CN115041138A - Phosphorylated peptide adsorbent using nano diamond as substrate and preparation and application thereof - Google Patents

Abstract

The present invention relates to a reusable phosphorylated peptide adsorption material with nano-diamond as matrix and its preparation and application. Specifically, a nano-diamond with amino groups on the surface is used as the matrix, 3,4,5-trihydroxybenzaldehyde is used as a modifier, and an amino-aldehyde condensation reaction is used to introduce a pyrogallol group on the surface of the diamond particles, and the group is used as a ligand. It can be chelated with zirconium ion (Zr ⁴⁺ ), and the obtained material can be used as an adsorbent for immobilized metal ion affinity chromatography (IMAC) to enrich phosphopeptides in complex biological samples . Since the chelation force between Zr ⁴⁺ and biphloroglucinol is greater than that between it and the phosphate group, after the enrichment is completed, by using an appropriate alkaline eluent, the phosphopeptide can be removed from the adsorption. The chelated Zr ⁴⁺ on the sorbent is retained while eluting on the sorbent, so the sorbent can be used again after re-equilibration with an acidic solution.

Description

A kind of phosphorylated peptide adsorbent with nano-diamond as matrix and its preparation and application

technical field

The invention relates to a reusable Zr ⁴⁺ -IMAC adsorption material. Specifically, a nano-diamond with amino groups on the surface is used as a matrix, and 3, 4, 5-trihydroxybenzaldehyde and Zr ⁴⁺ are sequentially modified to prepare A reusable immobilized metal affinity chromatography (IMAC) adsorbent for separation and enrichment of phosphorylated peptides in complex samples.

Background technique

As a very important protein post-translational modification, phosphorylation modification is involved in almost all life activities, such as cell proliferation, differentiation, apoptosis and neural activities. Abnormal phosphorylation usually leads to the occurrence of certain diseases, such as malignant tumors, senile dementia, etc. Therefore, it is of great significance to study the process and mechanism of protein phosphorylation modification for the prevention, control and treatment of diseases (Literature 1, Humphrey S.J., et.al "High-throughput phosphoproteomics reveals in vivo insulin signaling dynamics" Nat. Biotechnol. 2015, 33, 990-995; Reference 2, Jiang, Y., et al. "Proteomics identifies new therapeutic targets of early-stage hepatocellular carcinoma". Nature 2019, 567, 258–261). The proteomic analysis strategy based on the "shotgun method" is the main method for the study of phosphorylated proteins at present. In this strategy, protein samples are first enzymatically digested into peptide fragments, enriched, and then subjected to tandem liquid-mass spectrometry analysis. Due to the extremely low abundance of phosphopeptides in the sample enzymatic hydrolysate, serious interference of non-phosphopeptides, and low ionization efficiency of phosphopeptides, high requirements for enrichment before mass spectrometry analysis are put forward. (Document 3, Lawrence R T, et al. "Plug-and-play analysis of the human phosphoproteome by targeted high-resolution massspectrometry". Nat. methods, 2016, 13, 431-434; Document 4, Zhang Chuanjing et al. "MALDI-TOF MS-based Application of tandem enrichment strategy in phosphorylated proteomics" Journal of Analytical Testing, 2017, 36, 1061-1068)

Immobilized metal ion affinity chromatography (IMAC) is a widely used method for enrichment of phosphopeptides. The method utilizes the immobilized metal ions on the adsorbent, such as Fe ³⁺ , Zr ⁴⁺ , Ti ⁴⁺ , which can chelate with the phosphate group in the phosphopeptide, thereby capturing the phosphopeptide in the enzymatic hydrolysis solution. (Document 5, Han Bin et al. "Research Progress on Immobilized Metal Ion Affinity Chromatography" Science and Technology Review, 2017, 35, 92-10). During the elution of phosphopeptides, traditional IMAC adsorbents may use strong alkaline solvents that may destroy the material composition, so most adsorbents are disposable and not reusable.

