A kind of amino-modified reduced graphene oxide and its preparation method and application

Abstract

The present invention relates to a kind of amino-modified reduced graphene oxide and its preparation method and application, by first modifying the amino linker on the surface of reduced graphene oxide (rGO) to obtain a first product, then removing the Fmoc protecting group from the first product, obtaining the amino-modified reduced graphene oxide, having an ultra-large specific surface area and a flexible structure, which can increase the solid loading of N-sugar chains and reduce the contact steric hindrance between it and the enriched N-sugar chains, therefore, the amino-modified reduced graphene oxide of the present invention is combined with MALDI-TOF MS to realize the specific enrichment and identification of protein N-glycopeptides or N-sugar chains in complex biological systems. The amino-modified reduced graphene oxide material of the present invention is rich in N groups and has excellent hydrophilic properties. And the material can be directly combined with a MALDI matrix and a MALDI laser source to realize the effective release and identification of the enriched N-sugar chains. Compared with other protein N-glycan enrichment methods in the prior art, the material enrichment method based on hydrophilic interaction chromatography (HILIC) has obvious advantages, such as high biocompatibility, simple operation, short time consumption, high recovery rate, and can achieve efficient capture and purification of N-glycans in biological samples.

Description

Technical Field

[0001] The invention belongs to the technical field of materials, and specifically relates to amino-modified reduced graphene oxide and a preparation method and application thereof. Background Art

[0002] As an important type of glycosylation modification, N-glycosylation modification is involved in almost all important life processes. Studies have shown that the development of many diseases is accompanied by changes in the composition and structure of glycoprotein N-glycans. Efficient and specific analysis and detection of disease-related N-glycan composition and structural changes in complex biological samples are of great significance for clarifying the role of sugar chains in various physiological processes and the development of diseases, as well as for the discovery of clinical diagnostic biomarkers. However, the content of N-glycans themselves is limited and slightly heterogeneous, and glycosylation can occur at different amino acid sites; and they are susceptible to severe interference from high-abundance proteins, nucleic acids and salts, resulting in signal suppression during mass spectrometry detection. These factors place higher demands on glycopeptide enrichment and glycan separation technologies.

[0003] In terms of glycopeptide / chain enrichment, various methods such as lectin affinity chromatography, boric acid chemistry, and hydrophilic interaction liquid chromatography (HILIC) have been developed and used. However, each lectin only has an enrichment effect on a specific glycoform, making it difficult to achieve enrichment detection of all sugar chains; the boric acid chemistry method has a low affinity; HILIC has attracted more and more attention due to its significant advantages such as unbiased enrichment of various glycoproteins / chains, good compatibility with mass spectrometry, high throughput and low cost. However, the current HILIC method generally requires the separation of sugar chains from the enriched material before subsequent identification. Summary of the invention

[0004] In order to solve the above problems existing in the prior art, the present invention provides an amino-modified reduced graphene oxide and its preparation method and application. The amino-modified reduced graphene oxide of the present invention is combined with MALDI-TOF MS to realize the specific enrichment and identification of protein N-glycopeptides or N-sugar chains in complex biological systems. The amino-modified reduced graphene oxide material of the present invention is rich in N groups and has excellent hydrophilic properties. And the material can be directly used as a MALDI matrix and combined with a MALDI laser source to realize the effective release and identification of the enriched N-sugar chains. Compared with other protein N-sugar chain enrichment methods in the prior art, the enrichment method of the material of the present invention based on hydrophilic interaction chromatography (HILIC) has obvious advantages, high biocompatibility, simple operation, short time consumption, high recovery rate, and can realize efficient capture and purification of N-sugar chains in biological samples.

[0005] The technical solution adopted by the present invention is:

[0006] A method for preparing amino-modified reduced graphene oxide comprises the following steps:

[0007] (1) modifying an amino linker on the surface of reduced graphene oxide to obtain a first product;

[0008] (2) removing the Fmoc protecting group from the first product to obtain the amino-modified reduced graphene oxide. The reduced graphene oxide has a carbon content of >65wt.%, and a nitrogen content of >8wt.%.

[0009] In step (1), the amino linker is 4{4-[1-(9-fluorenylmethoxycarbonylamino)ethyl]-2-methoxy-5-nitrophenoxy}butyric acid.

[0010] The amino group of the reduced graphene oxide undergoes a condensation reaction with the carboxyl ester bond of the amino connector; the condensation agent used is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide / 1-hydroxybenzotriazine (EDCI / HoBt), the dosage is 100 eq, and the reaction time for the heterogeneous reaction system is 7 days.

