CC-TIP one-stop device and method

Abstract

The invention discloses a CC-TIP one-stop device and method, and belongs to the technical field of trace glycome sample preparation. The CC-TIP one-stop device disclosed by the invention comprises a reaction chamber, a conical reaction tube and a gasket, the reaction chamber is communicated with the large-head end of the conical reaction tube through a gasket; a protein enrichment layer, a sugar chain enrichment and purification layer and a supporting layer are sequentially arranged in the conical reaction tube from one end of the reaction chamber to the small end; according to the method, the steps of glycoprotein denaturation, reduction, alkylation, enzymolysis release, sugar chain enrichment and purification and the like can be smoothly integrated in the same device, in the operation process, a sample does not need to be transferred, good stability and reproducibility are achieved, sample loss is reduced, and compared with a traditional sugar chain preparation method, the method is faster and more efficient.

Description

Technical Field

[0001] The present invention belongs to the technical field of micro-glycomic sample preparation, and particularly relates to a CC-TIP one-stop device and method. Background Art

[0002] Post-translational modification (PTM) is a chemical modification process that occurs after protein translation, including acetylation, phosphorylation, methylation, glycosylation, and other types. Glycosylation is the most common PTM and plays a key role in numerous life processes, including cell recognition, antigen recognition, immune responses, development, aging, and the development and progression of various diseases. Glycosylation is an important foundation of cellular immunity. Differences in sugar chain structure enable cells to recognize different ligands and activate specific immune responses. Currently, protein drugs and protein drug targets in clinical use are commonly glycosylated, and glycosylation has a significant impact on drug efficacy and safety. For example, glycosylation can affect drug pharmacokinetics, pharmacodynamics, and toxicity. Glycosylation characterization is a key quality control step for glycoprotein drugs. Therefore, given the role of glycosylation in regulating the function of proteins and glycoprotein drugs, glycosylation research is of great significance in the fields of biology and pharmacy.

[0003] Glycosylation is one of the most complex and highly dynamic post-translational modification processes in eukaryotes. Its research involves multiple dimensions, including the analysis of glycoprotein sugar chain structure, the identification of glycosylation sites, and the exploration of the biological functions of sugar chains. The sugar chains on the surface of glycoproteins are formed through a non-template-dependent biosynthetic pathway. This synthesis mechanism leads to multi-level heterogeneity in glycosylation modification. At the macro level, it manifests as differences in the occupancy of glycosylation sites among glycoprotein molecules (i.e., some sites may be fully glycosylated, partially glycosylated, or completely unglycosylated); at the micro level, it manifests as microscopic heterogeneity in the structure of sugar chains attached to specific glycosylation sites (including differences in sugar chain branching patterns, monosaccharide composition sequences, stereochemical configurations, and monosaccharide modifications). This structural complexity is further exacerbated by the fact that the biosynthesis of sugar chains is coordinated by multiple glycosyltransferases and glycosidases, resulting in a single glycoprotein molecule potentially carrying dozens or even hundreds of different glycoforms. In addition, the dynamic range of glycoprotein abundance in biological samples can span 6-8 orders of magnitude, and the signals of low-abundance glycoproteins are often masked by high-abundance non-glycosylated proteins; and the inherent low ionization efficiency of glycosylation modification makes its response value in mass spectrometry detection significantly lower than that of unmodified peptides. These factors together constitute the technical bottleneck for deep coverage of the glycoproteome, especially for the detection of trace glycoproteins with important biological functions. Current research strategies mainly focus on enrichment techniques (such as lectin affinity chromatography, hydrophilic interaction chromatography and boric acid chemical immobilization), derivatization methods (glycan or glycopeptide level) and bioinformatics algorithm development (such as glycopeptide spectrum analysis and glycoform quantification), in order to achieve systematic characterization and precise analysis of glycosylation modifications in complex samples, thereby revealing the regulatory laws of glycosylation networks in physiological and pathological processes.

[0004] With the advancement of protein composition analysis in single cells and trace tissue samples, the analysis of related protein glycosylation has become an important direction for in-depth glycoprotein research and biomarker exploration, and it also puts forward higher requirements for the efficient preparation of trace glycoprotein samples. At present, the glycosylation research of trace samples is still in its infancy, and there is an urgent need to develop targeted and efficient analytical techniques. In recent years, the research on the preparation of sugar chains using trace starting proteins has gradually attracted attention. Kim et al. published a semi-automatic method for solid phase extraction 96-well plates using vacuum operation. This method uses polyvinylidene fluoride (PVDF) membrane to fix 5μL serum and enzymatically release sugar chains, and then transfer them to porous graphitic carbon (PGC) for purification. The time from pretreatment to quantitative and qualitative analysis is shortened to about two days compared with traditional methods, but the results show that there is a problem of loss of sialylated sugar chains during the treatment process. Professor Thomas Oates's research group proposed a filtration-assisted N-glycan separation method for 3.8×10 5The method relies on molecular weight cutoff ultrafiltration tubes to enrich and prepare N-glycans in individual cells. Organic or inorganic impurities with smaller molecular weights are first washed out, followed by the release and elution of N-glycans. The sugar chains released by enzymatic hydrolysis are then transferred to a new device for derivatization and purification. However, this method was later proven to cause a certain degree of sample loss and deviation in sugar chain abundance due to molecular retention. In 2019, Qian Xiaohong and others developed a GPAT technology that achieved one-stop proteolysis, N-glycan release, and enrichment of glycoprotein samples as low as 10 μg by filling C18 and hydrophilic interaction chromatography (HILIC) fillers in the pipette tip. This method combines multiple steps of sample preparation into a single step, reduces the starting amount of sample preparation, and improves sample utilization.

