CN107727480B - Ultrathin lamellar functionalized molybdenum disulfide nano composite material and application thereof in glycopeptide enrichment - Google Patents
Ultrathin lamellar functionalized molybdenum disulfide nano composite material and application thereof in glycopeptide enrichment Download PDFInfo
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Abstract
The invention discloses an ultrathin lamellar functionalized molybdenum disulfide nano composite material and application thereof in glycopeptide enrichment. The glycopeptide enrichment material takes an ultrathin two-dimensional lamellar structure molybdenum disulfide nanomaterial as a matrix, and after nanogold is modified on the surface, a glycopeptide enrichment functional group containing sulfydryl is loaded through a simple and stable Au-S bond chemical reaction. Experimental results show that the functional molybdenum disulfide-nano noble metal composite material provided by the invention realizes high-selectivity enrichment of the functional material on glycopeptides through hydrophilic interaction with the glycopeptides or through reversible reaction between boric acid groups loaded on the surface of the material and sugar chains of the glycopeptides. The functionalized molybdenum disulfide-nano noble metal composite material has the advantages of simple preparation process and mild reaction conditions.
Description
Technical Field
The invention belongs to the field of materials, and relates to an ultrathin lamellar functionalized molybdenum disulfide nano composite material and application thereof in glycopeptide enrichment.
Background
Glycosylation modification of proteins is one of the most common and complex post-translational modifications of proteins. It can endow precursor protein with some new functions, such as specificity and stability of its combination. Glycoproteins are involved in many important functions of living cells, such as cell recognition, cell differentiation, signal transduction, and immune response. Glycoproteins are now used as biomarkers for cancer diagnosis and monitoring. Thus, deep coverage and accurate identification of glycoproteins is crucial for a thorough study of various functions of proteins.
The current glycoproteomics strategy based on the biological mass spectrometry technology becomes a very common research means. However, since the abundance of glycosylated proteins is very low and the dynamic range is wide, and proteins subjected to glycosylation modification are not easily ionized to form molecular ions and are inhibited by non-glycopeptides during mass spectrometry, the glycosylated proteins cannot be detected, so that the research on the glycosylated proteins still faces more challenges.
In order to solve the problem, the existing research method is to firstly carry out enzymolysis on glycosylated protein into glycosylated peptide fragments and then carry out identification. However, because a large number of non-glycosylated peptide sections exist in the enzyme digestion product, the enzyme digestion product has a strong inhibiting effect on response signals of the glycosylated peptide sections in mass spectrum detection. Therefore, mass spectrometry analysis after glycopeptide enrichment is necessary in the sample treatment process.
A number of different glycopeptide enrichment strategies have been developed, including lectin affinity chromatography, boronic acid chemistry, hydrophilic interaction chromatography and hydrazine chemistry. The lectin affinity chromatography is the glycoprotein and glycopeptide enrichment method which is widely applied at present, but has very good effect on specific glycoform glycopeptide/glycoprotein, and is lack of universality. The hydrazine chemical method is based on oxidizing ortho-dihydroxy on a sugar chain into aldehyde group by using sodium periodate, and then covalently bonding an acyl functional material with the aldehyde group, so that glycopeptide/glycoprotein is enriched, and therefore, the method has high enrichment selectivity, but sugar chain structures are difficult to release due to the generation of covalent bonds, and glycoform analysis on the enriched glycoprotein and glycopeptide cannot be realized.
Disclosure of Invention
The invention aims to provide an ultrathin lamellar functionalized molybdenum disulfide nano material and application thereof in glycopeptide enrichment.
The functionalized molybdenum disulfide-nano noble metal composite material provided by the invention is composed of molybdenum disulfide nanosheets, nano noble metals and functional molecules;
the functional molecule is glycopeptide enrichment functional molecule containing sulfhydryl; that is, the functional molecule contains sulfhydryl and also has the function of glycopeptide enrichment.
The nano noble metal is fixed on the surface of the molybdenum disulfide nanosheet through a coordination bond;
the functional molecule is connected with the nano noble metal through sulfydryl.
In the functionalized molybdenum disulfide-nano noble metal composite material, the longest side length of the molybdenum disulfide nanosheet is 50-500 nm; the thickness is 0.69 nm;
the nano noble metal is nano gold, nano silver or nano platinum;
the nano noble metal is in the form of nano particles or nano wires;
the particle size of the nano particles is 2-20 nm; the length of the nanowire is 2-5 nm;
the functional molecule is L-cysteine or mercapto-phenylboronic acid.
