CN105381786B - A kind of MOF materials of dendrimer modification and its preparation method and application - Google Patents

Abstract

The invention belongs to the field of biotechnology, and specifically relates to a dendrimer-modified MOF material, a preparation method thereof, and an application in glycopeptide enrichment. The present invention utilizes a hydrothermal method to synthesize a highly stable aminated Mil‑101 (Cr) MOF material, and then grafts hydrophilic dendritic polyamide‑amine (PAMAM) on the On the surface of MOF, dendrimer-modified MOF materials are obtained. The modified dendritic molecule PAMAM has a long chain and a large number of amino groups, which greatly enhances the hydrophilicity of this composite material and is used for the enrichment of glycopeptides, which can improve the enrichment efficiency. In the enrichment of the standard glycoprotein enzymatic peptide horseradish peroxidase (HRP), 13 glycopeptides were successfully enriched, with a selectivity of 1:100 and a detection limit as low as 1 fmol/µL. The invention is novel and convenient, practical and efficient, good in repeatability, high in stability and has broad application prospects.

Description

A dendrimer-modified MOF material and its preparation method and application

technical field

The invention belongs to the field of biotechnology, and in particular relates to a dendrimer-modified MOF material, a preparation method thereof and an application in glycopeptide enrichment.

technical background

Protein glycosylation is a very important post-translational modification, such as protein folding, intracellular sorting, recognition and transport and other functions. In recent studies, biological mass spectrometry has been successfully applied to the analysis of glycoproteins as an effective method. However, since glycoproteins are basically low-abundance expressed proteins and the microscopic heterogeneity of sugar chains, at the same time, the chemical nature of sugar chains is polyhydroxy aldehydes, polyhydroxy ketones and their derivatives, lacking functional groups that can produce ultraviolet absorption And because of its strong hydrophilicity, the response signal in mass spectrometry is low. Therefore, it is very important to specifically enrich glycoproteins or glycopeptides. At present, for the enrichment of glycoproteins, glycopeptides and N-glycans in complex biological samples, the commonly used strategies mainly include the following: (1) boric acid enrichment method; (2) hydrazine chemical method; (3) lectin affinity and enrichment method; (4) hydrophilic interaction chromatography, etc. And each of these methods also has its own advantages and disadvantages. The boric acid method can enrich N-sugar and O-sugar at the same time, but it has strict pH requirements, and the enrichment efficiency is low; the hydrazine chemical method requires very tedious experiments. steps, and the same lectin can only enrich the same type of sugar chains. Among these methods, the method of hydrophilic interaction has been applied more and more. Due to its high efficiency enrichment and mild experimental conditions, many functionalized hydrophilic nanomaterials have been applied to the enrichment of glycopeptides. Concentrates, including the application of maltose, zwitterions, and some hydrophilic celluloses.

In recent years, due to its large specific surface area, adjustable pore size, and modifiable properties of surface groups, MOF materials have been extensively studied in catalysis, drug loading, electrochemistry, and nanomaterials. In the field of bioanalysis research, more and more MOF materials have also received extensive attention.

Combined with this goal, the present invention designs a method that utilizes Mil-101 (Cr) MOF material as a substrate, and grafts a hydrophilic dendritic polymer PAMAM on its surface, thereby achieving separation and enrichment of glycopeptides. . This method has successfully improved the enrichment efficiency of glycopeptides, has higher selectivity and lower detection limit, and has also achieved good results in the enrichment of glycopeptides in actual sample serum.

Contents of the invention

The purpose of the present invention is to propose a glycopeptide enrichment method with simple operation, low sample loss rate, high efficiency and low detection limit, based on the mechanism of hydrophilic enrichment.

The glycopeptide enrichment method proposed in the present invention is a glycopeptide enrichment method based on dendrimer-modified MOF materials.

In the present invention, a Cr MOF with amino groups: NH ₂ -Mil-101 is used as the substrate, and a hydrophilic dendritic polyamide polyamide is synthesized by glutaraldehyde cross-linking method by utilizing its surface modification characteristics. -Amine (PAMAM) was grafted on the surface of MOF to obtain MOF materials modified by dendrimers. The modified dendritic molecule PAMAM has a long chain and a large number of amino groups, which greatly enhances the hydrophilicity of this composite material. This new type of porous material has a large specific surface area and strong hydrophilicity, and is used for glycopeptide enrichment. set, which can improve the enrichment efficiency of glycopeptides.