[0005] Although the technology for preparing trace glycoprotein samples has made significant progress in recent years, especially driven by high-sensitivity mass spectrometry detection technology and highly selective enrichment materials, its analytical throughput and detection limit have been greatly improved, the field still faces several key technical bottlenecks. These limiting factors have seriously restricted the depth and breadth of glycoproteomics research. First, the problem of sample loss during trace sample preparation is particularly prominent, which is mainly due to various technical challenges: in the sample pretreatment stage, due to the complexity of the operation process, it usually involves multiple steps such as protein extraction, reductive alkylation, enzymatic digestion, sugar chain release, solid-phase extraction enrichment and desalting purification, and each step may introduce irreversible sample loss; especially when dealing with nanogram or even picogram trace samples, sample molecules are easily retained on the inner wall of the polypropylene centrifuge tube, the surface of the filter membrane or the gap between the solid-phase extraction fillers through nonspecific adsorption. This adsorption effect follows the Langmuir adsorption isotherm under low concentration conditions, resulting in a nonlinear decrease in sample recovery rate; in addition, in an open operating system, the volatilization loss of trace liquids and the inevitable dilution effect will further aggravate the attenuation of sample concentration. Especially in steps such as high-temperature incubation or vacuum centrifugation, the volume change caused by solvent evaporation can significantly affect the stoichiometric ratio of the reaction system. For example, conventional methods based on solid-phase extraction (SPE) rely on multiple load-wash-elution cycles, which improve purification efficiency. However, each liquid transfer step results in approximately 5%-15% sample carryover, with cumulative losses potentially exceeding 50%. This poses a significant challenge for glycoprotein analysis at the already scarce scale of clinical biopsy samples or single cells. Furthermore, the discrete operational units in traditional approaches not only increase overall time but also introduce batch-to-batch variability due to frequent manual intervention. These issues combine to ensure that the accuracy and reproducibility of final mass spectrometry results cannot meet the stringent requirements of quantitative glycoproteomics. Innovative approaches such as integrated design or in situ reaction strategies are urgently needed to improve analytical efficiency. Furthermore, glycan structures are unstable and easily degraded under conditions such as acidity, alkalinity, high temperature, and enzymatic hydrolysis, affecting structural and conformational analysis. Furthermore, sample preparation can easily introduce impurities from reagents, consumables, and the environment, compromising the sensitivity and specificity of subsequent analyses. These issues severely hinder the preparation and analysis of trace glycoprotein samples, limiting the advancement of glycoprotein research. Summary of the Invention

[0006] The object of the present invention is to provide a CC-TIP one-stop device and method to solve the technical problem of sample loss in existing preparation methods leading to inaccurate subsequent analysis results.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention discloses a CC-TIP one-stop device, comprising: a reaction chamber, a conical reaction tube and a gasket; the reaction chamber is connected to the large end of the conical reaction tube via the gasket; the conical reaction tube is provided with a protein enrichment layer, a sugar chain enrichment and purification layer and a support layer in sequence from one end of the reaction chamber to the small end.

[0009] Furthermore, the material of the protein enrichment layer is C18; the material of the sugar chain enrichment and purification layer is PGC; the supporting layer is a sieve plate layer; and the material of the sieve plate layer is C8.

[0010] The present invention also discloses a method for preparing the CC-TIP one-stop device, comprising the following steps:

[0011] The reaction chamber is connected to the large end of the conical reaction tube through a gasket; then the protein enrichment layer, the sugar chain enrichment and purification layer and the support layer are filled in the conical reaction tube in sequence.

[0012] Furthermore, the method specifically includes the following steps:

[0013] Connect the reaction chamber to the large end of the conical reaction tube through a gasket; then fill the small end of the conical reaction tube with C8 membrane as a sieve plate layer;

[0014] Weigh PGC in a centrifuge tube, mix with acetonitrile, and transfer to a conical reaction tube using a pipette. Centrifuge and fix it as a sugar chain enrichment and purification layer;

[0015] Weigh C18 in a centrifuge tube, mix with acetonitrile, and transfer to a conical reaction tube using a pipette. Centrifuge and fix it as the protein enrichment layer;

[0016] Then, the centrifugal activation solution is added into the reaction chamber for centrifugal activation;

[0017] In terms of volume percentage, the centrifugal activation solution includes 99.9% ACN + 0.1% TFA, 50% ACN + 0.1% TFA + 49.9% H2O, 5% ACN + 0.1% TFA + 94.5% H2O, and 0.1% TFA + 99.9% H2O;

[0018] Each 20 μL conical reaction tube was filled with 200-400 μg of C8 membrane;

[0019] The mass ratio of the PGC to the glycoprotein sample is 100 to 1,000;

[0020] The mass ratio of the C18 to the glycoprotein sample is greater than 1,000.