[0011] The specific operation of step (1) is as follows: 500 mg of 4{4-[1-(9-fluorenylmethoxycarbonylamino)ethyl]-2-methoxy-5-nitrophenoxy}butyric acid is added to 20 mL of DMF. Then 100 mg of the raw material, 5 g of EDCI, and 5 g of HOBt are added to the reaction system in sequence. The reaction is carried out at room temperature for 7 days under vigorous stirring.

[0012] In step (2), pyridine is used to remove the Fmoc protecting group. The specific operation is as follows: after the reaction of step (1) is completed, suction filtration is performed, and the filter cake is washed with DMF and dichloromethane respectively. After the obtained solid is dried, the Fmoc removal reaction is directly performed without further treatment. That is, 258 mg of the first product is added to 10 mL of DMF, and then 10 mL of pyridine is added. Under vigorous stirring, the reaction is carried out at room temperature for 1 day. After the reaction is completed, suction filtration is performed, and the filter cake is washed with DMF and dichloromethane respectively. After the obtained solid is dried, 203 mg of the product is obtained.

[0013] The amino-modified reduced graphene oxide prepared by the method.

[0014] The use of the amino-modified reduced graphene oxide in the following (I) or (II) or (III);

[0015] (Ⅰ) Extraction of intact N-glycopeptides from human immunoglobulin G;

[0016] (II) Extraction of protein N-glycans from human serum;

[0017] (Ⅲ) N-glycomic analysis of serum proteins in healthy subjects and HCC patients.

[0018] The specific operation of extracting protein N-glycans from human serum is as follows:

[0019] After adding PNGase F enzyme to the serum to release N-sugar chains, ethanol is added to precipitate proteins, and the supernatant is freeze-dried; the amino-modified reduced graphene oxide is added to the freeze-dried supernatant, incubated, centrifuged, and the precipitate is collected to obtain the protein N-sugar chains in the serum.

[0020] Prior to the enzymatic hydrolysis, the method further comprises the steps of centrifuging the serum to remove cell debris and / or adding a buffer to dilute the serum sample;

[0021] The ratio of the PNGase F enzyme to the serum is: 500U: 5-30μL, specifically 500U: 10μL;

[0022] In the protein precipitation step, the ratio of the serum to ethanol is: 10 μL: 900 μL-1000 μL, specifically 10 μL: 900 μL; the ratio of the amino-modified reduced graphene oxide to the serum is: 0.5 mg-1 mg: 10 μL, specifically 1 mg: 10 μL.

[0023] The method further comprises the step of washing the precipitate with a cleaning solution to obtain the protein N-sugar chain, wherein the cleaning solution is a mixed solution of ACN, H2O, and TFA in a volume ratio of 86:13.9:0.1.

[0024] The specific operation of the serum protein N-glycomics analysis of healthy subjects and liver cancer patients is as follows:

[0025] Serum protein N-glycans of healthy subjects and liver cancer patients were extracted respectively, and the precipitates were collected after centrifugal separation. The precipitates were added to a MALDI target plate, and after natural drying, DHB matrix was added. After the matrix was naturally dried, MALDI-TOFMS analysis was performed to complete the N-glycomic analysis of serum proteins of healthy subjects and liver cancer patients.

[0026] The method for enriching standard protein N-glycopeptides and complex biological sample protein N-glycans includes the following steps:

[0027] 1) FASP enzymatic hydrolysis of IgG protein: add lysis buffer to cleave IgG protein, and reduce and alkylate it to open the disulfide bonds of the protein; add Trypsin, digest overnight, and collect the filtrate by centrifugation, which is the IgG digestion product (peptide fragment); the FASP enzymatic hydrolysis method of bovine serum albumin (BSA) is consistent with the FASP enzymatic hydrolysis method of IgG protein.

[0028] 2) The amino-modified reduced graphene oxide is mixed and incubated with the standard protein IgG enzyme digestion product in a binding buffer, washed with a cleaning buffer to remove non-specific adsorption, eluted with an elution buffer or not eluted, and then entered into MALDI-TOFMS identification and analysis.

[0029] 3) The amino-modified reduced graphene oxide was incubated with the serum sample, washed with a washing buffer to remove non-specific adsorption, and then identified and analyzed by MALDI-TOF MS.

[0030] The present invention also provides a method for enriching standard protein glycopeptides using amino-modified reduced graphene oxide, comprising the following steps:

[0031] The IgG proteolysis product is dissolved in a binding buffer and mixed with the broken amino-modified graphene to fully bind the sugar chain to the new material. After incubation, the unbound peptides in the supernatant are removed by centrifugation, and then a washing buffer solution is added to remove the unadsorbed and non-specific adsorbed components. The precipitated part is the nanomaterial adsorbed with glycopeptides, which can be directly spotted on the target plate and, after drying, identified by MALDI-TOF MS.

