A lectin affinity-based flow analysis method for serum glycosylation

Abstract

The present application belongs to the technical field of glycosylation detection and analysis, and particularly relates to a serum glycosylation flow analysis method based on lectin affinity. The present application enriches target molecules in a sample to be measured by coupling a polystyrene microsphere with a decoy molecule, and then incubates the polystyrene microsphere with a fluorescently labeled lectin, and directly analyzes the glycosylation information of the target molecules by a flow cytometer. In the present application, the decoy molecule of the target protein is coupled to the polystyrene microsphere to enrich the target protein, and the target protein is focused for subsequent analysis, which can effectively reduce the field of view and improve the specificity. After the lectin is combined with the target protein, the fluorescence analysis of the flow cytometry is directly performed, which can effectively reduce the damage of the washing operation to the affinity of the lectin, and thus overcomes the weakness of the weak affinity between the lectin and the sugar chain. Moreover, only a small amount of sample is required to realize the analysis of multiple lectins, and the washing-free real-time analysis can be realized, which provides a new way for the analysis of glycosylation by using lectin, and has important potential application value.

Description

Technical Field

[0001] This invention belongs to the field of glycosylation detection and analysis technology, specifically relating to a flow cytometry method for serum glycosylation based on lectin affinity. Background Technology

[0002] Glycosylation is an important post-translational modification of proteins, playing a wide role in many life activities such as human physiology and pathology. In cells, the glycosylation of most proteins occurs along the secretory pathway. Glycoproteins secreted in body fluids are considered a characteristic window of an individual's health status. Because glycan modification has no specific template and its secondary structure is complex, especially given the wide dynamic range of serum protein abundance, glycosylation modification is highly heterogeneous at both the macroscopic and microscopic levels, making the analysis of serum glycosylation modification still quite challenging.

[0003] Currently, liquid chromatography-tandem mass spectrometry (LC-MS / MS) has become the mainstream method for glycosylation analysis due to its accuracy and sensitivity, simultaneously acquiring information on glycans, peptides, and glycosylation sites. However, this method is highly dependent on mass spectrometry technology and suffers from drawbacks such as high cost, cumbersome operation, long processing time, significant sample loss, low throughput, and low ionization efficiency of glycosylated peptides. Furthermore, when processing serum samples, the large dynamic range of protein abundance can lead to high-abundance proteins significantly masking low-abundance proteins. Therefore, the widespread adoption of LC-MS / MS in clinical and research applications is limited.

[0004] Furthermore, phytolectins, as highly specific binding proteins to sugars on glycoproteins, are frequently used to analyze protein glycosylation. Through the interaction between different lectins and specific glycans, the separation and purification of glycopeptides and affinity imaging of glycan structures can be achieved. Compared to LC-MS / MS, lectin analysis does not rely on mass spectrometry, is simple to operate, and saves time and effort. Moreover, nearly 1000 phytolectins have been discovered, widely distributed in many plant groups such as legumes and solanaceae. However, because the glycan structures corresponding to most lectins are not well-defined, and the affinity between lectins and glycans is not as high as that of traditional antibody-antigen assays, only a few lectins, such as ConA, can currently be directly applied to glycoprotein enrichment among the thousands of phytolectins. Therefore, exploring new approaches to glycosylation analysis using lectins has become a research focus for biotechnology researchers both domestically and internationally. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, this invention provides a serum glycosylation analysis method based on lectin affinity. It utilizes polystyrene microspheres, EDC cross-linking reactive groups, decoy molecules that capture target molecules, and fluorescently labeled lectins for glycosylation analysis. It can perform multiple lectin analyses with only a small amount of sample and can achieve wash-free real-time analysis, overcoming the disadvantage of weak affinity between lectins and sugar chains.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] This invention provides a flow cytometry method for serum glycosylation based on lectin affinity. Specifically, the method involves first covalently crosslinking decoy molecules onto polystyrene microspheres using EDC and EDC crosslinking reactive groups to prepare decoy microbeads. The decoy molecules contain primary amine groups -NH2. Then, the decoy microbeads are co-incubated with the test sample and a lectin fluorescent probe in a binding buffer. Finally, the fluorescence signal of the decoy microbeads is directly read using flow cytometry to reflect the affinity abundance of lectin. By analyzing the correspondence between lectin and glycosylation, the glycosylation modification status of the target molecule can be determined.

