A method and application for detecting N-glycans based on in-gel digestion and electrophoresis.

Abstract

This invention discloses a method and application for detecting N-glycans based on in-gel enzymatic digestion and electrophoresis. The method includes the following steps: separating glycoprotein samples and cutting target bands by gel electrophoresis; decolorizing and dehydrating the target glycoprotein particles; adding enzyme solution for direct enzymatic release of N-glycans within the gel; transferring the enzyme digestion solution after sonication, drying, and labeling the N-glycan sample; and finally, separating, detecting, and analyzing the N-glycans using capillary electrophoresis. This invention innovatively combines in-gel enzymatic digestion with capillary electrophoresis, avoiding the complex sample transfer and pretreatment steps of traditional methods, significantly simplifying the operation process, reducing sample loss, and making it particularly suitable for the efficient detection of low-abundance N-glycans. This method features high sensitivity, high resolution, and high throughput, and can accurately analyze N-glycans in complex biological samples, with wide applications in disease biomarker screening, clinical diagnosis, and glycomics research.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method and application for detecting N-glycan chains based on in-gel enzyme digestion and electrophoresis. Background Technology

[0002] Glycosylation is one of the important post-translational modifications of proteins, with N-glycosylation playing a crucial role in various biological processes and disease development. N-glycosylation-modified glycans exhibit diverse structures, influencing protein stability, function, folding, and intercellular signaling. Aberrant N-glycosylation patterns are often closely associated with diseases, particularly in research on cancer, neurodegenerative diseases, and immune disorders, where N-glycans are considered potential biomarkers. Therefore, efficient detection and analysis of N-glycans are of great significance for elucidating disease mechanisms, discovering diagnostic biomarkers, and developing therapeutic approaches.

[0003] Currently, common methods for N-glycan detection mainly include mass spectrometry (MS), liquid chromatography (LC), and capillary electrophoresis (CE). MS, with its high sensitivity and high resolution, has become the primary tool for N-glycan analysis, enabling precise identification of the composition and structure of glycans. However, MS typically requires complex sample pretreatment steps, such as glycan release, purification, and derivatization. This is especially true for glycoproteins in complex biological samples (such as serum and cell lysates), requiring prior enrichment of specific glycoproteins or glycopeptides. This process results in significant sample loss, cumbersome procedures, and reduced analytical efficiency, particularly for the detection of N-glycans in low-abundance glycosylated proteins.

[0004] To simplify the N-glycan detection process, gel electrophoresis techniques (such as SDS-PAGE) have become a crucial preliminary separation step. SDS-PAGE allows proteins to be separated according to molecular weight and visualized within the gel. This process not only effectively reduces sample complexity but also enables the separation and enrichment of specific target proteins, providing a foundation for subsequent N-glycan analysis. However, despite the widespread application of SDS-PAGE in protein-related analyses, the technology for directly detecting N-glycans from the gel remains immature. How to efficiently release and detect N-glycans directly from the gel after protein separation has become a key challenge in glycomics research.

[0005] In-gel digestion is a widely used technique in proteomics research. It involves enzymatically digesting proteins within a gel to generate peptide fragments for subsequent mass spectrometry analysis. This technique avoids the difficulty of protein redissolution in solution digestion, and is particularly suitable for the analysis of hydrophobic proteins. However, despite its widespread application in proteomics, its application in N-glycan detection has not been fully developed, mainly due to the following technical challenges: (1) Complex sample pretreatment: Although in-gel digestion has fewer difficulties in protein redissolution, the separation and detection of N-glycans still face significant challenges due to their different distribution in glycoproteins. Existing in-gel digestion techniques require complex sample pretreatment steps (such as removing protein contaminants and purifying glycans) when processing glycoproteins, which increases the complexity and time cost of the detection process. (2) Limited detection of low-abundance glycans: Although in-gel digestion has advantages in peptide generation, the detection of low-abundance glycans remains a challenge in glycan analysis. Existing methods are not sensitive enough to low-abundance N-glycans, especially in complex biological samples. The extraction and enrichment of glycans are often limited by the sample type and glycan species, affecting the detection efficiency. (3) Limitations of mass spectrometry: In-gel digestion technology usually requires mass spectrometry analysis. However, mass spectrometry has high requirements for sample pretreatment, especially in glycan analysis, where the sensitivity and resolution of the mass spectrometer are affected by sample purity, impurities, and detection conditions. Therefore, existing technologies may cause signal overlap or background noise when processing complex glycoprotein samples, affecting the accuracy and efficiency of the analysis.