SUMMARY OF THE INVENTION

The present invention relates to a reusable Zr ⁴⁺ -IMAC adsorbent material. The material uses nano-diamonds with amino functional groups as the matrix and 3,4,5-trihydroxybenzaldehyde as the modifier. The amino-aldehyde condensation reaction is used to introduce the bipyloroglucinol group on the diamond surface to form a Schiff base. After sodium hydride is reduced, it is chelated with zirconium ions to obtain an affinity chromatography adsorption material for enriching phosphopeptides. After the phosphopeptide has been adsorbed, it can be eluted from the sorbent using a basic eluent and then equilibrated with an acidic solution, and the sorbent can be used again to enrich the phosphopeptide from the sample solution.

Its structural schematic is as follows:

The specific preparation process of Zr ⁴⁺ -IMAC adsorption material includes the following steps:

First, 100-300 mg of nano-diamonds were dispersed in 10-20 mL of ethanol solution of 3,4,5-trihydroxybenzaldehyde with a concentration of 0.02-0.05 g/mL in a container, under the condition of 60-80 ℃, with Magnetic stirring at a rate of 200-300 rpm, heating to reflux for 4-12 h, the product was washed with ethanol; then the product was dispersed in 10-20 mL of aqueous sodium borohydride solution with a concentration of 0.005-0.02 g/mL, at room temperature at a rate of 200-300 rpm Magnetic stirring for 2 to 10 hours; after the reaction, the material was washed with water until neutral to obtain bipyralol-functionalized nano-diamond particles;

Disperse the above materials once again in 10-20 mL of zirconium chloride (ZrCl ₄ ) solution with a concentration of 0.5-1.0 g/mL, at room temperature at a frequency of 100-150 rpm, shake and incubate the reaction for 4-8 h, and wash with water after the reaction is complete to neutrality, and vacuum drying at 50-80°C for 6-12 hours to obtain Zr ⁴⁺ -IMAC functional material.

The present invention is characterized in that:

(1) Zr ⁴⁺ -IMAC functional material is prepared by using nanodiamond with amino group as matrix, pyrogallol as ligand, and Zr ⁴⁺ as chelated metal ion;

(2) The material can be used as an adsorbent for immobilized metal ion affinity chromatography to enrich phosphorylated peptides in biological samples with high selectivity;

(3) The material can be reused as a phosphopeptide enrichment material.

In traditional IMAC adsorbents with phosphate groups as ligands, metal ions will fall off the adsorbent along with the phosphopeptides in the alkaline eluent, so they can only be used once. However, in the IMAC adsorbent prepared by the present invention, due to the strong chelating force between the ligand and the metal ion, the metal ion will not dissociate from the surface of the adsorbent in the alkaline eluent. Therefore, after elution of the phosphopeptide, the material can be used again after re-equilibration with an acidic solution.

The preparation method of the invention has mild reaction conditions, simple operation steps, and the obtained Zr ⁴⁺ -IMAC functional material has good enrichment effect on phosphopeptides, strong selectivity, can be reused, is green and environmentally friendly, and is processed before the sample. showed a good application prospect.

Description of drawings

Figure 1. Schematic diagram of (A) preparation and (B) use of Zr ⁴⁺ -IMAC materials in the examples.

Figure 2 TEM image of the nanodiamonds used in the examples.

Figure 3. X-diffraction photoelectron spectrum of the Zr ⁴⁺ -IMAC-1 material in Example 1. (A) Full spectrum, (B) Zr 3d high-resolution spectrum.

Figure 4. MALDI-TOF mass spectrometry analysis of β-casein hydrolysate in Example 1. (A) Direct detection, (B) Zr ⁴⁺ -IMAC-1 material after first enrichment. (*) represents phosphopeptides, (·) represents dephosphorylated fragments.