[0021] The present invention also discloses a method for using the CC-TIP one-stop device. The CC-TIP one-stop device is used to prepare N-glycans, comprising the following steps:

[0022] Adjust the filling amount of PGC and C18 to form the sugar chain enrichment and purification layer and the support layer;

[0023] Subsequently, a glycoprotein sample, ammonium bicarbonate, and DTT are added to the reaction chamber to obtain a mixed solution; after the mixed solution enters the conical reaction tube through the gasket, the large end of the conical reaction tube is sealed, and the conical reaction tube is then placed in a water bath for incubation to perform a reduction reaction. After the reaction is completed, IAA is added to the conical reaction tube, and a glycoprotein alkylation reaction is performed in a dark environment. After the reaction is completed, ammonium bicarbonate and PNGase F are further added to the conical reaction tube, and the conical reaction tube is placed in a water bath for incubation to perform an enzymatic hydrolysis reaction. After the reaction is completed, a reaction product solution is obtained;

[0024] The reaction product solution is sequentially separated, purified and eluted to obtain N-sugar chains.

[0025] Furthermore, the mass ratio of the C18 to the glycoprotein sample is greater than 1000:1; the mass ratio of the PGC to the glycoprotein sample does not exceed 500:1.

[0026] Furthermore, in the mixed solution, the molar concentrations of ammonium bicarbonate, DTT, and IAA are 50 mM, 10 mM, and 20 mM, respectively;

[0027] The reduction reaction temperature is 70°C and the time is 45 to 60 minutes;

[0028] The concentration of the ammonium bicarbonate is 50 mM; the concentration of the DTT is 10 mM.

[0029] Furthermore, the glycoprotein alkylation reaction is carried out at room temperature for 0.5 to 1 hour.

[0030] Furthermore, ammonium bicarbonate and PNGase F were added to the conical reaction tube until the concentrations of DTT and IAA in the mixed solution were diluted to 1 / 2 of the original concentrations; the amount of PNGase F used was 2U PNGase F per 1 μg of glycoprotein sample.

[0031] Furthermore, the enzymatic hydrolysis reaction temperature is 56-60°C in a water bath and the time is 2-3h;

[0032] The elution treatment is carried out using a mixed solution of acetonitrile, trifluoroacetic acid and H2O.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The present invention discloses a CC-TIP one-stop device. By sequentially arranging a protein enrichment layer, a sugar chain enrichment and purification layer, and a support layer in a conical reaction tube connected to a reaction chamber, the device can smoothly integrate the steps of glycoprotein denaturation, reduction, alkylation, enzymatic release, sugar chain enrichment and purification into the same device. During operation, there is no need to transfer samples, the device has good stability and reproducibility, and sample loss is eliminated. Compared with traditional sugar chain preparation methods, the device is faster and more efficient.

[0035] The present invention also discloses a method for N-glycan enrichment using the CC-TIP one-stop device. This device can concentrate multiple reaction steps in one device. According to relevant experimental results, the enzymatic hydrolysis time for N-glycan enrichment using this device is shortened from 16 hours in traditional methods to 2-3 hours, and the N-glycan preparation of 1 μg of IgG can be achieved. Compared with the existing trace sample pretreatment technology GPAT, which can prepare N-glycans from a starting amount of 10 μg of IgG, this method can achieve the preparation of N-glycans from even smaller amounts of protein, and is faster and more efficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a schematic structural diagram of the CC-TIP one-stop device of the present invention;

[0037] Figure 2 This is the sugar chain preparation process based on the CC-TIP one-stop device of the present invention;

[0038] Wherein: 1-reaction chamber; 2-conical reaction tube; 3-gasket; 4-protein enrichment layer; 5-sugar chain enrichment and purification layer; 6-support layer;

[0039] Figures 3 to 5 The N-glycan analysis test data obtained using Examples 1 to 3 of the present invention;

[0040] Figure 6 The data are the analytical test data of N-glycans obtained by using the device of the present invention in combination with traditional methods. DETAILED DESCRIPTION

[0041] To facilitate understanding of the features and effects of the present invention by those skilled in the art, the following provides a general description and definition of the terms and expressions used in the specification and claims. Unless otherwise indicated, all technical and scientific terms used herein have the ordinary meanings as understood by those skilled in the art regarding the present invention. In the event of conflict, the definitions in this specification shall prevail.

[0042] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.

[0043] Herein, all features such as values, amounts, amounts, and concentrations defined in numerical ranges or percentage ranges are for brevity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to include and specifically disclose all possible subranges and individual values ​​within the range (including integers and fractions).

[0044] In this document, unless otherwise specified, “include,” “including,” “contains,” “has” or similar terms cover the meanings of “consisting of” and “mainly consisting of,” for example, “A includes a” covers the meanings of “A includes a and other” and “A only includes a.”

[0045] In this document, for the sake of brevity, not all possible combinations of the various technical features in each embodiment or example are described. Therefore, as long as there are no contradictions in the combination of these technical features, the various technical features in each embodiment or example can be combined in any way, and all possible combinations should be considered to be within the scope of this specification.

[0046] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0047] The following examples were prepared using conventional instruments and equipment in the art. Experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or according to the conditions recommended by the manufacturer. The various raw materials used in the following examples, unless otherwise specified, were conventional commercially available products, with specifications conventional in the art. In the present specification and the following examples, unless otherwise specified, "%" indicates percentage by weight, "part" indicates parts by weight, and "ratio" indicates weight ratio.

[0048] like Figure 1 As shown, the present invention discloses a CC-TIP one-stop device, which consists of three parts: a reaction chamber 1, a conical reaction tube 2, and a gasket 3. The conical reaction tube 2 is filled with a support layer 6, a sugar chain enrichment and purification layer 5, and a protein enrichment layer 4 from bottom to top. The support layer 6 mainly serves as a support to prevent the column material and sample from directly flowing out. The protein enrichment layer 4 can better interact with the protein to separate the N-glycans from the protein after enzymatic hydrolysis. The sugar chain enrichment and purification layer 5 is used for enriching and purifying N-glycans. The reaction chamber 1 is connected to the large end of the conical reaction tube 2 through the gasket 3.