[0006] Although gel electrophoresis and in-gel enzyme digestion have significant advantages in protein analysis, combining them for N-glycan detection still faces some challenges: (1) Complex operation process and high risk of sample loss: Traditional in-gel enzyme digestion requires extracting peptide fragments from the gel, transferring them to a solution for enzymatic digestion, and then performing glycan analysis. However, such steps increase the complexity of sample processing, and each sample transfer and extraction process will result in some sample loss. For complex samples (such as serum, cell lysates, etc.), these steps will significantly affect the recovery rate of glycans, thereby affecting the accuracy and reliability of glycan analysis. (2) Poor technical compatibility, affecting high-throughput analysis: After separating proteins using gel electrophoresis, additional protein transfer and purification steps are usually required before subsequent glycan analysis. These steps not only increase experimental time and operational difficulty, but also affect the throughput of the technology. In practical applications, especially when a large number of samples need to be processed, the poor compatibility of existing methods makes rapid, high-throughput glycan detection difficult. (3) Combining separation and glycan analysis is challenging: Gel electrophoresis and intragel enzyme digestion are primarily for protein analysis, while glycan separation and analysis typically require different technical support. Combining these two techniques to effectively release glycans from proteins and ensure their successful subsequent separation, identification, and quantitative analysis remains a technical challenge. Existing technologies often face issues with separation efficiency and the accuracy of glycan quantitative analysis, especially given the wide variety of glycan types and significant molecular weight differences.

[0007] In summary, existing N-glycan detection techniques suffer from problems such as complex sample preparation, low efficiency, and insufficient sensitivity, particularly in the detection of low-abundance glycosylated proteins and the effective release, separation, and identification of glycans on intragel proteins. Therefore, developing a simplified sample pretreatment method that improves detection efficiency and sensitivity is of great significance. Although gel electrophoresis and intragel enzyme digestion techniques are widely used in protein analysis, their application in N-glycan detection still requires further exploration and optimization. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a method and application for N-glycan detection based on in-gel digestion and electrophoresis. By innovatively combining in-gel digestion and electrophoresis, it offers a highly efficient, sensitive, and easy-to-operate method for N-glycan detection. This method significantly simplifies the operational process, improves detection efficiency and accuracy, and is particularly suitable for the analysis of complex biological samples, showing broad application potential in disease research and clinical diagnosis.

[0009] This invention is achieved through the following technical solution:

[0010] A method for detecting N-glycan chains based on in-gel enzyme digestion and electrophoresis includes the following steps:

[0011] Step 1) Separate the glycoprotein samples using gel electrophoresis to prepare a gel;

[0012] Step 2) Cut the target glycoprotein bands on the gel prepared in step 1) to obtain target glycoprotein particles;

[0013] Step 3) Place the target glycoprotein particles obtained in Step 2) into an EP tube, add decolorizing solution to decolorize the target glycoprotein particles, repeat the decolorization 1 to 3 times until the blue color disappears completely;

[0014] Step 4) After completely discarding the decolorizing solution in the EP tube, add 100% acetonitrile solution to the EP tube to dehydrate the target glycoprotein particles.

[0015] Step 5) After completely discarding the 100% acetonitrile solution in the EP tube, add the enzyme solution to the EP tube and incubate at 37°C overnight to release the N-glycan chains.

[0016] Step 6) Place the EP tube treated in step 5 into an ultrasonic cleaner. After ultrasonic treatment, transfer the enzyme digestion solution into a new EP tube, dry and label it to prepare an N-glycan sample.