[0032] In the enrichment method, the usage ratio of the IgG proteolysis product, the binding buffer, and the amino-modified reduced graphene oxide is 1 μg: 20-60 μL: 0.5 mg, specifically 1 μg: 50 μL: 0.5 mg;

[0033] The binding buffer and the washing buffer are both composed of a mixed solution of ACN, H2O, and TFA in a volume ratio of 86:13.9:0.1, and the elution buffer is a mixed solution of ACN, H2O, and TFA in a volume ratio of 30:69.9:0.1;

[0034] The incubation may be performed under shaking at 1000-1500 rpm, specifically 1500 rpm; the incubation temperature may be 25°C-37°C, specifically 37°C; the incubation time may be 20-40 min, specifically 30 min;

[0035] The washing condition may be vortex washing with 200 μL of washing buffer for 3 to 5 times, specifically 3 times.

[0036] The centrifugation condition may be 10000-15000 g for 3-5 min, specifically 12000 g for 3 min;

[0037] In the step of eluting and releasing the glycopeptide with the eluent, 10 to 50 μL of elution buffer may be added for elution twice, specifically 20 μL; the elution conditions may be 25° C. to 37° C., with shaking for 5 to 10 minutes, specifically eluting at 37° C. for 10 minutes. The material not eluted, i.e., the material to which the glycopeptide is attached, may enter the identification step with the glycopeptide, and the glycopeptide is released under the MALDI laser during the identification process.

[0038] The present invention also provides a method for enriching serum protein N-sugar chains using amino-modified reduced graphene oxide, comprising the following steps:

[0039] Whole blood was drawn from the human elbow vein, centrifuged to discard cells, and the upper serum was retained; the serum was centrifuged again to discard cell debris, and the upper serum was retained; the serum was diluted with 50mM NH4HCO3 buffer, heated to denature serum proteins, and PNGase F was added after natural cooling to release protein N-sugar chains. Serum proteins were precipitated with ethanol, and the supernatant was collected by centrifugation and freeze-dried; amino-modified reduced graphene oxide was weighed, washed, resuspended with binding buffer, and transferred to the above-mentioned freeze-dried sample. After shaking incubation, the unadsorbed impurities in the supernatant were removed by centrifugation and non-characteristic adsorbed components were washed to remove the precipitated part, which was the amino-modified reduced graphene oxide that captured the N-sugar chains. Serum protein N-sugar chains were identified by MALDI-TOF MS.

[0040] In the above-mentioned N-glycan enrichment method, the whole blood centrifugation condition can be 2000-3000g centrifugation for 5-10 minutes, specifically 3000g centrifugation for 5 minutes;

[0041] The serum centrifugation to discard cell debris can be performed at 4°C, 9000-10000g for 15-30 min, specifically 9600g for 30 min;

[0042] The ratio of the serum to the NH4HCO3 buffer may be 10 μL:50-100 μL, specifically 10 μL:90 μL;

[0043] The serum protein denaturation condition may be placed in a metal bath at 65°C to 90°C for 10 to 30 minutes, specifically 90°C for 10 minutes;

[0044] The binding buffer and the washing buffer are both mixed solutions composed of ACN, H2O, and TFA in a volume ratio of 86:13.9:0.1;

[0045] The amino-modified reduced graphene oxide can be washed 2 to 3 times with 100 to 500 μL of binding buffer, specifically 3 times with 200 μL of binding buffer;

[0046] The incubation may be performed under shaking at 1000-1500 rpm, specifically 1500 rpm; the incubation temperature may be 25°C-37°C, specifically 37°C; the incubation time may be 20-40 min, specifically 30 min;

[0047] The washing condition may be vortex washing with 400 μL of washing buffer for 3 to 5 times, specifically 5 times.

[0048] The centrifugation condition may be 10000-14000 g for 3-5 min, specifically 12000 g for 3 min;

[0049] Before the serum sample is mixed and incubated with graphene, the steps of PNGase F enzymatic hydrolysis of serum protein N-sugar chains and serum protein ice ethanol precipitation are also included, wherein the ratio of PNGase F to serum can be 300-500U:5-30μL serum, specifically 500U:10μL; the ratio of ice ethanol to serum can be 900-1000μL:10-30μL, specifically 900μL:10μL;

[0050] Before the MALDI-TOF MS identification step, the method also includes the steps of sample re-dissolution / re-suspending and target spotting. The sample can be re-dissolved / re-suspended with 1 to 5 μL of deionized water, specifically 3 μL, and 1 μL of the sample can be used for target spotting. After the sample is naturally dried, 1 μL of DHB matrix is ​​added. The DHB matrix solution is 20 mg / mL of 2,5-dihydroxybenzoic acid (prepared with ACN, 0.1% TFA, and phosphoric acid in a volume ratio of 30:70:1), and the target plate is an MTP 384 target plate.