[0049] Preferably, the material of the protein enrichment layer 4 is C18; the material of the sugar chain enrichment and purification layer 5 is porous graphite carbon (PGC); the supporting layer 6 is a sieve plate layer; the material of the sieve plate layer is C8.

[0050] The present invention also discloses a method for preparing the CC-TIP one-stop device, comprising the following steps:

[0051] Step 1: Take 200-400 μg of C8 membrane and fill it at the top of a 20 μL tip as the screening layer (support layer 6);

[0052] Step 2: Accurately weigh PGC in a centrifuge tube, mix thoroughly with acetonitrile, and transfer to the Tip (conical reaction tube 2) using a pipette. Centrifuge and fix to form the sugar chain enrichment and purification layer 5;

[0053] Step 3: Accurately weigh C18 in a centrifuge tube, mix thoroughly with acetonitrile, and transfer to the tip using a pipette. Centrifuge and fix it as protein enrichment layer 4;

[0054] Step 4: Use 100 μL each of 99.9% ACN + 0.1% TFA, 50% ACN + 0.1% TFA + 49.9% H2O, 5% ACN + 0.1% TFA + 94.5% H2O, 0.1% TFA + 99.9% H2O and pure water for centrifugal activation in sequence.

[0055] The present invention also discloses a method for enriching N-glycans using the CC-TIP one-stop device, such as Figure 2 As shown, the following steps are included:

[0056] S1: Device construction and activation

[0057] Adjust the filling amount of PGC and C18 according to the amount of glycoprotein sample to be processed; generally, the ratio of C18 to glycoprotein sample is greater than 1000:1, and the ratio of PGC to glycoprotein sample is determined according to the degree of glycosylation modification of the glycoprotein sample, generally not exceeding 500:1 (if the sugar chain accounts for 1 / 10 of the total mass of the glycoprotein, 1ug ​​of the glycoprotein should be filled with more than 0.5mg but not more than 10mg of PGC); fill the tips in the following order: frit → PGC → C18. After filling, activate them by centrifugation using 99.9% ACN + 0.1% TFA, 50% ACN + 0.1% TFA + 49.9% HO, 5% ACN + 0.1% TFA + 94.5% HO, 0.1% TFA + 99.9% HO, and pure water, respectively, and then add the glycoprotein sample;

[0058] S2: Reduction reaction

[0059] a) Carefully add 50 mM ammonium bicarbonate (freshly prepared, pH 7.5-8.0) to the top of the tip (reaction chamber 1), preventing the liquid from dripping during the process;

[0060] b) adding 500 mM DTT (dithiothreitol) to a final concentration of 10 mM dithiothreitol (DTT);

[0061] c) After adding the glycoprotein sample, seal the top of the tip with sealing film to ensure a tight seal;

[0062] d) Place the tip in a water bath at 70°C and incubate for 45-60 minutes.

[0063] S3: Alkylation

[0064] a) After the reaction is completed, cool to room temperature and remove the sealing film;

[0065] b) adding 1 M IAA (iodoacetamide) (to a final concentration of 20 mM) and incubating in the dark at room temperature for 0.5 to 1 hour to allow for glycoprotein alkylation reaction;

[0066] S4: Enzymatic hydrolysis

[0067] a) After the reaction is complete, add an appropriate amount of 50 mM ammonium bicarbonate to the Tip;

[0068] b) adding PNGase F (peptide N-glycosidase F) for enzymatic hydrolysis at a dosage of 2 U per 1 μg of glycoprotein sample;

[0069] c) Seal the tip with parafilm and transfer to a water bath at 56°C for 2–3 hours.

[0070] S5: Isolation and purification

[0071] a) Separation of sugar chains and proteins: After the reaction is completed, centrifuge thoroughly, remove the sealing film, add the centrifuge solution back to the tip, and centrifuge again. Repeat this process twice.

[0072] b) Purification: Add pure water to the tip and centrifuge to remove impurities;

[0073] c) Elution: elution of sugar chains with 20% ACN + 0.1% TFA + 79.9% H2O;

[0074] d) Collect the centrifuged liquid and dry it using a vacuum centrifugal concentrator for subsequent testing or storage.

[0075] Example 1

[0076] The method for enriching N-glycans from 1 μg of IgG using the CC-TIP one-stop device includes the following steps:

[0077] S1: Device construction and activation

[0078] Step 1: Take 400 μg of C8 membrane and fill it at the top of the 20 μL tip as the screening layer (support layer 6);

[0079] Step 2: Accurately weigh PGC in a centrifuge tube, mix thoroughly with acetonitrile, and then use a pipette to transfer 0.2 mg of PGC to the tip (conical reaction tube 2). After centrifugation and fixation, it serves as the sugar chain enrichment and purification layer 5;

[0080] Step 3: Accurately weigh C18 in a centrifuge tube, mix thoroughly with acetonitrile, and then use a pipette to transfer 1 mg of C18 to the tip. Centrifuge and fix it as protein enrichment layer 4;

[0081] Step 4: Centrifuge and activate the mixture using 100 μL each of 99.9% ACN + 0.1% TFA, 50% ACN + 0.1% TFA + 49.9% H2O, 5% ACN + 0.1% TFA + 94.5% H2O, 0.1% TFA + 99.9% H2O, and pure water.