Figure 5. In Example 1, the Zr ⁴⁺ -IMAC-1 material was repeatedly used (A) the signal intensity change curve of the three characteristic phosphopeptides enriched in the β-casein hydrolyzate, (B) 10 enrichment MALDI-TOF mass spectrometry after collection. (*) represents phosphopeptides, (·) represents dephosphorylated fragments.

Figure 6. The mixed enzymatic hydrolysis solution of β-casein and bovine serum albumin (1/100, mass ratio) in Example 1 was enriched by Zr ⁴⁺ -IMAC-1 material (A) before and (B) after enrichment Comparison of MALDI-TOF mass spectrometry analysis. (*) represents phosphopeptides, (·) represents dephosphorylated fragments.

Figure 7. In Example 2, (A) MALDI-TOF mass spectrometry analysis of the β-casein hydrolyzate after the first enrichment by Zr ⁴⁺ -IMAC-2 material, (B) during the 10 enrichment process, three characteristic phosphate Graph of changes in signal intensity of peptides. (*) represents phosphopeptides, (·) represents dephosphorylated fragments.

Figure 8. In the comparative example, the MALDI-TOF mass spectrometry analysis of the β-casein hydrolyzate after enrichment with the commercial SPE-Ti-IMAC material. (A) First enrichment. (B) Second enrichment. (*) represents phosphopeptides, (·) represents dephosphorylated fragments.

Detailed ways

Example 1 Zr ⁴⁺ -IMAC-1 functional materials prepared based on nanodiamonds are used for separation and enrichment of phosphopeptides

Preparation of Zr ⁴⁺ -IMAC-1 functional material: 100 mg of nanodiamonds (particle size range: 2-40 nm) were dispersed in 10 mL of 3,4,5-trihydroxybenzaldehyde with a concentration of 0.02 g/mL in a round-bottomed flask In the ethanol solution of the product, in an oil bath of 70 °C, magnetic stirring at a rate of 250 rpm, heating to reflux for 8 h, the product was washed with ethanol for 3 times; Magnetic stirring was carried out at a rate of 250 rpm for 4 h; after the reaction, the material was washed with water to neutrality to obtain bipyralol-functionalized nano-diamond particles;

Disperse the above materials again in 10 mL of zirconium chloride (ZrCL ₄ ) solution with a concentration of 0.5 g/mL, incubate and shake for 8 h at a frequency of 150 rpm at room temperature, and wash with deionized water after the reaction to neutrality, 60 Vacuum drying at ℃ for 12 h to obtain Zr ⁴⁺ -IMAC functional material.

Preparation of enzymatic hydrolysis samples: First, 1 mg of β-casein/bovine serum albumin was dissolved in 100 mM ammonium bicarbonate aqueous solution containing 8 M urea, and the pH was adjusted to 8.2. Then, 80 μmol of dithiothreitol was added, and the mixture was heated in a constant temperature water bath at 60 °C for 1 h. Then, 40 μmol of iodoacetamide was added, and the reaction was performed in the dark for 40 min. Next, after diluting the urea concentration to 1 M with a 100 mM ammonium bicarbonate aqueous solution, trypsin was added at a ratio of protein to trypsin mass ratio of 25:1. The mixture was incubated in a 37°C water bath for 16h. Finally, the obtained enzymolysis solution was desalted with a C18 SPE column, freeze-dried and stored in a refrigerator at -20°C for future use.

Enrichment of phosphopeptides: First, 5 mg of Zr ⁴⁺ -IMAC-1 material was weighed into a centrifuge tube and equilibrated with a loading solution (80% ACN, 6% TFA). The lyophilized proteolysis solution was then reconstituted with 200 μL of loading solution and transferred to a centrifuge tube containing Zr ⁴⁺ -IMAC material. After shaking at room temperature for 30 min, the supernatant solution was removed by centrifugation at 14,000 rpm. The material was rinsed three times with washing solution A (200 mM NaCl, 50% ACN, 6% TFA) to remove non-specifically adsorbed peptides, and then washed three times with washing solution B (30% ACN, 0.1% TFA) to remove the material contained in the material. Salt, shake for 15min each time. Finally, the phosphopeptide adsorbed on the material was eluted with 100 μL of 8% ammonia solution by mass. The eluate was analyzed by MALDI-TOF MS.