The dosage ratio of the molybdenum disulfide, the noble metal and the functional molecule is 10 mg: 1.5-5 mg: 40-100 mg.
The structure of the composite material is schematically shown in figure 1.
The invention provides two methods for preparing the functionalized molybdenum disulfide-nano noble metal composite material, wherein the first method is based on a boric acid chemical method and comprises the following steps:
the molybdenum disulfide nanosheets and the nano noble metal are connected through coordination bonds to obtain molybdenum disulfide-noble metal, then the molybdenum disulfide-noble metal and functional molecules are dispersed in a solvent, the mixture is stirred and uniformly mixed to carry out a sulfur-noble metal reaction, and after the reaction is finished, the precipitate is centrifugally collected, so that the functionalized molybdenum disulfide-nano noble metal composite material is obtained;
the functional molecule is a molecule containing sulfydryl and having glycopeptide enrichment function.
In the step of the sulfur-noble metal reaction, the temperature is 10-60 ℃, in particular to room temperature; the time is 12-24 h;
the solvent is absolute ethyl alcohol;
the dosage ratio of the molybdenum disulfide-noble metal, the functional molecule and the solvent is 5-20 mg: 30-100 mg: 2-5 mL;
in the centrifugation step, the centrifugal force is 1000-10000g, in particular 4500 g; the time is 5-30 minutes, specifically 15 minutes.
The second method is based on the principle of hydrophilic enrichment and comprises the following steps:
the molybdenum disulfide nanosheets and the nano noble metal are connected through coordination bonds to obtain molybdenum disulfide-noble metal, the molybdenum disulfide-noble metal is stirred with an ethanol water solution of functional molecules, precipitates are collected centrifugally, and the precipitates are sequentially washed with ethanol and water and then taken to obtain the molybdenum disulfide-noble metal;
the functional molecule is a molecule containing sulfydryl and having glycopeptide enrichment function.
In the ethanol aqueous solution of the functional molecules of the method, the volume percentage concentration of the ethanol aqueous solution is 50-80 percent, and is specifically 75 percent;
the dosage ratio of the functional molecules to the ethanol water solution is 40-100 mg: 4mL, specifically 100 mg: 4 mL;
in the stirring step, the time is 12-24 hours;
in the centrifugation step, the centrifugal force is 1000-10000g, in particular 4500 g; the time is 5-30 minutes, in particular 15 minutes;
in the washing step, the times of washing with ethanol and water are 3-10 times, specifically 3 times.
The schematic diagram of the two methods is shown in fig. 2.
In the two methods, the molybdenum disulfide nanosheet and the nano noble metal are connected through a coordination bond, and can be prepared according to various conventional methods, for example, the method can be realized through a method comprising the following steps:
mixing the molybdenum disulfide normal hexane suspension with the precious metal salt and oleylamine normal hexane solution, carrying out ultrasonic treatment, adding a reducing agent, uniformly mixing, standing at room temperature, adding absolute ethyl alcohol, carrying out centrifugation to collect precipitate, adding normal hexane for dissolving, adding absolute ethyl alcohol, uniformly mixing, and carrying out centrifugation to collect precipitate;
specifically, the noble metal salt is gold tetrachloride trihydrate, silver nitrate or potassium tetrachloroplatinate;
the reducing agent is Triisopropylsilane (TIPS);
in the molybdenum disulfide normal hexane suspension, the dosage ratio of molybdenum disulfide to normal hexane is 10-50 mg: 1-5 mL;
in the n-hexane solution of the noble metal salt and the oleylamine, the dosage ratio of the noble metal salt to the oleylamine to the n-hexane is 3-15 mg: 100-500 μ L: 2-8 mL, specifically 3 mg: 100 μ L of: 1.5 mL;
the dosage ratio of the reducing agent to the molybdenum disulfide is 100-500 mu L: 10-50 mg, specifically 150 μ L: 10 mg;
in the standing step, the time is 4-5 hours;
in the centrifugation step, the centrifugal force is 1000-10000g, in particular 4500 g; the time is 5-30 minutes, specifically 15 minutes.
In addition, the application of the functionalized molybdenum disulfide-nano noble metal composite material provided by the invention in enrichment and/or detection of glycosylated peptide segments also belongs to the protection scope of the invention.