[0082] S2: Reduction reaction

[0083] a) Carefully add 50 μL of 50 mM ammonium bicarbonate (freshly prepared, pH 7.5-8.0) to the top of the tip (reaction chamber 1), preventing the liquid from dripping.

[0084] b) Add 1 μL of 500 mM DTT to the reaction chamber to make the final DTT concentration reach 10 mM;

[0085] c) Add 1 μg of IgG (glycoprotein sample) to the reaction chamber and seal the top of the tip with sealing film to ensure a tight seal;

[0086] d) Place the tip in a water bath at 70°C for 45 minutes;

[0087] S3: Alkylation

[0088] a) After the reaction is completed, cool to room temperature and remove the sealing film;

[0089] b) adding 1 μL of 1 M IAA to the reaction chamber (to a final concentration of 20 mM) and incubating in the dark at room temperature for 30 minutes to allow for glycoprotein alkylation;

[0090] S4: Enzymatic hydrolysis

[0091] a) After the reaction is completed, add 50 μL of 50 mM ammonium bicarbonate to the tip;

[0092] b) adding 2 U of PNGase F to the reaction chamber for enzymatic hydrolysis;

[0093] c) Seal the tip with parafilm and transfer to a water bath at 56°C for 3 h.

[0094] S5: Isolation and purification

[0095] a) Separation of sugar chains and proteins: After the reaction is completed, centrifuge thoroughly, remove the sealing film, add the centrifuge solution back to the tip, and centrifuge again. Repeat this process twice.

[0096] b) Purification: Add 200 μL of purified water to the tip and centrifuge to remove impurities and discard the centrifuge solution. Repeat 4 times.

[0097] c) Elution: Replace the centrifuge tube with a new one and elute the sugar chains with 40 μL of 20% ACN + 0.1% TFA + 79.9% H2O (v / v, mixed solution). Repeat 4 times.

[0098] d) collecting the centrifuged liquid and drying it using a vacuum centrifugal concentrator to obtain the N-glycans for subsequent detection or storage.

[0099] Example 2

[0100] The method for enriching N-glycans from 1 μg of IgG using the CC-TIP one-stop device includes the following steps:

[0101] S1: Device construction and activation

[0102] Step 1: Take 400 μg of C8 membrane and fill it at the top of the 20 μL tip as the screening layer (support layer 6);

[0103] Step 2: Accurately weigh PGC in a centrifuge tube, mix thoroughly with acetonitrile, and then use a pipette to transfer 0.2 mg of PGC to the tip (conical reaction tube 2). After centrifugation and fixation, it serves as the sugar chain enrichment and purification layer 5;

[0104] Step 3: Accurately weigh C18 in a centrifuge tube, mix thoroughly with acetonitrile, and then use a pipette to transfer 1 mg of C18 to the tip. Centrifuge and fix it as protein enrichment layer 4;

[0105] Step 4: Centrifuge and activate the mixture using 100 μL each of 99.9% ACN + 0.1% TFA, 50% ACN + 0.1% TFA + 49.9% H2O, 5% ACN + 0.1% TFA + 94.5% H2O, 0.1% TFA + 99.9% H2O, and pure water.

[0106] S2: Reduction reaction

[0107] a) Carefully add 50 μL of 50 mM ammonium bicarbonate (freshly prepared, pH 7.5-8.0) to the top of the tip (reaction chamber 1), preventing the liquid from dripping.

[0108] b) Add 1 μL of 500 mM DTT to the reaction chamber to make the final DTT concentration reach 10 mM;

[0109] c) Add 1 μg of IgG (glycoprotein sample) to the reaction chamber and seal the top of the tip with sealing film to ensure a tight seal;

[0110] d) Place the tip in a water bath at 70°C for 45 minutes;

[0111] S3: Alkylation

[0112] a) After the reaction is completed, cool to room temperature and remove the sealing film;

[0113] b) adding 1 μL of 1 M IAA to the reaction chamber (to a final concentration of 20 mM) and incubating in the dark at room temperature for 30 minutes to allow for glycoprotein alkylation;

[0114] S4: Enzymatic hydrolysis

[0115] a) After the reaction is completed, add 50 μL of 50 mM ammonium bicarbonate to the tip;

[0116] b) adding 2 U of PNGase F to the reaction chamber for enzymatic hydrolysis;

[0117] c) Seal the tip with parafilm and transfer to a water bath at 56°C for 2.5 hours.

[0118] S5: Isolation and purification

[0119] a) Separation of sugar chains and proteins: After the reaction is completed, centrifuge thoroughly, remove the sealing film, add the centrifuge solution back to the tip, and centrifuge again. Repeat this process twice.

[0120] b) Purification: Add 200 μL of purified water to the tip and centrifuge to remove impurities and discard the centrifuge solution. Repeat 4 times.

[0121] c) Elution: Replace the centrifuge tube with a new one and elute the sugar chains with 40 μL of 20% ACN + 0.1% TFA + 79.9% H2O (v / v, mixed solution). Repeat 4 times.

[0122] d) collecting the centrifuged liquid and drying it using a vacuum centrifugal concentrator to obtain the N-glycans for subsequent detection or storage.