[0017] Step 7) Separate, detect and analyze the N-glycan sample prepared in step 6) using a capillary electrophoresis apparatus.

[0018] Preferably, the specific steps for preparing the gel in step 1) are as follows: After separating the glycoprotein sample according to its molecular weight, the gel is placed on a shaker and stained with 2-5 mL of Coomassie Brilliant Blue staining solution for at least 2 hours. Then, the stained gel is destained with 2-5 mL of destaining solution for 30-60 minutes. The destaining is repeated 3-4 times until the protein bands are clearly visible.

[0019] Preferably, in step 2), after the target glycoprotein band is cut, it needs to be cut again into 1-2 mm pieces. 2 The gel particles were transferred into EP tubes and stored at 4°C.

[0020] Preferably, the specific steps of decolorization in step 3) are as follows: 100-300 μL of decolorizing solution is added to an EP tube containing the target glycoprotein particles, and the tube is placed in a constant temperature mixer at 800 rpm and 37°C for 30-60 min to decolorize. The liquid is then discarded, and the process is repeated 1-3 times until the blue color fades completely.

[0021] Preferably, the specific steps of the dehydration treatment in step 4) are as follows: Add 100-300 μL of 100% acetonitrile solution to the EP tube containing the target glycoprotein particles, shake at 37°C for about 10-30 minutes until the particles are dehydrated and turn white, discard the liquid, and evaporate the remaining acetonitrile solution.

[0022] Preferably, the specific steps of incubation in step 5) are as follows: Add 100-300 μL of enzyme solution to an EP tube containing the target glycoprotein particles, incubate at 37°C for 30-60 min. If the particles cannot be completely submerged at this time, add 25 mM NH4HCO3 solution to completely submerge the particles, and incubate at 37°C overnight.

[0023] Preferably, the enzyme solution is formulated as follows: PNGase F enzyme and sialidase are dissolved in 25mM NH4HCO3 solution, the final concentration of PNGase F enzyme is 1000-1500 U / mL, and the final concentration of sialidase is 100-150 U / mL.

[0024] Preferably, the specific steps for preparing the N-glycan sample in step 6) are as follows: Place the overnight digested EP tube in an ultrasonic cleaner, sonicate for 30–60 min, vortex for 10–30 s, centrifuge for 10–30 s, and repeat this operation twice; transfer all the liquid from the sonicated EP tube to a new EP tube, centrifuge for 10–30 s, and dry at 80°C for 1–2 h until completely dry, then cool to 4°C; then add 10–20 μL to the dried EP tube. Purified water is repeatedly blown around the inner wall until completely dry. After blowing, the tube is vortexed for 1-2 minutes, centrifuged for 10-30 seconds, and dried at 80°C for 30-60 minutes until completely dry. Then, the tube is cooled to 4°C. 2-5 μL of fluorescent labeling solution is added to the dried EP tube, the tube is capped, numbered, centrifuged for 10-30 seconds, and placed at 90°C for 1-2 hours. After cooling to 4°C, 10-30 μL of stop solution is added, the tube is vortexed for 10-30 seconds to mix thoroughly, and centrifuged for 10-30 seconds to obtain the N-glycan sample.

[0025] Preferably, the amount of N-glycan sample used in step 7) is 5-10 μL, and the capillary electrophoresis instrument is an ABI 3500dx gene sequencer.

[0026] The above-mentioned N-glycan detection method based on in-gel digestion and electrophoresis is applied in the preparation of N-glycan detection kits.

[0027] The beneficial effects of this invention are as follows:

[0028] (1) This invention innovatively performs enzymatic cleavage of N-glycans directly within the gel, ensuring the efficient release of N-glycans for subsequent separation, identification, and quantitative analysis, while avoiding frequent sample transfers and complex pretreatment steps in traditional methods. This integrated technology significantly simplifies the operation process and reduces sample loss, especially for the detection of low-abundance N-glycans. By combining enzymatic cleavage with gel electrophoresis separation, this invention achieves an integrated operation from sample separation to glycan release, greatly improving detection efficiency.