The invention also provides a method for enriching glycopeptides, which comprises the following steps:
1) washing the functionalized molybdenum disulfide-nano noble metal composite material by using an enrichment buffer solution, then carrying out co-shaking incubation on a glycopeptide sample to be separated and the functionalized molybdenum disulfide-nano noble metal composite material in the enrichment buffer solution, and centrifugally collecting precipitates;
2) washing the precipitate obtained in the step 1) by using an enrichment buffer solution, placing the precipitate in an elution buffer solution, oscillating and incubating, centrifuging and collecting supernatant, thus completing the enrichment of the glycopeptide sample to be separated.
In the incubation step in the step 1) of the method, the time is 30-60 minutes; step 2), in the incubation step, the time is 10-30 minutes; the incubation temperature in the steps 1) and 2) is room temperature;
when the functionalized molybdenum disulfide-nanogold composite material is molybdenum disulfide-nanogold-L-cysteine, the volume ratio of the enrichment buffer solution is 86:13.9:0.1 of acetonitrile, water and trifluoroacetic acid;
the elution buffer solution is prepared by mixing a solution prepared from 30:69:1 of acetonitrile, water and trifluoroacetic acid;
when the functionalized molybdenum disulfide-nanogold composite material is molybdenum disulfide-nanogold-mercaptophenylboronic acid, the enrichment buffer solution is 50mM ammonium bicarbonate aqueous solution;
the elution buffer solution is prepared by mixing 50:49:1 of acetonitrile, water and trifluoroacetic acid;
the glycopeptide sample to be separated can be specifically an IgG trypsin enzyme digestion peptide segment.
A schematic of this enrichment process is shown in figure 3.
The invention is based on that a molybdenum disulfide nanometer material with a two-dimensional ultrathin sheet layer structure is used as a substrate, precious metals such as nanogold and the like are loaded on the surface of the molybdenum disulfide nanometer material, finally, a group which contains sulfydryl and has a glycopeptide enrichment function is grafted in one step, and the nanometer material which contains sulfydryl and has the glycopeptide enrichment function is prepared through an Au-S bond.
The invention has the advantages that:
1) the specific surface area of the material is improved by the ultrathin sheet layer of molybdenum disulfide, the active sulfur atoms exposed on the surface of the ultrathin sheet layer can be reduced in situ to generate nanogold, the defect that the nanogold is easy to agglomerate is overcome, a stable Au-S bond can be generated through a chemical reaction, a large number of functional groups are loaded on the novel ultrathin two-dimensional nanosheet material, and the material synthesis method is simple, easy, clean and rapid.
2) The selective enrichment of glycopeptides is achieved by hydrophilic interaction with the glycopeptides.
3) The selective enrichment of glycopeptides is realized by forming a stable and reversible covalent bond between the boronic acid group on the surface and the glycopeptide.
4) Can realize the high-sensitivity enrichment detection of the glycopeptide with low concentration, and can detect the glycopeptide with the lowest detectable concentration of 0.1 fmol/mu L.
Drawings
FIG. 1 is a characteristic structure diagram of a functionalized molybdenum disulfide-nano gold composite material.
FIG. 2 is a schematic diagram of the synthesis of a functionalized molybdenum disulfide-nanogold composite material. (a) Based on the principle of hydrophilic enrichment, (b) based on the boric acid chemistry.
Fig. 3 is a schematic view of glycopeptide enrichment of the functionalized molybdenum disulfide-nanogold composite material.
FIG. 4 is a MALDI-TOF mass spectrum of example 1. (a) Directly analyzing IgG trypsin enzyme digestion peptide (6.7pmol), (b) enriching the IgG trypsin enzyme digestion peptide (6.7pmol) of the functionalized molybdenum disulfide-nano gold composite material
FIG. 5 is a MALDI-TOF mass spectrum in example 2. The IgG trypsin enzyme digestion peptide fragment after the enrichment of the functionalized molybdenum disulfide-nanogold composite material: (a)0.67 pmol; (b)20 fmol; (c)10 fmol.
FIG. 6 is a MALDI-TOF mass spectrum of example 3, (a) peptide fragment cleaved by trypsin with IgG (IgG: BSA,1:200, w/w,2 μ g) after enrichment of functionalized molybdenum disulfide-nanogold composite; (b) and (3) carrying out enzyme digestion on the enriched BSA and IgG trypsin peptide fragment (IgG: BSA,1:500, w/w,2 mu g) of the functionalized molybdenum disulfide-nanogold composite material.
FIG. 7 is a MALDI-TOF mass spectrum of example 4, (a) direct analysis of HRP trypsin cleaved peptide (6.7 pmol); (b) and (3) digesting the peptide fragment (6.7pmol) by using HRP trypsin after the enrichment of the functionalized molybdenum disulfide-nanogold composite material.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples, but the present invention is not limited to the following examples. The method is a conventional method unless otherwise specified. The starting materials are commercially available from the open literature unless otherwise specified.
