to dissolve The formamide is stirred to obtain a reaction solution, solidified by reaction to obtain a solid material, and then washed to obtain an immobilized metal ion affinity chromatography column packing.
  2. 根据权利要求1所述的制备方法，其特征在于，所述的钾水玻璃、γ-缩水甘油醚氧丙基三甲氧基硅烷、三磷酸腺苷二钠盐和甲酰胺的用量比是500-2000:1-10:2-50:20-130。preparation method according to claim 1, is characterized in that, the consumption ratio of described potassium water glass, γ-glycidyl ether oxypropyl trimethoxysilane, adenosine triphosphate disodium salt and formamide is 500-2000:1 -10:2-50:20-130.
  3. 根据权利要求2所述的制备方法，其特征在于，所述的钾水玻璃、γ-缩水甘油醚氧丙基三甲氧基硅烷、三磷酸腺苷二钠盐和甲酰胺的用量质量比是1000:6:7.5:68。preparation method according to claim 2, is characterized in that, the consumption mass ratio of described potassium water glass, γ-glycidyl ether oxypropyl trimethoxysilane, adenosine triphosphate disodium salt and formamide is 1000:6: 7.5:68.
  4. 根据权利要求1所述的制备方法，其特征在于，所述的钾水玻璃的模数范围2-4，波美度范围为20-50。The preparation method according to claim 1, wherein the modulus range of the potassium water glass is 2-4, and the Baumé degree range is 20-50.
  5. 根据权利要求4所述的制备方法，其特征在于，所述的钾水玻璃的模数3.3，波美度为40。The preparation method according to claim 4, wherein the modulus of the potassium water glass is 3.3, and the Baume degree is 40.
  6. 根据权利要求1所述的制备方法，其特征在于，所述的固化是在温度100℃下固化10小时；所述的洗涤是先后用1M硝酸铵、0.1M硝酸和水洗涤。The preparation method according to claim 1, wherein the curing is curing at a temperature of 100° C. for 10 hours; the washing is successively washing with 1M ammonium nitrate, 0.1M nitric acid and water.
  7. 一种按照权利要求1-6任一制备方法制备得到的固定化金属离子亲和色谱柱填料。An immobilized metal ion affinity chromatography column filler prepared according to any one of the preparation methods of claims 1-6.
  8. 一种固定化金属离子亲和色谱柱的制备方法，其特征在于，是将色谱柱插入权利要求1-6任一中的反应液中，使反应液注满色谱柱，然后注满的色谱柱的反应液反应固化，再经洗涤，获得ATP修饰的固定化金属离子亲和色谱柱。A method for preparing an immobilized metal ion affinity chromatographic column, wherein the chromatographic column is inserted into the reaction solution according to any one of claims 1-6, the reaction solution is filled with the chromatographic column, and then the filled chromatographic column is filled with the chromatographic column. The reaction solution was reacted and solidified, and then washed to obtain an ATP-modified immobilized metal ion affinity chromatography column.
  9. 根据权利要求8所述的制备方法，其特征在于，所述的色谱柱是弹性石英毛细管。The preparation method according to claim 8, wherein the chromatographic column is an elastic quartz capillary.
  10. 一种按照权利要求8或9所述的制备方法制备得到的ATP修饰的固定化金属离子亲和色谱柱。An ATP-modified immobilized metal ion affinity chromatographic column prepared according to the preparation method of claim 8 or 9.
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CN116328740A (en) * 2023-02-27 2023-06-27 无锡萃纯生物材料科技有限公司 Centrifugal extraction process and solid phase extractant for purifying biomacromolecule

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