[0029] (2) This invention combines in-gel digestion and capillary electrophoresis (CE) techniques to achieve efficient separation and sensitive detection of N-glycans. CE technology, with its advantages of high resolution and low sample consumption, can accurately distinguish N-glycans with different structures. Furthermore, the introduction of fluorescent labeling further improves the detection sensitivity, making the analysis of low-abundance glycans more accurate. This combination of high sensitivity and high resolution significantly improves the accuracy of N-glycan detection.

[0030] (3) The detection method of this invention is particularly suitable for the detection of N-glycans in complex biological samples (such as serum, tissue samples, etc.). Through gel electrophoresis, the target glycoprotein can be effectively enriched and N-glycans released, thereby achieving highly selective and sensitive detection in complex samples. This technology provides a powerful tool for disease biomarker screening, clinical diagnosis, and glycomics research, and has broad application potential.

[0031] (4) Traditional N-glycan detection methods are prone to sample loss during sample pretreatment and transfer, especially for low-abundance glycans. This invention, by performing the enzymatic digestion reaction directly within the gel, avoids the sample transfer step, significantly reducing sample loss and improving glycan recovery. This improvement is particularly suitable for the detection of low-abundance glycans, providing more reliable technical support for related research.

[0032] (5) The detection method of the present invention features high throughput and high compatibility, and can process multiple samples simultaneously, making it suitable for large-scale glycomics research. Furthermore, this method is compatible with existing mass spectrometry analysis techniques, and can be further combined with mass spectrometry for precise identification of glycan structures, providing a more comprehensive solution for glycomics research.

[0033] (6) Because this invention can efficiently and sensitively detect N-glycans in complex biological samples, it has important application value in the screening of disease biomarkers and clinical diagnosis. Abnormal N-glycosylation patterns are closely related to a variety of diseases (such as cancer, neurodegenerative diseases, immune diseases, etc.), and the technology of this invention provides a new research tool for the early diagnosis and treatment of these diseases. Attached Figure Description

[0034] Figure 1 These are the four different molecular weight bands obtained by SDS-PAGE gel electrophoresis in Example 1;

[0035] Figure 2 The N-glycan maps are those of serum (A) in the range of 35–100 kDa and four different molecular weight bands (B, C, D, E) in Example 1. Detailed Implementation

[0036] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0037] Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and the experimental methods without specific conditions are all conventional methods in the art.

[0038] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0039] Example 1

[0040] A method for detecting N-glycans based on in-gel enzyme digestion and electrophoresis is disclosed. This method detects N-glycan patterns in human serum by detecting four different molecular weight bands in the range of 35–100 kDa. The specific steps are as follows:

[0041] (1) After separating human serum samples according to molecular weight using SDS-PAGE, the gels were placed on a shaker and stained with 2–5 mL of Coomassie Brilliant Blue staining solution for at least 2 hours. Subsequently, the stained gels were destained with 2–5 mL of destaining solution A (water: anhydrous ethanol: glacial acetic acid = (25–50):(20–40):(5–10), volume ratio) for 30–60 minutes. This destaining process was repeated 3–4 times until protein bands were clearly visible. Figure 1 The image shows a decolorized human serum protein gel, displaying bands of four different molecular weights.

[0042] (2) After cutting the target glycoprotein band from the gel, it needs to be cut again into 1-2 mm pieces. 2 Transfer the approximately 100g gel particles into an EP tube and store at 4°C.

[0043] (3) Add 100-300 μL of decolorizing solution B (100% ACN: 50-100 mM NH4HCO3 = 1:1, volume ratio) to an EP tube containing the target glycoprotein particles, place it in a constant temperature mixer, decolorize at 800 rpm and 37°C for 30-60 min, discard the liquid, and repeat 1-3 times until the blue color disappears.