[0123] Example 3

[0124] A method for enriching N-glycans from 100 nL of normal human serum using a CC-TIP one-stop device comprises the following steps:

[0125] S1: Device construction and activation

[0126] Step 1: Take 400 μg of C8 membrane and fill it at the top of a 20 μL tip as the screening layer (support layer 6);

[0127] Step 2: Accurately weigh PGC in a centrifuge tube, mix thoroughly with acetonitrile, and then use a pipette to transfer 0.2 mg of PGC to the tip (conical reaction tube 2). After centrifugation and fixation, it serves as the sugar chain enrichment and purification layer 5;

[0128] Step 3: Accurately weigh C18 in a centrifuge tube, mix thoroughly with acetonitrile, and then use a pipette to transfer 1 mg of C18 to the tip. Centrifuge and fix it as protein enrichment layer 4;

[0129] Step 4: Centrifuge and activate the mixture using 100 μL each of 99.9% ACN + 0.1% TFA, 50% ACN + 0.1% TFA + 49.9% H2O, 5% ACN + 0.1% TFA + 94.5% H2O, 0.1% TFA + 99.9% H2O, and pure water.

[0130] S2: Reduction reaction

[0131] a) Carefully add 50 μL of 50 mM ammonium bicarbonate (freshly prepared, pH 7.5-8.0) to the top of the tip (reaction chamber 1), preventing the liquid from dripping.

[0132] b) Add 1 μL of 500 mM DTT to the reaction chamber to make the final DTT concentration reach 10 mM;

[0133] c) Add 100 nL of normal human serum to the reaction chamber and seal the top of the tip with sealing film to ensure a tight seal;

[0134] d) Place the tip in a water bath at 70°C for 45 minutes;

[0135] S3: Alkylation

[0136] a) After the reaction is completed, cool to room temperature and remove the sealing film;

[0137] b) adding 1 μL of 1 M IAA to the reaction chamber (to a final concentration of 20 mM) and incubating in the dark at room temperature for 30 minutes to allow for glycoprotein alkylation;

[0138] S4: Enzymatic hydrolysis

[0139] a) After the reaction is completed, add 50 μL of 50 mM ammonium bicarbonate to the tip;

[0140] b) adding 2 U of PNGase F to the reaction chamber for enzymatic hydrolysis;

[0141] c) Seal the tip with parafilm and transfer to a water bath at 56°C for 3 h.

[0142] S5: Isolation and purification

[0143] a) Separation of sugar chains and proteins: After the reaction is completed, centrifuge thoroughly, remove the sealing film, add the centrifuge solution back to the tip, and centrifuge again. Repeat this process twice.

[0144] b) Purification: Add 200 μL of purified water to the tip and centrifuge to remove impurities and discard the centrifuge solution. Repeat 4 times.

[0145] c) Elution: Replace the centrifuge tube with a new one and elute the sugar chains with 40 μL of 20% ACN + 0.1% TFA + 79.9% H2O (v / v, mixed solution). Repeat 4 times.

[0146] d) collecting the centrifuged liquid and drying it using a vacuum centrifugal concentrator to obtain the N-glycans for subsequent detection or storage.

[0147] Comparative Example 1

[0148] The method for enriching N-glycans from 1 μg of IgG using the CC-TIP one-stop device combined with traditional methods includes the following steps:

[0149] S1: Reduction reaction of glycoprotein samples

[0150] a) Pour 50 μL of 50 mM ammonium bicarbonate (freshly prepared, pH 7.5-8.0) into a new 1.5 mL EP tube;

[0151] b) Add 1 μL of 500 mM DTT to the EP tube to make the final DTT concentration reach 10 mM;

[0152] c) Add 1 μg of IgG (glycoprotein sample) to the EP tube and cap the tube;

[0153] d) Place the EP tube in a water bath at 60°C and incubate for 45 minutes;

[0154] S2: Alkylation of glycoprotein samples

[0155] a) After the reaction is completed, cool to room temperature;

[0156] b) Add 1 μL of 1 M IAA to the EP tube (to a final concentration of 20 mM) and incubate in the dark at room temperature for 30 minutes to allow for glycoprotein alkylation.

[0157] S3: Enzymatic hydrolysis

[0158] a) After the reaction is completed, add 50 μL of 50 mM ammonium bicarbonate to the EP tube;

[0159] b) adding 2 U of PNGase F to the reaction chamber for enzymatic hydrolysis;

[0160] c) Seal the tip with parafilm and transfer to a water bath and incubate at 37°C for 16 hours.

[0161] S4: CC-TIP device construction and activation

[0162] Step 1: Take 400 μg of C8 membrane and fill it at the top of the 20 μL tip as the screening layer (support layer 6);

[0163] Step 2: Accurately weigh PGC in a centrifuge tube, mix thoroughly with acetonitrile, and then use a pipette to transfer 0.2 mg of PGC to the tip (conical reaction tube 2). After centrifugation and fixation, it serves as the sugar chain enrichment and purification layer 5;

[0164] Step 3: Accurately weigh C18 in a centrifuge tube, mix thoroughly with acetonitrile, and then use a pipette to transfer 1 mg of C18 to the tip. Centrifuge and fix it as protein enrichment layer 4;

[0165] Step 4: Centrifuge and activate the mixture using 100 μL each of 99.9% ACN + 0.1% TFA, 50% ACN + 0.1% TFA + 49.9% H2O, 5% ACN + 0.1% TFA + 94.5% H2O, 0.1% TFA + 99.9% H2O, and pure water.