[0051] The MALDI-TOF mass spectrometry analysis was performed on a Bruker Ultrafex III MALDI-TOF / TOF mass spectrometer (Bruker, Daltonics, Germany) in positive ion reflection mode on a 337 nm nitrogen laser. The spectrum was acquired using an available accelerating voltage of 20 kV, and the laser intensity of all samples was kept constant. The mass spectrum covering the mass range from m / z 1000 to 4000 was recorded. The spectrum was analyzed by flexAnalysis software (version 3.3).

[0052] The present invention has the following specific beneficial effects:

[0053] (1) The amino-modified reduced graphene oxide described in the present invention has an ultra-large specific surface area and a flexible structure, which can increase the immobilization amount of N-sugar chains and reduce the contact steric hindrance between it and the enriched N-sugar chains. Therefore, the amino-modified reduced graphene oxide described in the present invention, as an enrichment carrier of N-sugar chains, can improve the enrichment scale and efficiency of N-sugar chains in complex biological samples. The amino-modified reduced graphene oxide described in the present invention has good energy absorption, storage and transfer properties, and can be directly used as a MALDI matrix to solve the interference of conventional enrichment materials on mass spectrometry detection, and can also further improve the adsorption / ionization efficiency of N-sugar chains;

[0054] (2) The amino-modified reduced graphene oxide material of the present invention contains abundant NH3 groups and has excellent hydrophilic properties, and can realize efficient separation of N-glycans in biological samples based on HILIC chromatography. The mobile phase used in HILIC is mainly water and organic solvents, which have good compatibility with biological samples and mass spectrometry. Since N-glycans are adsorbed on the surface of the material based on HILIC action, the on-target release of the enriched protein N-glycans can be directly achieved using a MALDI laser source (337nm or 355nm). This solves the problem of effective release of the enriched target in the HILIC enrichment method.

[0055] (3) The amino-modified reduced graphene oxide described in the present invention can not only enrich free N-glycochains, but also can be used to capture intact N-glycopeptides in biological samples, providing complete glycopeptide information for biological analysis, and is widely applicable to samples, and has excellent enrichment sensitivity and selectivity. Based on the reduced graphene oxide described in the present invention, the enrichment of N-glycochains or the capture of intact N-glycopeptides has fewer operation steps, which can effectively reduce sample loss and further improve work efficiency. Therefore, the amino-modified reduced graphene oxide described in the present invention can increase the identification scale of N-glycochains in complex biological samples, and provide new technical means for biological research, clinical diagnosis and treatment, and new drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0057] Figure 1 The synthetic route of the amino-modified reduced graphene oxide of the present invention is as follows;

[0058] Figure 2The present invention is a flow chart of enriching protein N-glycans based on the HILIC enrichment method of the amino-modified reduced graphene oxide;

[0059] Figure 3 The infrared and XPS characterization spectra of reduced graphene oxide and the synthetic product amino-reduced graphene oxide; Figure 3 a and 3b are the infrared spectra of reduced graphene oxide before and after amino modification. Figure 3 c and 3d are the full XPS spectra of reduced graphene oxide before and after amino modification. Figure 3 e~3g are the XPS fine spectra of C, N, and O of reduced graphene oxide. Figure 3 h~3j are the XPS fine spectra of C, N, and O of amino-modified reduced graphene oxide;

[0060] Figure 4 The MALDI-TOF MS spectra of 1 μg of IgG peptide fragments released under different conditions before and after enrichment with the amino-modified reduced graphene oxide; wherein, Figure 4 a is the MALDI-TOF MS spectrum of the IgG peptide before enrichment with the amino-modified reduced graphene oxide, Figure 4 b is the MALDI-TOF MS spectrum of the N-glycopeptides released after enrichment of IgG peptides and elution with eluent; Figure 4 c is the MALDI-TOF MS spectrum of IgG peptides after enrichment without elution;

[0061] Figure 5 The MALDI-TOF MS spectra of amino-modified reduced graphene oxide enriched N-glycopeptides from different moles of IgG protease cleavage products; Figure 5 a is 1 pmol, Figure 5 b is 100 fmol, Figure 5 c is 50 fmol, Figure 5 d is 10 fmol;

[0062] Figure 6 The MALDI-TOF MS identification spectra of IgG and BSA protease cleavage products mixed at different molar ratios without any enrichment and after enrichment with amino-modified reduced graphene oxide; Figure 6 a means without any enrichment, Figure 6 b is the molar ratio of IgG:BSA protease cleavage product is 1:50, Figure 6 c is the molar ratio of IgG:BSA protease cleavage product 1:100, Figure 6 d is the molar ratio of IgG:BSA protease cleavage product is 1:200.