[0044] (4) Add 100-300 μL of 100% acetonitrile (ACN) solution to the EP tube containing the target glycoprotein particles, and shake at 37°C for about 10-30 minutes until the particles dehydrate and turn white. Discard the liquid and evaporate the remaining ACN (it can be heated appropriately in a constant temperature incubator).

[0045] (5) Add 100-300 μL of enzyme solution to an EP tube containing the target glycoprotein particles and incubate at 37°C for 30-60 min. If the particles cannot be completely submerged at this time, add 25 mM NH4HCO3 solution to completely submerge the particles and incubate at 37°C overnight.

[0046] The enzyme solution is formulated as follows: PNGase F enzyme (final concentration 1000-1500 U / mL) and sialidase (final concentration 100-150 U / mL) are dissolved in 25 mM NH4HCO3 solution.

[0047] (6) Place the overnight enzyme-digested EP tubes in an ultrasonic cleaner and sonicate for 30–60 min. Vortex for 10–30 s, then centrifuge for 10–30 s. Repeat this operation twice. Transfer all the liquid in the sonicated EP tubes to new EP tubes, centrifuge for 10–30 s, and dry at 80°C for 1–2 h until completely dry. Then cool to 4°C. Next, add 10–20 μL of purified water to the dried EP tubes and repeatedly blow water around the inner wall. After complete blowing, vortex for 1–2 min. Centrifuge for 10–30 s, dry at 80°C with the cap off for 30–60 min until completely dry, then cool to 4°C; add 2–5 μL of fluorescent labeling solution (5–10 mM 8-aminopyrene-1,3,6-trisulfonic acid) to the dried EP tube, cap, label, centrifuge for 10–30 s, place at 90°C for 1–2 h, cool to 4°C, add 10–30 μL of stop solution (ultrapure water), vortex for 10–30 s to mix thoroughly, centrifuge for 10–30 s to prepare N-glycan samples.

[0048] (7) Place 5-10 μL of the obtained glycan sample into an ABI 96-well plate and then detect it using an ABI 3500dx sequencer.

[0049] like Figure 2 As shown, the sugar chains include P1(NGA2F), P2(NGA2FB), P3(NG1A2F-1), P4(NG1A2F-2), P5(NA2), P6(NA2F), P7(NA2FB), P8(NA3), P9(NA3Fb), P10(NA4) and P11(NA4Fb), wherein P3(NG1A2F-1) and P4(NG1A2F-2) are isomers, and the peak height represents the relative content of the sugar chains.

[0050] Through the Figure 2 Analysis yielded the following conclusions: (1) The distribution characteristics of glycans in human serum were clarified: the total glycans were classified by bands, further clarifying the proportion of each glycan in different molecular weight bands. Taking P9 as an example, its peak proportions in bands 1, 2, 3, and 4 were 5.4%, 7.4%, 23.3%, and 52.4%, respectively. This result indicates that P9 in human serum mainly comes from proteins in bands 3 and 4, which can provide a basis for subsequent glycan tracing. (2) Detection and enrichment of low-abundance glycans: Taking P11 (NA4Fb) as an example, its abundance in total serum is low, with a peak value of only 159. However, after further purification and enrichment of the glycans in band 4 using the detection method of this invention, the peak value of P11 in the spectrum of band 4 was significantly increased to 1217, and the peak signal was enhanced by about 7 times.

[0051] The above results fully demonstrate the high sensitivity and high resolution advantages of this invention in the detection of low-abundance glycans. This invention successfully achieves high-sensitivity, high-resolution analysis of multiple glycans in human serum samples, particularly the significant enrichment and detection of low-abundance glycans (such as P11), fully demonstrating its technical advantages. These results provide important experimental evidence for glycosylation research and lay a solid foundation for subsequent glycan function research and clinical applications.