Example 1, the enrichment and detection of glycopeptides by a functionalized molybdenum disulfide-nanogold composite material based on a hydrophilic enrichment principle includes the following steps:
firstly, preparing molybdenum disulfide-nano gold:
(1) 10mg of molybdenum disulfide powder was ultrasonically dispersed in 1mL of n-hexane.
(2) 3mg of gold tetrachloride trihydrate and 100. mu.L of oleylamine were dissolved in 1.5mL of n-hexane.
(3) And (3) mixing the product obtained in the step (1) with the product obtained in the step (2) and then carrying out ultrasonic dispersion.
(4) To the product mixture obtained in step (3), 150. mu.L of Triisopropylsilane (TIPS) was added, mixed well, and then allowed to stand for 4 hours.
(5) And (4) adding absolute ethyl alcohol into the product obtained in the step (4), uniformly mixing, and centrifuging at 4,500g for 15 minutes.
(6) And (3) taking the precipitate obtained in the step (5), adding n-hexane to dissolve the precipitate, then adding absolute ethyl alcohol, uniformly mixing, and centrifuging at 4,500g for 15 minutes.
(7) And (5) repeating the step (6) for 2 times, and taking the precipitate as molybdenum disulfide-nanogold.
Secondly, preparing a functionalized molybdenum disulfide-nano gold composite material:
(8) 100 mgL-cysteine was dissolved in 4mL of 75% (v/v) ethanol aqueous solution.
(9) And (4) mixing the molybdenum disulfide-nano gold obtained in the step (7) with the solution obtained in the step (8). The mixture was stirred and mixed for 24 hours at room temperature using a mixer.
(10) The product obtained in step (9) was centrifuged at 4,500g for 15 minutes and the supernatant was discarded to take out the precipitate. And washing the obtained product with ethanol and water for 3 times in sequence, and taking the precipitate to obtain the functionalized molybdenum disulfide-nano noble metal composite material provided by the invention.
Thirdly, enriching glycopeptides:
(11) weighing 1mg of the material obtained in the step (10), shaking and dispersing in 1mL of enrichment buffer (acetonitrile/water/trifluoroacetic acid, 86:13.9:0.1, v/v/v), taking 20 μ L of the suspension, centrifuging, taking the lower layer precipitate, and washing 3 times with the enrichment buffer.
(12) Groups 2 (denoted as groups 1 and 2) each containing 100. mu.L of enrichment buffer (acetonitrile/water/trifluoroacetic acid, 86:13.9:0.1, v/v/v) of IgG tryptic peptide were taken, and the IgG tryptic peptide in each group was 6.7 pmol.
(13) After suspending the precipitate obtained in step (11) in the group 1 solution in step (12), shaking and incubating at room temperature for 30 min. Then, the liquid was discarded by centrifugation, washed thoroughly with 100. mu.L of enrichment buffer, and repeated 3 times, and the precipitate was retained.
(14) The pellet obtained in step (13) was suspended in 20. mu.L of elution buffer (acetonitrile/water/trifluoroacetic acid, 30:69:1, v/v/v), incubated with shaking at room temperature for 10min, and the supernatant was collected by centrifugation. Meanwhile, the resulting pellet was resuspended in 20. mu.L of elution buffer (acetonitrile/water/trifluoroacetic acid, 30:69:1, v/v/v), incubated at room temperature with shaking for 10min, and after centrifugation, the supernatants were collected and combined.
Fourthly, detection of glycopeptides:
mu.L of the collected supernatant was dropped onto a MALDI target plate, and after it was naturally air-dried, a certain amount (1. mu.L) of DHB matrix (25mg/mL2, 5-dimethylbenzoic acid, acetonitrile/water/phosphoric acid, 70:29:1, v/v/v) was dropped, and after it was volatilized and crystallized, MALDI-TOF MS analysis was performed.
Meanwhile, a certain amount (1 μ L) of the group 2 solution in the step (12) is dropped on a MALDI target plate, after the solution is naturally air-dried, a certain amount (1 μ L) of DHB matrix (25mg/mL of 2, 5-dimethylbenzoic acid, acetonitrile/water/phosphoric acid, 70:29:1, v/v/v) is dropped, after the solution is volatilized and crystallized, MALDI-TOF MS analysis is carried out as a blank control.