The preparation method of the dendrimer modified MOF material proposed by the present invention, the specific steps are as follows:

(1) Synthesis of MOF of Cr with amino groups: NH ₂ -Mil-101(Cr): Mix 400-500 mg chromium nitrate nonahydrate [Cr(NO ₃ ) ₃ 9H ₂ O] with 200-250 mg 2 - Dissolve aminoterephthalic acid in approximately 10 -13mL deionized water; after stirring at room temperature for 1-2 hours, transfer the mixture to a stainless steel autoclave, react at 100-130°C for 24-28 hours, then cool to room temperature; The final solution was centrifuged to obtain a green precipitate, washed 4-5 times with ethanol, and dried in a vacuum oven at 50-80°C to obtain NH ₂ -Mil-101(Cr);

(2) NH ₂ -Mil-101(Cr) surface-grafted dendrimer polyamide-amine (PAMAM): take dry NH ₂ -Mil-101(Cr) 1-2 mg in EP tube, add 4 %-5% glutaraldehyde aqueous solution; activate at 30-50°C for 1-2 h, centrifuge and wash 3-4 times, add 100-200 µL of deionized water and 4-6 µL of PAMAM; react at 30-50°C for 3-4 After 2 h, continue to centrifuge and wash 3-4 times, add 150-200 µL of 10-15 mg/mL sodium cyanoborohydride solution, react for 2-3 h, centrifuge, wash, and dry to obtain dendrimer-modified MOF materials. Denote as NH ₂ -Mil-101(Cr)@PAMAM.

In the present invention, the operation steps of using dendrimer-modified MOF materials for glycopeptide enrichment are as follows:

Take 400-500 µg of NH ₂ -Mil-101(Cr)@PAMAM, and wash 3-4 times with 90%-95% acetonitrile and 1-2% TFA solution. Add this solution and an appropriate amount of glycopeptide mixture after enzymatic hydrolysis to make the total volume 95-110 µL; enrich at 30-50°C for 50 min-60 min; separate and remove the supernatant, and then use 90%-95 Wash 3-4 times with % acetonitrile and 1-2% TFA solution; add 5-10 µL eluent (20%-30% ACN+0.1-0.2% TFA), and elute at 30-37°C for 20-25 min , the glycopeptide was eluted, and the eluate was analyzed and detected by MALDI-TOF MS.

The glycopeptide enrichment method based on dendrimer-modified MOF materials proposed by the present invention has achieved good results in the separation and enrichment of standard glycopeptides and glycopeptides in human serum.

As shown in Figure 2, through the method of the present invention, 13 glycopeptides were successfully enriched in the enrichment of the standard glycoprotein enzymatic peptide horseradish peroxidase (HRP), and its enrichment selection is shown in Figure 3 The specificity reached 1:100, and the detection limit in Figure 4 was also as low as 1 fmol/μL. These results all indicate that our material has a very high efficiency in the enrichment of glycopeptides, which can also prove that this material can be used in future glycopeptides. It has a good application prospect in proteomics research.

Description of drawings

Fig. 1 Enrichment flow chart of NH ₂ -Mil-101(Cr)@PAMAM material.

Fig. 2 MALDI-TOF mass spectrum of NH ₂ -Mil-101(Cr)@PAMAM enriched HRP (1 μg/μL).

Fig. 3 MALDI-TOF mass spectrum of the detection limit of NH ₂ -Mil-101(Cr)@PAMAM material enriched with HRP. Among them, (a) 10 fmol/μL HRP, (b) 1 fmol/μL HRP.

Fig. 4 Selective MALDI-TOF mass spectrum of NH ₂ -Mil-101(Cr)@PAMAM material enrichment, the molar ratio of HRP and BSA is 1:100. Among them, (a) before enrichment, (b) after enrichment.