[0063] Figure 7The MALDI-TOF MS spectra of serum N-glycans enriched in amino-modified reduced graphene oxide in healthy subjects and patients with liver cancer; Figure 7 a~7c are the N-glycan spectra in healthy human serum. Figure 7 d~7f are the N-glycan spectra in the serum of liver cancer patients. DETAILED DESCRIPTION

[0064] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0065] Example 1 Preparation of amino-modified reduced graphene oxide nanomaterials

[0066] This embodiment provides a method for preparing an amino-modified reduced graphene oxide nanomaterial, specifically, Figure 1 As shown, the specific steps are as follows:

[0067] (1) Preparation of reduced graphene oxide with surface modified amino groups

[0068] 500 mg of 4{4-[1-(9-fluorenylmethoxycarbonylamino)ethyl]-2-methoxy-5-nitrophenoxy}butyric acid was added to 20 mL of DMF. Then 100 mg of the raw material, 5 g of EDCI, and 5 g of HOBt were added to the reaction system in sequence. The reaction was carried out at room temperature for 7 days under vigorous stirring. After the reaction was completed, the filter cake was filtered and washed with DMF and dichloromethane respectively, and the obtained solid was dried.

[0069] (2) Removal of Fmoc protecting group from amino-modified reduced graphene oxide

[0070] 258 mg of intermediate 1 was added to 10 mL of DMF, and then 10 mL of pyridine was added. The reaction was carried out at room temperature for 1 day under vigorous stirring to remove the Fmoc protecting group. After the reaction was completed, the filter cake was filtered and washed with DMF and dichloromethane respectively. The obtained solid was dried to obtain 203 mg of the product.

[0071] The obtained products were characterized by infrared and XPS. The infrared characteristics of the reduced graphene oxide before and after modification are as follows: Figure 3 As shown in a and b, the infrared spectrum characteristics of rGO are relatively simple, mostly showing broad peaks, such as 3412cm -1 The absorption peak at 1641cm is mainly attributed to the stretching vibration mode of OH and NH. The peak is stretched due to the van der Waals force and hydrogen bonding between graphene oxide molecules. -1and 1617cm -1 The absorption peak at 1530cm is attributed to the bending vibration mode of the hydroxyl group of water molecules and the stretching vibration mode of the C=C skeleton of the unoxidized graphene aromatic ring. The other absorption peaks overlap more seriously, which is mainly due to the fact that the free movement of graphene oxide is hindered during crystallization and the change of crystal form. It may also be the quantum size effect caused by the uneven particle size of graphene oxide. -1 and 1438cm -1 The absorption peak at 1122cm is mainly related to the structures of C=C, NH and CN. -1 The absorption peak at 3325cm is mainly derived from the CO stretching vibration mode in the oxygen-containing groups of graphene oxide. Compared with the unmodified reduced graphene oxide sample, the absorption peak composition becomes more complex and the resolution is significantly improved, indicating that the modification process changes the sample morphology and increases the richness of surface groups. -1 The absorption peak at 3034cm -1 The absorption peak at 2928cm is attributed to the CH stretching vibration mode of unsaturated aliphatic hydrocarbons / aromatic hydrocarbons; -1 and 2849cm -1 The absorption peak at 1625cm is mainly related to the CH stretching vibration mode in the saturated long-chain alkane structure; -1 The absorption peak at 1572cm is a typical characteristic of the stretching vibration of the C=O of the amide group; -1 The absorption peak at 1449cm is mainly derived from the NH bending vibration and CN stretching vibration modes; -1 The absorption peak at 1088cm is related to the methylene scissor bending vibration mode; -1 The absorption peak at 892 cm is mainly related to the stretching vibration mode of oxygen-containing groups; -1 The absorption peak at is attributed to the out-of-plane bending vibration mode of the benzene ring CH. In summary, amino, amide, aromatic hydrocarbon and long-chain aliphatic hydrocarbon structures were successfully introduced into the surface of the modified reduced graphene oxide sample.