MALDI-TOF MS analysis process: First, drop 0.5 μL of the eluate on the MADLI target and let it dry naturally. Then, 0.5 μL of 2,5-dihydroxybenzoic acid solution with a concentration of 25 mg/mL was covered on the sample point, and after it was completely dried, it was sent to the mass spectrometer for analysis.

Example 2 Zr ⁴⁺ -IMAC-2 functional materials prepared based on nanodiamonds are used for separation and enrichment of phosphopeptides

Preparation of Zr ⁴⁺ -IMAC-2 functional material: 100 mg of nanodiamonds (particle size range: 2-40 nm) were dispersed in 3.3 mL of 3,4,5-trihydroxybenzene with a concentration of 0.02 g/mL in a round-bottomed flask In an ethanol solution of formaldehyde, in an oil bath at 70 °C, magnetic stirring at a rate of 250 rpm, heating to reflux for 8 h, the product was washed three times with ethanol; magnetic stirring at a rate of 250 rpm for 4 h; after the reaction, the material was washed with water until neutral to obtain bipyralol-functionalized nano-diamond particles;

The modification and application of bipyralol-functionalized nanodiamond particles are the same as in Example 1

Comparative Example Commercial IMAC Microspheres for Separation and Enrichment of Phosphorylated Peptides

Commercially available IMAC microsphere material chelated titanium: according to the manufacturer's instructions, take 1.0g of commercial IMAC microspheres (trade name: SPE-Ti-IMAC, purchased from Bailingwei Technology Co., Ltd.), and add 100g of it at a ratio of 1:100. Titanium sulfate was chelated in an aqueous solution for 12 h, followed by washing the unchelated titanium ions on the microspheres with 10 mL of 0.1% TFA, 200 mM NaCl aqueous solution and deionized water, and vacuum drying at 50 °C for 6 h to obtain a commercial Ti-IMAC material .

Commercial Ti-IMAC material enriches phosphorylated peptides: The process of phosphorylated peptide enrichment and mass spectrometry analysis is the same as that in Example 1.

Material Synthesis and Characterization:

Figure 1 is a schematic diagram of the preparation of Zr ⁴⁺ -IMAC materials in the examples. The surface of the nano-diamond has a large number of amino groups, which can undergo amino-aldehyde condensation reaction with the aldehyde group in the monomer 3,4,5-trihydroxybenzaldehyde, and introduce the bipyrogallol group in the monomer into the surface of the nanoparticle. This group can act as a ligand to chelate with zirconium ions and chelate zirconium ions on the surface of nanoparticles. Utilizing the chelation of zirconium ions with phosphate groups in phosphopeptides, the nanoparticles can be used as adsorbents for IMAC to enrich phosphopeptides in the sample solution.

FIG. 2 is a transmission electron microscope image of the nanodiamond used in the examples. The material is amorphous particles, most of which are below 20 nm in size.

3A is a full spectrum of the X-diffraction photoelectron spectrum of the Zr ⁴⁺ -IMAC-1 material in Example 1, and FIG. 3B is a high-resolution spectrum of Zr 3d. The spectrum shows that the material contains C, O, N and Zr elements, which confirms the synthetic route. Zirconium ions were successfully chelated on the surface of Zr ⁴⁺ -IMAC material.

Material application

Since the chelation force between the Zr ⁴⁺ ion and the pyrogallol is greater than the chelation force between it and the phosphate group, after the enrichment is completed, only the phosphopeptide can be removed from the adsorbent by the alkaline eluent. Elution without dissociating Zr ⁴⁺ from the adsorbent, so after the elution of the phosphopeptide is completed, it is re-equilibrated with an acidic solution, and the adsorbent material can be used again.