[0008] Preferably, as a preferred embodiment of the present invention, the above-described serum glycosylation flow cytometry analysis method based on lectin affinity includes the following steps:

[0009] S1. Mix the polystyrene microsphere suspension with EDC and EDC crosslinking reaction groups, and react in a molecular hybridization apparatus for 1-3 hours;

[0010] S2. After removing the supernatant, resuspend the microspheres, add bait molecules and incubate overnight at 4°C in the dark. After removing the supernatant again, resuspend the microspheres to obtain bait microbeads.

[0011] S3. Add bait beads, test sample and lectin fluorescent probe to binding buffer, mix well and incubate for molecular hybridization in a molecular hybridization instrument for 1-2 hours.

[0012] S4. After incubation, flow cytometry was used for analysis. Microspheres were selected by FSC / SSC, and the average fluorescence intensity was analyzed. The glycosylation modification status of the target molecule was obtained by the correspondence between lectin and glycosylation.

[0013] Preferably, the binding buffer is an LBB buffer, which includes 20 mM HEPES, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2, and 1 mM ZnCl2.

[0014] This invention provides an analytical scheme for target protein enrichment combined with lectin affinity flow cytometry. First, the target protein is captured by cross-linking a bait molecule containing a primary amine group (-NH2) onto polystyrene microspheres. Then, fluorescein-labeled lectin is used in conjunction with flow cytometry to read the glycosylation modification information of the target molecule. This method utilizes the liquid-phase enrichment effect of the cross-linked polystyrene microspheres to "concentrate" the target protein on the surface of the microspheres, increasing the probability of lectin binding to the glycan chains of the target protein and facilitating subsequent wash-free detection. Furthermore, the incubation environment for the lectin probe (i.e., LBB buffer) is further optimized by adding a high concentration of divalent metal ions (including Ca2+). 2+ Mg 2+ Zn 2+ Mn 2+ This further enhances the binding strength between lectins and sugar chains, enabling the analysis of multiple lectins with only a small amount of sample, and allowing for real-time analysis without washing, thus overcoming the disadvantage of weak affinity between lectins and sugar chains.

[0015] Preferably, the decoy molecule includes an antibody, nucleic acid, streptavidin, or Protein-AGL. More preferably, the decoy molecule is Protein-AGL.

[0016] Preferably, the EDC crosslinking reaction group is sulfo-NHS.

[0017] Preferably, the polystyrene microspheres are carboxylated polystyrene microspheres.

[0018] Preferably, the lectin fluorescent probes include ConA, DBA, PNA, RCA-1, SBA, UEA-1, WGA, DSL, ECL, GSL-II, Jacalin, LEL, and STL.

[0019] Preferably, the sample to be tested is serum or plasma.

[0020] Preferably, after overnight incubation in step S2, the microspheres are incubated with BSA at room temperature for 1-3 hours to block the cross-linking groups. More preferably, after blocking the cross-linking groups, the supernatant is removed, the microspheres are washed with PBS, and then the microspheres are resuspended in PBST (containing 0.1% Tween-20).

[0021] Preferably, in step S1, a polystyrene microsphere suspension is prepared using a protective liquid.

[0022] Preferably, in step S1, after preparing the polystyrene microsphere suspension, the microspheres are first washed with MES buffer, then resuspended with MES buffer, and finally mixed with EDC and EDC crosslinking reaction groups.

[0023] Preferably, before adding the decoy molecules in step S2, the microspheres are washed with PBS and then resuspended in PBS; after incubation overnight, the microspheres are also washed with PBS and then resuspended in PBS.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] This invention discloses a flow cytometry method for serum glycosylation based on lectin affinity. This method enriches the target molecule in the sample by coupling a decoy molecule to polystyrene microspheres, and then incubates it with fluorescently labeled lectins before directly analyzing the glycosylation information of the target molecule via flow cytometry. Because this invention enriches the target protein by coupling a decoy molecule to the polystyrene microspheres, focusing the analysis on the target protein, it effectively narrows the field of view and improves specificity. Simultaneously, direct flow cytometry fluorescence analysis after lectin binding to the target protein effectively reduces the damage to lectin affinity caused by washing operations, fully presenting the glycosylation modification information of the target protein. This overcomes the disadvantage of weak affinity between lectins and glycans. Furthermore, it requires only a small sample to achieve multiple lectin analyses and enables wash-free real-time analysis.