The result is shown in figure 4, and the mass spectrogram result of the blank control sample before enrichment and the supernatant after enrichment is compared, so that the functionalized molybdenum disulfide-nano gold composite material has specific enrichment performance on glycopeptide in the peptide fragment digested by IgG trypsin. As a result, in the case of not enriching the sample, only 5 glycopeptides can be identified on the MALDI-TOF MS spectrum, and a large amount of high-abundance non-glycopeptide signals appear in the molecular weight range of 1000-2000. And under the condition that the functionalized molybdenum disulfide-nano noble metal composite material provided by the invention is used for enriching glycopeptides of a sample, 32 glycopeptides can be identified on a MALDI-TOF MS spectrum, and a large amount of high-abundance non-glycopeptide signals with the molecular weight of 1000-2000 are removed. Therefore, the result shows that the prepared functionalized molybdenum disulfide-nanogold composite material has excellent performance on enrichment of glycosylated peptide segments.
Example 2, the detection of glycopeptide enrichment sensitivity of a functionalized molybdenum disulfide-nanogold composite material based on a hydrophilic enrichment principle includes the following steps:
firstly, enriching glycopeptides:
(11) weighing 1mg of the functionalized molybdenum disulfide-nano noble metal composite material obtained in example 1, shaking and dispersing in 1mL of enrichment buffer (acetonitrile/water/trifluoroacetic acid, 86:13.9:0.1, v/v/v), taking 20 mu L of the suspension, centrifuging, taking the lower-layer precipitate, and washing for 3 times by using the enrichment buffer.
(12) Groups 3 (designated as groups 1, 2 and 3) each contained 100. mu.L of enrichment buffer (acetonitrile/water/trifluoroacetic acid, 86:13.9:0.1, v/v/v) of IgG tryptic peptide fragments, wherein the IgG tryptic peptide fragment content in groups 1, 2 and 3 was 6.7pmol,20fmol and 10fmol, respectively.
(13) Suspending the precipitate obtained in the step (11) in the solutions of the group 1, the group 2 and the group 3 in the step (12), and then incubating the suspension at room temperature with shaking for 30 min. Then, the liquid was discarded by centrifugation, washed thoroughly with 100. mu.L of enrichment buffer, and repeated 3 times, and the precipitate was retained.
(14) The precipitates obtained in step (13) were suspended in 20. mu.L of each elution buffer (acetonitrile/water/trifluoroacetic acid, 30:69:1, v/v/v), incubated at room temperature with shaking for 10min, and then centrifuged to collect the supernatant as an eluate. Meanwhile, the obtained precipitate was resuspended in 20. mu.L of elution buffer (acetonitrile/water/trifluoroacetic acid, 30:69:1, v/v/v), incubated at room temperature with shaking for 10min, centrifuged to collect the supernatant as an eluate, and the above eluates were combined.
II, detection of glycopeptides:
mu.L of each of the above-collected supernatants was dropped on a MALDI target plate, and after the plate was naturally air-dried, a certain amount (1. mu.L) of DHB matrix (25mg/mL of 2, 5-dimethylbenzoic acid, acetonitrile/water/phosphoric acid, 70:29:1, v/v/v) was dropped, and after the crystals were volatilized, MALDI-TOF MS analysis was performed.
The result is shown in figure 5, and mass spectrometry analysis is performed on the eluate after the enrichment of the low-content IgG enzyme digestion peptide fragment, so that the functionalized molybdenum disulfide-nanogold composite material has high-sensitivity enrichment performance on glycopeptide in the IgG trypsin enzyme digestion peptide fragment. As a result, 28 glycopeptides were identified on the MALDI-TOF MS spectrum when the peptide fragment was cleaved with 6.7pmol IgG, and a large amount of the abundant non-glycopeptide signals in the molecular weight range of 1000-2000 was removed. When the content of IgG enzyme digestion peptide section is as low as 20fmol, 6 glycopeptides can be identified on the spectrum of MALDI-TOF MS, and a large amount of high abundance non-glycopeptide signals with the molecular weight of 1000-2000 are removed. When the peptide fragment is cut by IgG with the content of as low as 10fmol, the signal intensity of 1 glycopeptide can be identified on a MALDI-TOF MS spectrum, and a large amount of high-abundance non-glycopeptide signals with the molecular weight in the range of 1000-2000 are removed. Therefore, the result shows that the prepared functionalized molybdenum disulfide-nanogold composite material has high sensitivity on enrichment of low-abundance glycosylated peptide segments.