[0166] S5: Isolation and purification

[0167] a) Separation of sugar chains and proteins: After the reaction is completed, the reaction solution is transferred to a Tip and centrifuged. The centrifuged solution is re-added to the Tip and centrifuged again. This is repeated twice. The reaction EP tube is washed with 50 μL of purified water, and the washing solution is transferred to the Tip and centrifuged again. The number of washes is 3 times.

[0168] b) Purification: Add 200 μL of purified water to the tip and centrifuge to remove impurities and discard the centrifuge solution. Repeat 4 times.

[0169] c) Elution: Replace the centrifuge tube with a new one and elute the sugar chains with 40 μL of 20% ACN + 0.1% TFA + 79.9% H2O (v / v, mixed solution). Repeat 4 times.

[0170] d) collecting the centrifuged liquid and drying it using a vacuum centrifugal concentrator to obtain the N-glycans for subsequent detection or storage.

[0171] The prepared N-glycans were analyzed using MALDI-TOF MS. Before N-glycan detection, external calibration was performed using peptide standards (Bruker). 2,5-DHB was selected as the matrix (prepared in 50% ACN to a final concentration of 20 mg / mL). The N-glycan sample was reconstituted with 4 μL of 40% ACN. A 2 μL sample was pipetted onto a clean stainless steel target plate. After drying slightly, 1 μL of the matrix was applied to the same location on the target plate. Repeated aspiration was performed to mix the sample and matrix. The target plate was then dried under negative pressure to allow uniform crystallization of the sample and matrix. After the target plate was completely dry, it was inserted into the ion source for analysis. The optimized laser intensity was selected, 10,000 spectra were accumulated, and N-glycan analysis was performed in positive ion reflectron mode.

[0172] Figure 3 、 Figure 4 and Figure 5 The figure shows the analytical test data of N-glycans obtained using the device of the present invention. As can be seen from the figure, the device can be used for N-glycan enrichment, where the enzymatic hydrolysis time is shortened from 16 hours in the traditional method to 2-3 hours, and the N-glycan preparation of 1μg IgG can be achieved. Compared with the existing trace sample pretreatment technology GPAT, which can prepare N-glycans from a starting amount of 10μg IgG, this method can achieve N-glycan preparation from even smaller amounts of protein, and is faster and more efficient.

[0173] Figure 6 This is the analytical test data of N-glycans obtained by using the device of the present invention in combination with the traditional method. It can be seen from the figure that the results of N-glycan enrichment using the device combined with the traditional method are close to those obtained by directly using the device of the present invention.

[0174] The method of the present invention utilizes a micro-device fused with octadecyl bonded silica gel and porous graphene (CC-TIP) to successfully integrate the conventional N-glycan preparation process: glycoprotein denaturation, reduction, alkylation, enzymatic hydrolysis and release, and glycan enrichment and purification, all into a single device. The enzymatic hydrolysis time is shortened from 16 hours in traditional methods to 2-3 hours. Simultaneously, all conventional glycan preparation steps are centralized within the same device, making the entire process faster and more efficient than traditional glycan preparation methods. Furthermore, regarding the preparation of trace IgG samples, the micro-sample pretreatment technology GPAT developed by Qian Xiaohong et al. can prepare N-glycans from a starting amount of 10 μg of IgG. In comparison, the CC-TIP device has achieved N-glycan preparation from as little as 1 μg of IgG and has the potential to prepare glycoprotein N-glycans at the nanogram level.

[0175] Specifically, the present invention adopts an integrated reaction chamber-conical reaction tube-gasket structure, integrating the traditional multi-step sugar chain preparation process (including protein enrichment, reductive alkylation, enzymatic hydrolysis to release sugar chains, sugar chain purification, etc.) into a single device, avoiding sample transfer loss and cross-contamination risks. The tandem design of the protein enrichment layer 4 (C18) and the sugar chain enrichment and purification layer 5 (PGC) can complete glycoprotein capture and sugar chain purification in the same device, reducing intermediate steps and improving recovery rate. At the same time, the C18 material (protein enrichment layer) has a high affinity for glycoproteins, and its mass ratio with the glycoprotein sample ensures the effective enrichment of low-abundance glycoproteins, which is suitable for complex samples with a wide dynamic range. The PGC material (sugar chain enrichment and purification layer) has a specific adsorption capacity for sugar chains, and its mass ratio with the glycoprotein sample optimizes the sugar chain recovery efficiency while reducing interference from non-sugar impurities. The use of a C8 sieve plate layer as the support layer 6 not only ensures interlayer stability, but also prevents filler leakage and improves the durability of the device.

[0176] Furthermore, during use, the reductive alkylation reaction adopts a high-temperature (70°C) short-time (45-60 min) strategy to accelerate the breakage of disulfide bonds, which is more efficient than traditional room-temperature reactions (such as 37°C for 1 h); PNGase F enzymatic hydrolysis is carried out in a 56-60°C water bath, which promotes enzyme activity while shortening the reaction time (2-3 h), which is better than the conventional 37°C long-term incubation (such as overnight reaction); in general, the structure of the device is suitable for centrifugal operation, and can drive liquid flow through centrifugal force, reducing manual operation errors. It is suitable for use with an automated workstation, and the standardized design of the activation solution (acetonitrile + trifluoroacetic acid gradient) and eluent (acetonitrile / trifluoroacetic acid mixed solution) facilitates direct docking with a liquid chromatography-mass spectrometry (LC-MS) system, thereby improving subsequent analysis throughput.