FIG. 4 is a comparison diagram before and after the first enrichment of the β-casein hydrolyzate in Example 1. FIG. Before enrichment (Fig. 4A), the three characteristic phosphopeptides (2061, 2556, 3122, m/z) were difficult to detect due to their low abundance and severe signal interference. However, after enrichment with Zr ⁴⁺ -IMAC-1, the non-glycopeptide signal peaks basically disappeared, and three characteristic phosphopeptides and their dephosphorylated fragment peaks (1963, 2458, 3024, 2926, m/z) occupied the upper part of the spectrum. main location. This indicates that the material has a good enrichment ability for phosphopeptides.

In Example 1, in order to investigate the reusability of the Zr ⁴⁺ -IMAC-1 material, a 10-round cyclic enrichment experiment was performed. 10 mg of Zr ⁴⁺ -IMAC material was used as the adsorbent for recycling, and 10 μg of β-casein hydrolysate was enriched each time. After each enrichment process is completed, the phosphopeptide adsorbed on the material is eluted with 8% aqueous ammonia solution for mass spectrometry detection, and the adsorbent is then acidified and equilibrated with an acidic loading solution to enter the next round of phosphopeptide enrichment. Experiment, 10 cycles of experiments were performed in this way. The results are shown in Figure 5. Figure 5A shows that after the Zr ⁴⁺ -IMAC-1 material was reused ten times, the signal intensities of the three characteristic phosphopeptides enriched from the β-casein hydrolyzate had no obvious downward trend, indicating that the material was adsorbed as IMAC The agent has good reusability.

In Example 1, in order to investigate the specificity of the Zr ⁴⁺ -IMAC-1 material, we used the β-casein/bovine serum albumin mixed enzymatic hydrolyzate with a mass ratio of 1/100 as a sample, and carried out phosphopeptide enrichment. . Before enrichment (Fig. 6A), the detected peptides were almost all non-phosphopeptides, but after enrichment (Fig. 6B), no obvious signal of non-phosphopeptide appeared, and the obvious signal peaks in the spectrum were all attributed to phosphopeptides and their phosphopeptides. Dephosphorylation fragments. This indicates that the Zr ⁴⁺ -IMAC-1 material has high specificity for phosphopeptides.

In order to investigate the effect of the ligand 3,4,5-trihydroxybenzaldehyde on the enrichment performance of Zr ⁴⁺ -IMAC functional materials, in the preparation process of the material in Example 2, the amount of the ligand added was changed to one-third of that in Example 1. about one, and the rest of the conditions are the same as those in Example 1. The obtained Zr ⁴⁺ -IMAC-2 functional material was also subjected to 10 rounds of cyclic enrichment experiments using β-casein hydrolyzate as a sample, and the results are shown in FIG. 7 . Figure 7A shows that after the first round of enrichment, the non-phosphopeptide signal values in the mass spectrogram also basically disappeared, and the obvious signal peaks in the figure are all phosphopeptide signal peaks and their dephosphorylated fragment signal peaks, indicating that Zr ⁴⁺ -IMAC- 2 The material is highly selective for phosphopeptides. However, compared with the Zr ⁴⁺ -IMAC-1 prepared in Example 1, the signal peak intensity of the phosphopeptide generally decreased, which may be due to the ligand 3,4,5-trihydroxybenzaldehyde during the preparation of the material The reduction of the amount of used leads to the corresponding reduction of the sites available for Zr ⁴⁺ chelation, so the enrichment ability of the material decreases. However, similar to Example 1, after 10 rounds of enrichment, the peak intensities of the three characteristic phosphopeptides did not have a significant downward trend (Fig. 7B), indicating that the Zr ⁴⁺ -IMAC-2 material has excellent reusability.