Detailed ways

Example 1: Synthesis of NH ₂ -Mil-101(Cr)@PAMAM material

(1) Synthesis of NH ₂ -Mil-101(Cr): Dissolve 500 mg of chromium nitrate nonahydrate [Cr(NO ₃ ) ₃ 9H ₂ O] and 230 mg of 2-aminoterephthalic acid in 7 mL of deionized in water; after stirring at room temperature for 2 hours, transfer the mixture into a stainless steel autoclave, react at 130°C for 24 hours, and cool to room temperature; the cooled solution is centrifuged to obtain a green precipitate, which is washed five times with ethanol and placed in a vacuum drying oven Drying at 50℃, it is NH ₂ -Mil-101(Cr).

(2) NH ₂ -Mil-101(Cr) surface grafted dendrimer polyamide-amine (PAMAM): take dry NH ₂ -Mil-101(Cr) 2 mg in a 1.5 mLEP tube, add 5% Glutaraldehyde aqueous solution; after activation at 37°C for 2 h, centrifuge and wash three times, add 200 µL deionized water and 5 µL PAMAM; after reacting at 37°C for 4 h, continue to centrifuge and wash three times, add 10 mg/mL sodium cyanoborohydride The solution was 150 µL, reacted for 2 h, centrifuged, washed with water, and dried to obtain NH ₂ -Mil-101(Cr)@PAMAM.

Example 2: NH2-Mil-101(Cr)@PAMAM material is used for the enrichment of glycopeptides

Take 500 µg of NH2-Mil-101(Cr)@PAMAM and wash it three times with 95% acetonitrile+1%TFA solution. Add 95% acetonitrile + 1% TFA solution and an appropriate amount of enzymatically digested glycopeptide mixture to make the total volume 100 µL. Enrichment was carried out at 37°C for 50 min. The supernatant was separated and removed, and then washed three times with 95% acetonitrile+1% TFA solution. Add 10 µL of eluent (30% ACN, 0.1% TFA) and elute at 37°C for 20 min to elute the glycopeptide. The eluate is analyzed and detected by MALDI-TOF MS.

Claims (2)

1. A preparation method of a dendrimer-modified MOF material, characterized in that the MOF material is based on the MOF of Cr with amino groups: NH ₂ -Mil-101, and the hydrophilic The dendrimer polyamide-amine is grafted on the surface of MOF; the specific steps are: (1) Synthesis of MOF of Cr with amino groups: NH ₂ -Mil-101(Cr): Mix 400-500 mg chromium nitrate nonahydrate [Cr(NO ₃ ) ₃ 9H ₂ O] with 200-250 mg 2 - Dissolve aminoterephthalic acid in 10 -13mL deionized water; after stirring at room temperature for 1-2 hours, transfer the mixture to a stainless steel autoclave, react at 100-130°C for 24-28 hours, then cool to room temperature; The solution was centrifuged to obtain a green precipitate, which was washed 4-5 times with ethanol and dried in a vacuum oven at 50-80°C to obtain NH ₂ -Mil-101(Cr); (2) NH ₂ -Mil-101(Cr) surface-grafted dendrimer polyamide-amine (PAMAM): take dry NH ₂ -Mil-101(Cr) 1-2 mg in EP tube, add 4 %-5% glutaraldehyde aqueous solution; activate at 30-50°C for 1-2 h, centrifuge and wash 3-4 times, add 100-200 µL of deionized water and 4-6 µL of PAMAM; react at 30-50°C for 3-4 After 2 h, continue to centrifuge and wash 3-4 times, add 150-200 µL of 10-15 mg/mL sodium cyanoborohydride solution, react for 2-3 h, centrifuge, wash, and dry to obtain dendrimer-modified MOF materials. Denote as NH ₂ -Mil-101(Cr)@PAMAM. 2. The application of the dendrimer-modified MOF material prepared by the preparation method according to claim 1 in glycopeptide enrichment is characterized in that the specific operation steps are as follows: take 400-500 µg NH ₂ -Mil-101(Cr )@PAMAM, washed 3-4 times with 90%-95% acetonitrile and 1-2% TFA solution; add this solution and an appropriate amount of enzymatically digested glycopeptide mixture to make the total volume 95-110 µL; at 30 Enrich at -50°C for 50 min-60 min; separate and remove the supernatant, then wash with 90%-95% acetonitrile and 1-2% TFA solution for 3-4 times; add 5-10 µL eluent, After elution at 37°C for 20-25 min, the glycopeptide was eluted, and the eluate was analyzed and detected by MALDI-TOF MS.