[0177] Secondly, the present invention can adapt to different sample types (such as high-abundance serum glycoproteins or low-abundance cell lysates) by adjusting the filling ratio of C18 and PGC. The elution conditions (acetonitrile / trifluoroacetic acid) are compatible with subsequent mass spectrometry analysis or derivatization requirements such as fluorescent labeling, expanding the application scenarios of the method. The technical solution of the present invention significantly improves the efficiency, recovery rate and reproducibility of N-glycan preparation through integrated design, efficient material combination and optimized reaction conditions, while reducing the technical threshold and operational complexity, providing a reliable and high-throughput tool for glycosylation research.

[0178] The above content is only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A CC-TIP one-stop device, characterized in that: include: A reaction chamber (1), a conical reaction tube (2) and a gasket (3); the reaction chamber (1) is connected to the large end of the conical reaction tube (2) through the gasket (3); a protein enrichment layer (4), a sugar chain enrichment and purification layer (5) and a support layer (6) are sequentially arranged inside the conical reaction tube (2) from one end of the reaction chamber (1) to the small end.

2. The CC-TIP one-stop device according to claim 1, characterized in that: The material of the protein enrichment layer (4) is C18; the material of the sugar chain enrichment and purification layer (5) is PGC; the supporting layer (6) is a sieve plate layer; the material of the sieve plate layer is C8.

3. The method for preparing a CC-TIP one-stop device according to claim 1 or 2, characterized in that: The following steps are involved: The reaction chamber (1) is connected to the large end of the conical reaction tube (2) through a gasket (3); then the protein enrichment layer (4), the sugar chain enrichment and purification layer (5) and the support layer (6) are filled in the conical reaction tube (2) in sequence.

4. The method for preparing a CC-TIP one-stop device according to claim 3, characterized in that: The specific steps include: The reaction chamber (1) is connected to the large end of the conical reaction tube (2) via a gasket (3); a C8 membrane is then filled into the small end of the conical reaction tube (2) as a sieve plate layer; Weigh PGC in a centrifuge tube, mix with acetonitrile, and transfer to a conical reaction tube (2) using a pipette. After centrifugation and fixation, use it as a sugar chain enrichment and purification layer (5); Weigh C18 in a centrifuge tube, mix with acetonitrile, and transfer to a conical reaction tube (2) using a pipette. Centrifuge and fix to form a protein enrichment layer (4). Then, the centrifugal activation solution is added into the reaction chamber (1) for centrifugal activation; In terms of volume percentage, the centrifugal activation solution includes 99.9% ACN + 0.1% TFA, 50% ACN + 0.1% TFA + 49.9% H2O, 5% ACN + 0.1% TFA + 94.5% H2O, and 0.1% TFA + 99.9% H2O; Each 20 μL conical reaction tube (2) was filled with 200-400 μg of C8 membrane; The mass ratio of the PGC to the glycoprotein sample is 100 to 1,000; The mass ratio of the C18 to the glycoprotein sample is greater than 1,000.

5. The method for using the CC-TIP one-stop device according to claim 1 or 2, characterized in that: The CC-TIP one-stop device for preparing N-glycans includes the following steps: Adjust the filling amount of PGC and C18 to form a sugar chain enrichment and purification layer (5) and a support layer (6); Then, a glycoprotein sample, ammonium bicarbonate and DTT are added to the reaction chamber (1) to obtain a mixed solution; after the mixed solution enters the conical reaction tube (2) through the gasket (3), the large end of the conical reaction tube (2) is sealed, and then the conical reaction tube (2) is placed in a water bath for incubation to perform a reduction reaction. After the reaction is completed, IAA is added to the conical reaction tube (2) and a glycoprotein alkylation reaction is performed in a dark environment. After the reaction is completed, ammonium bicarbonate and PNGase F are added to the conical reaction tube (2), and the conical reaction tube (2) is placed in a water bath for incubation to perform an enzymatic hydrolysis reaction. After the reaction is completed, a reaction product solution is obtained; The reaction product solution is sequentially separated, purified and eluted to obtain N-sugar chains.

6. The method for using the CC-TIP one-stop device according to claim 5, characterized in that: The mass ratio of the C18 to the glycoprotein sample is greater than 1000:1; the mass ratio of the PGC to the glycoprotein sample is no more than 500:

1.

7. The method for using the CC-TIP one-stop device according to claim 5, characterized in that: In the mixed solution, the molar concentrations of ammonium bicarbonate, DTT, and IAA are 50 mM, 10 mM, and 20 mM, respectively; The reduction reaction temperature is 70°C in a water bath and the time is 45 to 60 minutes; The concentration of the ammonium bicarbonate is 50 mM; the concentration of the DTT is 10 mM.

8. The method for using the CC-TIP one-stop device according to claim 5, characterized in that: The glycoprotein alkylation reaction is carried out at room temperature for 0.5 to 1 hour.

9. The method for using the CC-TIP one-stop device according to claim 5, characterized in that: Ammonium bicarbonate and PNGase F were added to the conical reaction tube (2) until the concentrations of DTT and IAA in the mixed solution were diluted to 1 / 2 of the original concentrations; the amount of PNGase F used was 2U PNGase F per 1 μg of glycoprotein sample.

10. The method for using the CC-TIP one-stop device according to claim 5, characterized in that: The enzymatic hydrolysis reaction temperature is 56-60°C in a water bath and the time is 2-3h; The elution treatment is carried out using a mixed solution of acetonitrile, trifluoroacetic acid and water.

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