[0072] XPS characteristics of reduced graphene oxide before and after modification Figure 3 c~3j, in the XPS spectrum, compared with the unmodified reduced graphene oxide ( Figure 3 c), after amino modification ( Figure 3 d) It was found that the proportion of N1s increased; the XPS data processing software Avantage was used to perform peak fitting on the O1s, N1s, and C1s fine spectra of reduced graphene oxide before and after modification ( Figure 3 e~3j), the spectrum peak is calibrated with the C1s graphite carbon sp2 CC energy position 284.0eV. In the C1s fine spectrum, OC=O is rGO( Figure 3e) Unique functional group, after modification of amino group ( Figure 3 h) This functional group disappears, and the proportion of CC functional groups increases by 17.8%; in the N1s fine spectrum, rGO ( Figure 3 f) has CNC as its main functional group. After modification of amino groups ( Figure 3 i) Added a unique O=CN functional group; O1s fine spectrum ( Figure 3 g, Figure 3 j), the proportion of C=O double bonds increased by 36.37% after modification. XPS results show that amino linkers were successfully introduced on the surface of the modified reduced graphene oxide.

[0073] Example 2: Enrichment of N-glycopeptides from enzyme digestion products of standard protein IgG using amino-modified reduced graphene oxide

[0074] This embodiment provides a method for using the amino-modified reduced graphene oxide to enrich N-glycopeptides of the standard protein IgG cleavage product, and the specific steps are as follows:

[0075] (1) FASP cleavage of human immunoglobulin G (IgG) and bovine serum albumin (BSA)

[0076] Weigh 1 mg of IgG protein, add 200 μL of 8M urea (UA) solution to lyse, add dithiothreitol (DTT) to a final concentration of 10 mM, react at 37°C for 4 hours to reduce the disulfide bonds of the protein. Centrifuge to discard excess DTT in the filtrate, and wash 3 times with 8M UA. Then add chloroacetamide (CAA) to a final concentration of 50 mM, leave at room temperature for 40 minutes, alkylate the reduced free sulfhydryl groups, and prevent the re-formation of disulfide bonds. Centrifuge to discard excess CAA in the filtrate, and wash the protein 3 times with 50 mM NH4HCO3. Add 200 μL of 50 mM NH4HCO3 to resuspend the protein solution, add Trypsin according to the ratio of trypsin to protein of 1:50, digest overnight at 37°C for 16 hours, centrifuge at 14000g for 10 minutes to collect the peptide filtrate, heat dry and freeze at -80°C. The digestion steps of BSA protein are the same as those of IgG.

[0077] (2) Optimization of conditions for enriching IgG protease cleavage products N-glycopeptides by breaking amino graphene

[0078] Dissolve 1 μg of IgG protease cleavage product in 50 μL binding buffer ACN / H2O / TFA (86:13.9:0.1, v / v / v), mix with new materials and incubate, shake at 37°C for 30 min, centrifuge at 12000g for 3 min to remove the supernatant. Add 200 μL of washing buffer ACN / H2O / TFA (86:13.9:0.1, v / v / v) to wash and remove impurities such as non-glycopeptides, centrifuge and discard the supernatant, and repeat 3 times. The precipitate is amino-reduced graphene oxide adsorbed with N-glycopeptides. Resuspend the precipitate with 1 μL of deionized water, spot it on the MALDI target plate, let it dry naturally at room temperature, overlay 1 μL of DHB matrix, let it dry naturally, and then perform MALDI-TOF MS analysis and identification. Figure 4 Shown are the MALDI-TOF MS spectra of 1 μg of IgG peptide fragments released under different conditions before and after enrichment with the amino-modified reduced graphene oxide; wherein, Figure 4 a is the MALDI-TOF MS spectrum of the IgG peptide before enrichment with the amino-modified reduced graphene oxide, Figure 4 b is the MALDI-TOF MS spectrum of the N-glycopeptides released after enrichment of IgG peptides and elution with eluent; Figure 4 c is the MALDI-TOF MS spectrum of IgG peptides after enrichment without elution; it can be seen from the figure that after 1 μg of IgG enzyme-cleaved peptides were enriched with the new material, the non-glycopeptide signal was significantly reduced or even disappeared, the number of glycopeptides increased and the signal was significantly enhanced.

[0079] Different release conditions after the enrichment of glycopeptides by broken amino graphene were investigated: elution of released glycopeptides with eluent (30% ACN + 0.1% TFA) and glycopeptides without elution, and identification of targets together with nanomaterials. a) Add 20 μL of eluent, shake and elute at 37 ° C for 10 minutes, repeat twice, combine the glycopeptide eluent and heat dry. Use 2 μL of deionized water to re-dissolve the peptides, take 1 μL of the target, dry naturally and superimpose 1 μL of DHB matrix, and perform MALDI-TOF MS identification after drying. A total of 27 IgG N-glycopeptides were identified; b) No elution, direct identification: add 2 μL of deionized water to resuspend the precipitate, take 1 μL of the target, dry naturally and superimpose 1 μL of DHB matrix, and perform MALDI-TOF MS identification after drying. 37 IgG N-glycopeptides can be identified. The results show that the glycopeptides adsorbed by the material can be dissociated under the MALDI laser, and the amino-modified graphene material can be used as a matrix target. Glycopeptides were eluted from the new material and the eluate was collected for identification. Incomplete elution may cause loss of glycopeptides. However, when only amino-modified reduced graphene oxide was used as a matrix, the signal response was poor, so DHB matrix was added in the experiment to assist the transfer of laser energy.