[0052] The embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. The scope of protection of the present invention is determined by the scope claimed in the claims. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A method for detecting N-glycan chains based on in-gel enzyme digestion and electrophoresis, characterized in that, Includes the following steps: Step 1) Separate the glycoprotein samples using gel electrophoresis to prepare a gel; Step 2) Cut the target glycoprotein bands on the gel prepared in step 1) to obtain target glycoprotein particles; Step 3) Place the target glycoprotein particles obtained in Step 2) into an EP tube, add decolorizing solution to decolorize the target glycoprotein particles, repeat the decolorization 1 to 3 times until the blue color disappears completely; Step 4) After completely discarding the decolorizing solution in the EP tube, add 100% acetonitrile solution to the EP tube to dehydrate the target glycoprotein particles. Step 5) After completely discarding the 100% acetonitrile solution in the EP tube, add 100~300 µL of enzyme solution to the EP tube containing the target glycoprotein particles, and incubate at 37°C for 30~60 min to release N-glycan chains. If the particles cannot be completely submerged at this time, add 25 mM NH4HCO3 solution to completely submerge the particles, and incubate at 37°C overnight. The enzyme solution is formulated as follows: PNGase F enzyme and sialidase are dissolved in 25 mM NH4HCO3 solution, the final concentration of PNGase F enzyme is 1000~1500 U / mL, and the final concentration of sialidase is 100~150 U / mL. Step 6) Place the EP tube treated in Step 5) in an ultrasonic cleaner and sonicate for 30-60 min. Vortex for 10-30 s, then centrifuge for 10-30 s. Repeat this operation twice. Transfer all the liquid from the sonicated EP tube to a new EP tube, centrifuge for 10-30 s, and dry at 80°C with the cap open for 1-2 h until completely dry. Then cool to 4°C. Next, add 10-20 µL of purified water to the dried EP tube, repeatedly blow and agitate the inner wall until completely dry, then vortex for 1-2 min, centrifuge for 10-30 s, and dry at 80°C with the cap open for 30-60 min until completely dry. Then cool to 4°C. Add 2-5 µL of fluorescent labeling solution to the dried EP tube, cap, label, centrifuge for 10-30 s, place at 90°C for 1-2 h, cool to 4°C, add 10-30 µL of stop solution, vortex for 10-30 s to mix thoroughly, and centrifuge for 10-30 s. s, to obtain N-glycan chain samples; Step 7) Separate, detect and analyze the N-glycan sample prepared in step 6) using a capillary electrophoresis apparatus.

2. The method for detecting N-glycans based on in-gel digestion and electrophoresis according to claim 1, characterized in that, Step 1) The specific steps for preparing the gel are as follows: After separating the glycoprotein sample according to its molecular weight, place the gel on a shaker and stain it with 2-5 mL of Coomassie Brilliant Blue staining solution for at least 2 hours. Then, decolorize the stained gel with 2-5 mL of decolorizing solution for 30-60 minutes. Repeat the decolorization process 3-4 times until the protein bands are clearly visible.

3. The method for detecting N-glycans based on in-gel digestion and electrophoresis according to claim 1, characterized in that, In step 2), after the target glycoprotein band is cut, it needs to be cut again into 1-2 mm pieces. 2 The gel particles were transferred into EP tubes and stored at 4°C.

4. The method for detecting N-glycans based on in-gel digestion and electrophoresis according to claim 1, characterized in that, Step 3) The specific steps for decolorization are as follows: Add 100~300 µL of decolorizing solution to an EP tube containing the target glycoprotein particles, place it in a constant temperature mixer at 800 rpm and 37℃ for 30~60 min to decolorize, discard the liquid, and repeat 1~3 times until the blue color disappears.

5. The method for detecting N-glycans based on in-gel digestion and electrophoresis according to claim 1, characterized in that, Step 4) The specific steps of the dehydration treatment are as follows: Add 100~300 µL of 100% acetonitrile solution to the EP tube containing the target glycoprotein particles, shake at 37℃ for 10~30 min, wait for the particles to dehydrate and turn white, discard the liquid, and evaporate the remaining acetonitrile solution.

6. The method for detecting N-glycans based on in-gel digestion and electrophoresis according to claim 1, characterized in that, Step 7) The amount of N-glycan sample used is 5~10 µL, and the capillary electrophoresis instrument is an ABI 3500dx gene sequencer.

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