[0026] The reagents used in this invention mainly include polystyrene microspheres, EDC cross-linking reactive groups, decoy molecules for capturing target molecules, and fluorescently labeled lectins. The reagents are easy to assemble, highly customizable, simple to operate, low in cost, and require little sample (only 2 μL of serum is needed for each lectin indicator). At the same time, through molecular cross-linking, any antibody and nucleic acid aptamer decoy molecules can be cross-linked with polystyrene microspheres, which can quickly achieve the capture and glycosylation modification analysis of target molecules, providing a new approach for the analysis of glycosylation using lectins and has important potential application value. Attached Figure Description

[0027] Figure 1 The flowchart for the operation of serum glycosylation based on lectin affinity flow cytometry is as follows: (First, the decoy molecules are covalently cross-linked onto polystyrene microspheres using the EDC / sulfo-NHS two-step method to prepare the decoy microspheres; then, the decoy microspheres are used to capture the target molecules in the sample to be tested; finally, the lectin affinity abundance of the target molecules is analyzed using a lectin fluorescent probe).

[0028] Figure 2 Silver staining image showing successful capture of immunoglobulins from the sample by recombinant protein-AGL;

[0029] Figure 3 Serum lectin affinity profile analysis [using BSA (known to be unglycosylated) as a control, analyzed the abundance of affinity between immunoglobulins and 14 lectins in serum samples; "glycan structure" represents the glycan chain modification corresponding to the lectin, and "flow cytometry" is a bar chart of the average fluorescence intensity of lectin affinity between BSA and serum];

[0030] Figure 4 The results of the PNGase-F glycoside hydrolase experiment (affinity experiments of four lectins, ConAn, RAC-I, WGA and Jacalin, were performed after removing glycosylation modifications from serum samples using PNGase-F). Detailed Implementation

[0031] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0032] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0033] Example 1: A flow cytometry method for serum glycosylation based on lectin affinity

[0034] In this embodiment, recombinant protein-AGL lyophilized powder (purchased from GPLBIO Bioreactors, catalog number GP24249, reconstituted in ultrapure water to 1 mg / mL, stored at -80℃) was used as a bait molecule, and the antibodies in the serum sample were used as target molecules for flow cytometry analysis of serum glycosylation.

[0035] like Figure 1 As shown, this method mainly consists of two parts: loading decoy molecules and fluorescence analysis based on lectin affinity. First, using an EDC / sulfo-NHS two-step method, the decoy molecule protein-AGL is covalently cross-linked onto carboxylated polystyrene (PS) microbeads (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number P107809, CAS number 9003-53-6, particle size 5.0-5.9 μm, concentration 5% w / v. Thoroughly suspended before use and stored at -4℃). Unreacted molecules are washed away, and the cross-linking groups are blocked with BSA before subsequent use. In use, the decoy PS microspheres are co-incubated with the sample to be tested, unbound molecules are washed away, and then mixed with the lectin fluorescent probe. The mixture is then co-incubated in binding buffer LBB (20 mM HEPES, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2, 1 mM ZnCl2). No washing is required throughout the process. Finally, the fluorescence signal of the decoy PS microspheres is directly read using flow cytometry to reflect the affinity and abundance of the lectin. The glycosylation modification status of the target molecule is determined by the correspondence between the lectin and glycosylation. The specific method is as follows:

[0036] 1. Loading decoy molecules:

[0037] 1) Vortex mix the carboxylated PS microsphere suspension, take 1 mL, and centrifuge at 10000 rpm to remove the protective solution;

[0038] 2) Resuspend the microspheres in 500 μL of 4-morpholine ethanesulfonic acid buffer (MES, pH=4.5), wash the microspheres once, and remove the supernatant;

[0039] 3) Add 500 μL of MES resuspended microspheres;

[0040] 4) Weigh 25mg of activator Sulfo-HNS (Chinese name: N-hydroxythiosuccinimide, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number: H109337, CAS number: 106627-54-7), add 500uL of MES to dissolve, and mix thoroughly with the carboxylated PS microsphere suspension from the previous step.