[0080] (3) Sensitivity evaluation of amino-modified reduced graphene oxide materials

[0081] Weigh 2 mg of broken amino-modified reduced graphene oxide, add 200 μL binding buffer and ultrasonically disperse for 5 minutes. Wash 3 times and divide into four equal parts. Mix with 1 pmol / 100 fmol / 50 fmol / 10 fmol IgG protease cleavage products respectively, place in a Thermo-Shaker, shake and incubate at 37°C for 30 minutes, centrifuge at 12000g for 3 minutes to remove the supernatant. Add 200 μL of washing buffer ACN / H2O / TFA (86:13.9:0.1, v / v / v) to wash and remove non-glycopeptides, centrifuge and discard the supernatant, and repeat 3 times. The precipitate is the amino-modified reduced graphene oxide that captures glycopeptides. Resuspend the precipitate with 1 μL deionized water, spot it on the MALDI target plate, wait for it to dry naturally at room temperature, overlay 1 μL of DHB matrix, let it dry naturally at room temperature, and perform MALDI-TOF MS analysis and identification. Figure 5 Shown are the MALDI-TOF MS spectra of amino-modified reduced graphene oxide enriched N-glycopeptides from different molar IgG protease cleavage products; Figure 5 a is 1 pmol, Figure 5 b is 100 fmol, Figure 5 c is 50 fmol, Figure 5 d is 10fmol; it can be seen from the figure that when the IgG peptide segment is reduced to 10fmol, 20 IgG N-glycopeptides can still be identified, indicating that the new material has a high sensitivity and can capture N-glycopeptides from trace samples.

[0082] (4) Selective evaluation of amino-modified reduced graphene oxide materials

[0083] Different ratios of BSA protease cleavage products and IgG protease cleavage products were mixed to further increase the complexity of the sample. IgG and BSA protease cleavage products were mixed at a molar ratio of 1:50 / 1:100 / 1:200, respectively. Then, IgG N-glycopeptide was separated from the mixture using amino-modified reduced graphene oxide. The method was the same as in "Step (3) Sensitivity evaluation of amino-modified reduced graphene oxide materials". The results are shown in Figure 2. Figure 6 The following are MALDI-TOF MS identification spectra of IgG and BSA protease cleavage products mixed at different molar ratios without any enrichment and after enrichment with amino-modified reduced graphene oxide; Figure 6 a means without any enrichment, Figure 6 b is the molar ratio of IgG:BSA protease cleavage product is 1:50, Figure 6 c is the molar ratio of IgG:BSA protease cleavage product 1:100, Figure 6d is the molar ratio of IgG:BSA protease cleavage product 1:200. As can be seen from the figure, when the molar ratio of IgG:BSA protease cleavage product increased to 1:200, 13 IgG N-glycopeptides could still be identified. The signal of non-glycopeptide generated by BSA was significantly reduced or disappeared, which proved that the synthesized new material can selectively capture IgG N-glycopeptides from relatively complex samples.

[0084] Example 3 Application of amino-modified reduced graphene oxide in complex biological samples

[0085] This embodiment provides the application of the amino-modified reduced graphene oxide in complex biological samples, such as Figure 2 As shown, the specific operations are as follows:

[0086] (1) Release and separation of serum protein N-glycans

[0087] Venous blood was collected from healthy people and liver cancer patients in serum tubes, left to stand at room temperature for 30 minutes, and then centrifuged at 3000g for 5 minutes. The upper serum was collected in EP tubes and frozen at -80°C for later use.

[0088] Take 10μL human serum, centrifuge at 9600g for 30min, remove cell debris, dilute it 10 times with 90μL 50mMNH4HCO3 buffer containing 10mM DTT, heat at 90℃ for 10min to denature serum protein, cool naturally to room temperature, add 500UPNGase F, place in 50℃ Thermo-shaker for 2h to remove protein N-sugar chain. Add 900μL ice ethanol to the above system to make the final concentration of ice ethanol reach 90% or more, and place at -20℃ for 1h to precipitate serum protein. Remove protein precipitate by centrifugation at 4℃ (14800rpm, 15min), collect supernatant and freeze-dry for use or freeze at -80℃.