Example 3, the method for detecting glycopeptide enrichment selectivity and specificity of a functionalized molybdenum disulfide-nanogold composite material based on a hydrophilic enrichment principle comprises the following steps:
firstly, enriching glycopeptides:
(11) weighing 1mg of the functionalized molybdenum disulfide-nano noble metal composite material obtained in example 1, shaking and dispersing in 1mL of enrichment buffer (acetonitrile/water/trifluoroacetic acid, 86:13.9:0.1, v/v/v), taking 20 mu L of the suspension, centrifuging, taking the lower-layer precipitate, and washing for 3 times by using the enrichment buffer.
(12) Groups 3 (designated as groups 1 and 2) containing a mixture of IgG and Bovine Serum Albumin (BSA) cleavage products were taken in 100. mu.L enrichment buffer (acetonitrile/water/trifluoroacetic acid, 86:13.9:0.1, v/v/v), wherein the mass ratio of the mixture containing 2. mu.g of IgG and BSA cleavage products in groups 1 and 2 was 1:200 and 1:500, respectively.
(13) Suspending the precipitate obtained in the step (11) in the solutions of the group 1 and the group 2 in the step (12), and then incubating the suspension at room temperature with shaking for 30 min. Then, the liquid was discarded by centrifugation, washed thoroughly with 100. mu.L of enrichment buffer, and repeated 3 times, and the precipitate was retained.
(14) The precipitates obtained in step (13) were suspended in 20. mu.L of each elution buffer (acetonitrile/water/trifluoroacetic acid, 30:69:1, v/v/v), incubated at room temperature with shaking for 10min, and then centrifuged to collect the supernatant as an eluate. Meanwhile, the obtained precipitate was resuspended in 20. mu.L of elution buffer (acetonitrile/water/trifluoroacetic acid, 30:69:1, v/v/v), incubated at room temperature with shaking for 10min, centrifuged to collect the supernatant as an eluate, and the above eluates were combined.
II, detection of glycopeptides:
mu.L of each of the above-collected supernatants was dropped on a MALDI target plate, and after the plate was naturally air-dried, a predetermined amount (1. mu.L) of DHB matrix (25mg/mL2, 5-dimethylbenzoic acid, acetonitrile/water/phosphoric acid, 70:29:1, v/v/v) was dropped, and after the crystals were volatilized, MALDI-TOF MS analysis was performed.
The result is shown in figure 6, and the mass spectrogram result of the eluent after the mixture of IgG and BSA enzyme digestion products with different mass ratios is enriched shows that the functionalized molybdenum disulfide-nano gold composite material has high-sensitivity enrichment performance on glycopeptide in the IgG trypsin enzyme digestion peptide segment. The result was enrichment under interference from non-glycosylated peptide fragments in a large number of BSA trypsin cleavage products, with 7 and 5 glycopeptides identified on the MALDI-TOF MS spectra at IgG to BSA mass ratios of 1:200 and 1:500, respectively, and a large number of high abundance non-glycopeptide signals in the molecular weight range of 1000-2000 removed. Therefore, the result shows that the prepared functionalized molybdenum disulfide-nanogold composite material has high selectivity for enriching low-abundance glycosylated peptide segments.
Example 4, the selective and specific detection of glycopeptide enrichment of functionalized molybdenum disulfide-nanogold composite material based on boric acid chemistry method includes the following steps:
firstly, preparing a functionalized molybdenum disulfide-nanogold composite material:
(8) 5mg of the molybdenum disulfide-nanogold obtained in example 1 and 30mg of 4-mercaptophenylboronic acid were weighed and dispersed in 2mL of absolute ethanol, and magnetically stirred at room temperature for 24 hours.
(9) The product obtained in step (8) was centrifuged at 4,500g for 15 minutes and the precipitate was taken.
(10) And (4) adding 2mL of absolute ethyl alcohol into the precipitate obtained in the step (9), washing for 3 times, and taking the precipitate to obtain the functionalized molybdenum disulfide-nano noble metal composite material provided by the invention.
II, enriching glycopeptides:
(11) weighing 1mg of the material obtained in the step (10), shaking and dispersing in 1mL of enrichment buffer 50mMNH4HCO320 μ L of the suspension was centrifuged, the lower layer was precipitated, and washed 3 times with enrichment buffer.
(12) Take 2 groups (designated as group 1 and group 2) of 100. mu.L enrichment buffer (50 mMNH) containing HRP trypsin cleaved peptide fragments4HCO3) The amount of HRP trypsin cleaved peptide in each group was 6.7 pmol.