[0041] 5) Weigh 25 mg of crosslinking agent EDC (Chinese name: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number: E106172, CAS number: 25952-53-8), add it directly to the reaction system of the previous step, and place it in a molecular hybridization apparatus at 25℃ for crosslinking reaction for 2 h;

[0042] 6) Centrifuge at 10,000 rpm to remove the supernatant, and wash twice with PBS;

[0043] 7) Add 500uL of PBS to resuspend the microspheres, and then add 0.2mg of bait molecule (recombinant protein-AGL);

[0044] 8) Incubate overnight in a shaker at 4°C, away from light;

[0045] 9) Centrifuge at 10,000 rpm to remove the supernatant, and wash once with PBS;

[0046] 10) Add 500uL of PBS to resuspend the microspheres, then add 200uL of BSA (2mg / mL) and incubate with the microspheres at room temperature for 2h to block the cross-linking groups;

[0047] 11) Centrifuge at 10,000 rpm to remove the supernatant, and wash twice with PBS;

[0048] 12) Add 1 mL of PBST (1×PBS buffer containing 0.1% Tween-20) to resuspend the microspheres to obtain the bait PS microspheres, and store at 4℃.

[0049] 2. Fluorescence analysis:

[0050] 1) The vortex fully suspends the PS microspheres as decoys;

[0051] 2) Mix 2 μL of the sample to be tested (serum or plasma from a healthy person) and 10 μL of bait PS microspheres in 1 mL of PBS, and incubate on a shaker at 25 °C for 1 h;

[0052] 3) Centrifuge at 10000 rpm for 1 min, discard the supernatant, and resuspend the PS microspheres in 200 uL LBB (Lectin Binding Buffer) buffer (containing 20 mM HEPES, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2, and 1 mM ZnCl2) for later use.

[0053] 4) Add 0.2 μL of lectin fluorescent probes (ConA, DBA, PNA, RCA-1, SBA, UEA-1, WGA, DSL, ECL, GSL-II, Jacalin, LEL, STL, etc. All lectins were purchased from Vector Biotech. The lectin fluorescent probes are liquid reagents with a concentration of 2 mg / mL. In this example, the working concentration of lectin is 2 μg / mL.) to the above system and incubate at 4 °C for 1 h. Then, analyze the incubation using flow cytometry. Use FSC / SSC to select microspheres, with Ex / Em = 495 / 515 nm channels, and analyze the mean fluorescence intensity (MFI).

[0054] 3. Analysis Results

[0055] (1) Detection of serum antibodies enriched by recombinant Protein-AGL polystyrene microbeads

[0056] Staphylococcus aureus protein A (Protein-A), Streptococcus protein G (Protein-G), and Peptostreptococcus protein L (Protein-L) are typical immunoglobulin-binding proteins. They can bind to most mammalian immunoglobulins, but their binding modes and binding spectra differ. Recombinant protein Protein-AGL concentrates five Ig binding domains from protein-A, two from protein-G, and five from protein-L. These three proteins complement each other, exhibiting not only better binding ability but also a broader binding spectrum, showing great promise for applications. Using the bait loading scheme in this embodiment, recombinant protein-AGL was loaded onto PS microbeads and then incubated with serum and plasma samples, respectively, to capture the target molecule immunoglobulin in the samples. Silver staining analysis showed that the heavy chain (50 kDa) and light chain (25 kDa) of the immunoglobulin were clearly visible. Figure 2 This demonstrates that recombinant protein-AGL PS microspheres can significantly capture immunoglobulins in samples, and the loading method is reliable.

[0057] (2) Serum agglutinin analysis

[0058] Lectins are a class of molecules that bind to sugar chains; each lectin has a specific relationship with a sugar chain. Figure 4 Therefore, the degree of binding of different lectins can reflect the degree of glycosylation modification. This example demonstrates 14 commercially available lectin fluorescent probes (ConA, DBA, PNA, RCA-1, SBA, UEA-1, WGA, DSL, ECL, GSL-II, Jacalin, LEL, STL). The lectins bound to the test beads in LBB buffer for 1 hour, and without washing, serum immunoglobulin lectin affinity flow cytometry was performed to calculate the mean fluorescence intensity (MFI). Bovine serum albumin (without glycosylation) was used as a negative control. The results show ( Figure 3 Except for lectin STL, all other lectins showed strong fluorescence signals, indicating that the modified LBB buffer no-wash protocol is feasible.