In order to compare with the enrichment performance of Zr ⁴⁺ -IMAC material, in the comparative example, commercial SPE-Ti-IMAC was also used as IMAC adsorbent to enrich phosphopeptides. Figure 8A shows that although SPE-Ti-IMAC can also enrich three characteristic phosphopeptides from β-casein hydrolysate, the signal peaks of non-phosphopeptides cannot be completely ignored. After the phosphopeptide was eluted from the material, even if it was washed and equilibrated with an acidic solution, the material lost the ability to enrich the phosphopeptide, and the signal of the phosphopeptide could not be detected in the eluate (Fig. 8B), and the material could not be reused . The reason may be that during the elution of phosphopeptides, the alkaline ammonia eluent can not only destroy the chelation of phosphate groups in phosphopeptides and Ti ⁴⁺ , but also destroy the complexation between Ti ⁴⁺ and phosphoric acid on the adsorbent. At the same time, the chelation between the phosphopeptides and the adsorbent dissociates from the adsorbent, and the Ti ⁴⁺ immobilized on the surface of the adsorbent is also dissociated from the adsorbent. The above results show that the Zr ⁴⁺ -IMAC material prepared with nanodiamond as the matrix, bipyralol as the ligand, and zirconium (IV) as the chelated metal ion is more effective than the commercial SPE-Ti-IMAC in the complex sample. Phosphopeptides have higher enrichment efficiency and specificity, and the material can be reused, while commercial materials can only be used once.

Claims (5)

1. A phosphorylated peptide adsorbent using nano-diamond as a substrate is prepared by taking the nano-diamond with amino functional groups as the substrate and 3,4, 5-trihydroxybenzaldehyde as a modifier, introducing biphenyl triphenol groups on the surface of the diamond by adopting an ammonia-aldehyde condensation reaction to form Schiff bases, and chelating the Schiff bases with zirconium ions after reduction to obtain the reusable Zr ⁴⁺ -an IMAC adsorbent.

2. Zr according to claim 1 ⁴⁺ -a process for the preparation of an IMAC adsorbent, characterized in that:

nano diamond with amino functional groups is taken as a substrate (the particle size range is 2-40 nm);

3,4, 5-trihydroxy benzaldehyde is used as a modifier, ammonia-aldehyde condensation reaction is utilized to form Schiff base, and after reduction, a biphenyl triphenol functional group is introduced on the surface of diamond particles;

zirconium chloride is used as a metal ion source, zirconium ions and biphenyltriphenolChelating ligand to prepare Zr ⁴⁺ IMAC functional materials.

3. The production method according to claim 2, characterized in that: the method can be operated according to the following steps,

dispersing 100-300 mg of nano-diamond in 10-20 mL of ethanol solution of 3,4, 5-trihydroxybenzaldehyde with the concentration of 0.02-0.05 g/mL in a container, magnetically stirring at the speed of 200-300 rpm under the condition of 60-80 ℃, heating and refluxing for 4-12 h, and washing a product with ethanol; then dispersing the product in 10-20 mL of 0.005-0.02 g/mL sodium borohydride aqueous solution, and magnetically stirring at the speed of 200-300 rpm for 2-10 h at room temperature; after the reaction is finished, washing the material to be neutral by using water to obtain the biphenyltriphenol functionalized nano-diamond particles;

dispersing the above materials in 10-20 mL of zirconium chloride (ZrCl) with concentration of 0.5-1.0 g/mL ₄ ) In the solution, oscillating and incubating for 4-8 h at the room temperature at the frequency of 100-150 rpm, washing the solution to be neutral by water after the reaction is finished, and drying the solution in vacuum for 6-12 h at the temperature of 50-80 ℃ to obtain Zr ⁴⁺ IMAC functional materials.

4. Zr according to claim 1 ⁴⁺ -use of an IMAC functional material characterized in that:

can be used as adsorbent for immobilized metal ion affinity chromatography to enrich phosphorylated peptide in biological sample.

5. Use according to claim 4, characterized in that: the biological sample is one or more of body fluid, tissue and cell enzymolysis liquid of human body and/or animal.

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