(13) After suspending the precipitate obtained in step (11) in the group 1 solution in step (12), shaking and incubating at room temperature for 30 min. Then, the liquid was discarded by centrifugation, washed thoroughly with 100. mu.L of enrichment buffer, and repeated 3 times, and the precipitate was retained.
(14) The pellet obtained in step (13) was suspended in 20. mu.L of elution buffer (acetonitrile/water/trifluoroacetic acid, 50:49:1, v/v/v), incubated at room temperature with shaking for 10min, and the supernatant was collected by centrifugation. Meanwhile, the resulting pellet was resuspended in 20. mu.L of elution buffer (acetonitrile/water/trifluoroacetic acid, 50:49:1, v/v/v), and after incubation at room temperature with shaking for 10min, the supernatant was collected by centrifugation and combined.
Thirdly, detection of glycopeptides:
mu.L of the collected supernatant was dropped onto a MALDI target plate, and after it was naturally air-dried, a certain amount (1. mu.L) of DHB matrix (25mg/mL of 2, 5-dimethylbenzoic acid, acetonitrile/water/phosphoric acid, 70:29:1, v/v/v) was dropped, and after it was volatilized and crystallized, MALDI-TOF MS analysis was performed.
Meanwhile, a certain amount (1 μ L) of the group 2 solution in the step (12) is dropped on a MALDI target plate, after the solution is naturally air-dried, a certain amount (1 μ L) of DHB matrix (25mg/mL of 2, 5-dimethylbenzoic acid, acetonitrile/water/phosphoric acid, 70:29:1, v/v/v) is dropped, after the solution is volatilized and crystallized, MALDI-TOF MS analysis is carried out as a blank control.
The result is shown in figure 7, and the comparison of the mass spectrogram results of the blank control sample before enrichment and the eluate after enrichment shows that the functionalized molybdenum disulfide-nano noble metal composite material provided by the invention has specific enrichment performance on glycopeptides in the peptide fragment digested by HRP trypsin. In the case of not enriching the sample, only 5 glycopeptides can be identified on the MALDI-TOF MS spectrum, and a large amount of abundant non-glycopeptide signals appear. And under the condition that the functionalized molybdenum disulfide-nano noble metal composite material is used for enriching glycopeptides in a sample, 23 glycopeptides can be identified on a MALDI-TOF MS spectrum, and a large amount of high-abundance non-glycopeptide signals are removed. Therefore, the result shows that the functionalized molybdenum disulfide-nano noble metal composite material provided by the invention has excellent performance on enrichment of glycosylated peptide segments.
Claims (10)
1. A functionalized molybdenum disulfide-nano noble metal composite material is composed of molybdenum disulfide nanosheets, nano noble metals and functional molecules;
the functional molecule is glycopeptide enrichment functional molecule containing sulfhydryl;
the nano noble metal is fixed on the surface of the molybdenum disulfide nanosheet through a coordination bond;
the functional molecule is connected with the nano noble metal through sulfydryl;
the molybdenum disulfide nanosheet and the nano noble metal are connected through a coordination bond, and the method is realized through the following steps:
mixing the molybdenum disulfide normal hexane suspension with the precious metal salt and oleylamine normal hexane solution, carrying out ultrasonic treatment, adding a reducing agent, uniformly mixing, standing at room temperature, adding absolute ethyl alcohol, carrying out centrifugation to collect precipitate, adding normal hexane for dissolving, adding absolute ethyl alcohol, uniformly mixing, and carrying out centrifugation to collect precipitate.
2. The functionalized molybdenum disulfide-noble metal nanomaterial composite of claim 1, wherein: the length of the longest side of the molybdenum disulfide nanosheet is 50-500 nm; the thickness is 0.69 nm;
the nano noble metal is nano gold, nano silver or nano platinum;
the nano noble metal is in the form of nano particles or nano wires;
the particle size of the nano particles is 2-20 nm; the length of the nanowire is 2-5 nm;
the functional molecule is L-cysteine or mercapto phenylboronic acid;
the dosage ratio of the molybdenum disulfide, the noble metal and the functional molecule is 10 mg: 1.5-5 mg: 40-100 mg.
3. A method of making the functionalized molybdenum disulfide-nanonoble metal composite of claim 1 or 2, comprising the steps of:
the molybdenum disulfide nanosheets and the nano noble metal are connected through coordination bonds to obtain molybdenum disulfide-noble metal, then the molybdenum disulfide-noble metal and functional molecules are dispersed in a solvent, the mixture is stirred and uniformly mixed to carry out a sulfur-noble metal reaction, and after the reaction is finished, the precipitate is centrifugally collected, so that the functionalized molybdenum disulfide-nano noble metal composite material is obtained;
the functional molecule is a molecule containing sulfydryl and having glycopeptide enrichment function.