[0059] (3) PNGase-F glycoside hydrolase experiment

[0060] To further demonstrate the reliability of this protocol in analyzing the glycosylation modification of target molecules, serum samples were deglycosylated using the glycoside hydrolase PNGase-F (samples were divided into a control group (NC) and a treatment group (PNGase F), mixed with deglycosylation buffer to 10 μL, with the treatment group receiving 1000 U of PNGase F, and incubated at 37°C for 2 h; the PNGase F kit was purchased from New England Biolabs, catalog number: P0704, and specific operation was performed according to the reagent instruction manual). This eliminated glycosylation in the treatment group samples before subsequent lectin affinity analysis. The results showed ( Figure 4 The affinity of serum samples deglycosylated by PNGase F for ConA and RAC-I lectins decreased significantly, indicating that the MFI value of the lectin fluorescent probes does indeed reflect the glycosylation modification status of the samples, further demonstrating the authenticity and reliability of this method.

[0061] In summary, this invention enriches target molecules in serum or plasma by coupling decoy molecules to polystyrene microspheres, and then directly analyzes the glycosylation information of the target molecules by flow cytometry after incubation with fluorescently labeled lectins. The entire process can effectively reduce the damage to lectin affinity caused by washing operations, and multiple lectin analyses can be achieved with only a small amount of sample. Furthermore, it can achieve wash-free real-time analysis, thus overcoming the disadvantage of weak affinity between lectins and glycan chains.

[0062] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A flow cytometry method for serum glycosylation based on lectin affinity, characterized in that, First, decoy molecules were covalently crosslinked onto polystyrene microspheres using EDC and EDC crosslinking reactive groups to prepare decoy microbeads. The decoy molecules contained primary amine groups -NH2. Then, the decoy microbeads were co-incubated with the test sample and lectin fluorescent probe in binding buffer. Finally, the fluorescence signal of the decoy microbeads was directly read using flow cytometry to reflect the affinity abundance of lectin. By the correspondence between lectin and glycosylation, the glycosylation modification status of the target molecule was obtained. The serum glycosylation flow cytometry analysis method based on lectin affinity specifically includes the following steps: S1. Mix the polystyrene microsphere suspension with EDC and EDC crosslinking reactive groups, and react in a molecular hybridization apparatus for 1-3 hours; the polystyrene microspheres are carboxylated polystyrene microbeads, and the EDC crosslinking reactive groups are sulfo-NHS. S2. After removing the supernatant, resuspend the microspheres, add the decoy molecule, incubate overnight at 4°C in the dark, remove the supernatant again, and resuspend the microspheres to obtain decoy microbeads; the decoy molecule is Protein-AGL; S3. Add bait beads, the sample to be tested, and lectin fluorescent probes to the binding buffer, mix well, and incubate for molecular hybridization in a molecular hybridization instrument for 1-2 hours; the lectin fluorescent probes include ConA, DBA, PNA, RCA-1, SBA, UEA-1, WGA, DSL, ECL, GSL-II, Jacalin, and LEL. S4. After incubation, flow cytometry was used for analysis. Microspheres were selected by FSC / SSC, and the average fluorescence intensity was analyzed. The glycosylation modification status of the target molecule was obtained by the correspondence between lectin and glycosylation.

2. The serum glycosylation flow cytometry analysis method based on lectin affinity according to claim 1, characterized in that, The sample to be tested is serum or plasma.

3. The serum glycosylation flow cytometry analysis method based on lectin affinity according to claim 1, characterized in that, The binding buffer is an LBB buffer, which includes 20 mM HEPES, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2, and 1 mM ZnCl2.

4. The serum glycosylation flow cytometry analysis method based on lectin affinity according to claim 1, characterized in that, After incubating overnight in step S2, the microspheres are incubated with BSA at room temperature for 1-3 hours to block the cross-linking groups.

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