[0089] (2) Enrichment and purification of serum N-glycans by amino-modified reduced graphene oxide and MALDI identification

[0090] Weigh 1 mg of amino-modified reduced graphene oxide material, wash it twice with binding buffer ACN / H2O / TFA (86:13.9:0.1, v / v / v), then add 100 μL of binding buffer to resuspend the material and transfer it to the above-mentioned freeze-dried sample. Incubate with a 37°C Thermo-shaker for 1 hour, centrifuge and wash to remove supernatant impurities and non-specific adsorption. The precipitate is a complex of serum N-sugar chains and amino-modified reduced graphene oxide. Next, identify it using MALDI-TOF MS. First, prepare 25 mg·mL with ACN / H2O / H3PO4 (70:29:1, v / v / v). -1DHB solution was used as the matrix solution. 3 μL of deionized water was added to the above precipitate to resuspend it, 1 μL of sample was spotted on the target plate and dried at room temperature, and then 1 μL of DHB matrix solution was superimposed on the sample and allowed to dry naturally. The spectrum was acquired using an available accelerating voltage of 20 kV, and the laser intensity of all samples was kept constant. The mass spectrum covering the mass range from m / z 1000 to 4000 was recorded 500 times for each point. The spectrum was analyzed by flexAnalysis software (version 3.3). Figure 7 Shown is the enrichment and capture of healthy people by amino-modified reduced graphene oxide ( Figure 7 a~7c) and MALDI-TOF MS spectra of serum N-glycans from patients with liver cancer (7d~7f).

[0091] The hypothetical structures and corresponding molecular weights of N-glycans enriched by amino-modified reduced graphene oxide from the serum of healthy subjects and liver cancer patients are shown in Table 1.

[0092] Table 1 - Possible glycoform structures and their corresponding molecular masses

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099] Fucose; ○ Galactose; ● Mannose; □ N-acetylgalactosamine; ■ N-acetylglucosamine; ◆ Sialic acid

[0100] from Figure 7 As can be seen from Table 1, a total of 117 N-glycans were identified by MALDI-TOF MS, of which 44 were enriched and identified in the serum of both liver cancer patients and healthy controls; 24 N-glycans were stably identified in liver cancer patients but not in healthy controls, of which 7 were captured and identified only in the serum of liver cancer patients. There were 6 N-glycans that were stably identified in healthy controls but not in liver cancer patients; the number of sialylated N-glycans in the serum of liver cancer patients increased, which may be related to the occurrence and development of liver cancer.

[0101] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for preparing amino-modified reduced graphene oxide, characterized in that: The steps include: (1) modifying the surface of reduced graphene oxide with an amino linker to obtain a first product; The amino linker is 4{4-[1-(9-fluorenylmethoxycarbonylamino)ethyl]-2-methoxy-5-nitrophenoxy}butyric acid; The amino group of the reduced graphene oxide undergoes a condensation reaction with the carboxyl ester bond of the amino linker; the condensation agents used are 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 1-hydroxybenzotriazole; (2) Using pyridine to remove the Fmoc protecting group from the first product, thereby obtaining the amino-modified reduced graphene oxide.

2. The amino-modified reduced graphene oxide prepared according to the method of claim 1.

3. Use of the amino-modified reduced graphene oxide according to claim 2 in the following (I) or (II); (I) Extraction of intact N-glycopeptides from human immunoglobulin G; (II) Extraction of protein N-glycans from human serum.

4. The use according to claim 3, characterized in that: The specific operation of extracting protein N-glycans from human serum is as follows: After adding PNGase F enzyme to the serum to release N-sugar chains, ethanol is added to precipitate proteins, and the supernatant is freeze-dried; the amino-modified reduced graphene oxide is added to the freeze-dried supernatant, incubated, centrifuged, and the precipitate is collected to obtain the protein N-sugar chains in the serum.

5. The use according to claim 4, characterized in that: Prior to enzymatic hydrolysis, the method further comprises the steps of centrifuging the serum to remove cell debris and / or adding a buffer to dilute the serum sample; The ratio of the PNGase F enzyme to the serum is: 500U: 5-30μL; In the protein precipitation step, the ratio of serum to ethanol is: 10 μL: 900 μL~1000 μL; The ratio of the amino-modified reduced graphene oxide to the serum is: 0.5 mg~1 mg: 10 μL.

6. The use according to claim 4, characterized in that: The method further comprises the step of washing the precipitate with a washing buffer to obtain the protein N-sugar chain, wherein the washing buffer is a mixed solution of ACN, H2O, and TFA in a volume ratio of 86:13.9:0.1.

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