4. The method of claim 3, wherein: in the step of the sulfur-precious metal reaction, the temperature is 10-60 ℃; the time is 12-24 h;
the solvent is absolute ethyl alcohol;
the dosage ratio of the molybdenum disulfide-noble metal, the functional molecule and the solvent is 5-20 mg: 30-100 mg: 2-5 mL;
in the centrifugation step, the centrifugal force is 1000-10000 g; the time is 5-30 minutes.
5. A method of making the functionalized molybdenum disulfide-nanonoble metal composite of claim 1 or 2, comprising the steps of:
the molybdenum disulfide nanosheets and the nano noble metal are connected through coordination bonds to obtain molybdenum disulfide-noble metal, the molybdenum disulfide-noble metal is stirred with an ethanol water solution of functional molecules, precipitates are collected centrifugally, and the precipitates are sequentially washed with ethanol and water and then taken to obtain the molybdenum disulfide-noble metal;
the functional molecule is a molecule containing sulfydryl and having glycopeptide enrichment function.
6. The method of claim 5, wherein: in the ethanol aqueous solution of the functional molecules, the volume percentage concentration of the ethanol aqueous solution is 50-80%;
the dosage ratio of the functional molecules to the ethanol water solution is 40-100 mg: 4 mL;
in the stirring step, the time is 12-24 hours;
in the centrifugation step, the centrifugal force is 1000-10000 g; the time is 5 to 30 minutes;
in the washing step, the times of washing with ethanol and water are 3-10 times.
7. The method according to any one of claims 3-6, wherein: the noble metal salt is gold tetrachloride trihydrate, silver nitrate or potassium tetrachloroplatinate;
the reducing agent is triisopropylsilane;
in the molybdenum disulfide normal hexane suspension, the dosage ratio of molybdenum disulfide to normal hexane is 10-50 mg: 1-5 mL;
in the n-hexane solution of the noble metal salt and the oleylamine, the dosage ratio of the noble metal salt to the oleylamine to the n-hexane is 3-15 mg: 100-500 μ L: 2-8 mL;
the dosage ratio of the reducing agent to the molybdenum disulfide is 100-500 mu L: 10-50 mg;
in the standing step, the time is 4-5 hours;
in the centrifugation step, the centrifugal force is 1000-10000 g; the time is 5-30 minutes.
8. Use of the functionalized molybdenum disulfide-nano noble metal composite material of claim 1 or 2 in the enrichment and/or detection of glycosylated peptide segments.
9. A method for enriching glycopeptides, comprising the steps of:
1) washing the functionalized molybdenum disulfide-nano noble metal composite material of claim 1 or 2 with an enrichment buffer solution, then carrying out shaking incubation on the glycopeptide sample to be separated and the functionalized molybdenum disulfide-nano noble metal composite material in the enrichment buffer solution together, and centrifuging to collect precipitates;
2) washing the precipitate obtained in the step 1) by using an enrichment buffer solution, placing the precipitate in an elution buffer solution, oscillating and incubating, centrifuging and collecting supernatant, thus completing the enrichment of the glycopeptide sample to be separated.
10. The method of claim 9, wherein: in the incubation step in the step 1), the time is 30-60 minutes; step 2), in the incubation step, the time is 10-30 minutes; the incubation temperature in the steps 1) and 2) is room temperature;
when the functionalized molybdenum disulfide-nanogold composite material is molybdenum disulfide-nanogold-L-cysteine, the volume ratio of the enrichment buffer solution is 86:13.9:0.1 of acetonitrile, water and trifluoroacetic acid;
the elution buffer solution is prepared by mixing a solution prepared from 30:69:1 of acetonitrile, water and trifluoroacetic acid;
when the functionalized molybdenum disulfide-nanogold composite material is molybdenum disulfide-nanogold-mercaptophenylboronic acid, the enrichment buffer solution is 50mM ammonium bicarbonate aqueous solution;
the elution buffer solution is prepared by mixing 50:49:1 acetonitrile, water and trifluoroacetic acid.
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| CN111097038B (en) * | 2020-01-16 | 2022-04-29 | 长春工业大学 | Vancomycin-modified molybdenum disulfide/gold nanoneedle composite material and preparation method thereof |
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