BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
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在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
糖基化是最复杂的蛋白质翻译后修饰之一，哺乳动物体内有 50% 以上的蛋白质预测为糖基化修饰蛋白。目前富集纯化糖蛋白质的方法主要包括凝集素亲和法、硼酸亲和法、酰肼化学法及亲水作用色谱法。其中酰肼化学法对糖蛋白质的富集能力强且选择性高。阿拉丁提供粒径为 1 μm、具有酰肼表面基团的均匀超顺磁珠。该产品广泛用于通过形成稳定的腙键，共价偶联含有醛基的配体（如抗体、凝集素、糖蛋白、碳水化合物等）。这种偶联方法是一种高效的蛋白质固定方式，能够保留关键的活性位点不受影响。
酰肼磁珠偶联糖蛋白或聚糖可以通过高碘酸钠氧化多糖部分糖基中的顺式乙二醇基团实现。生成的醛与磁珠上的酰肼基自发反应，形成稳定的共价键。另外，也可以通过糖蛋白上释放的糖链的还原端与磁珠表面的酰肼发生反应，将糖链共价连接在磁珠上，通过酸的可逆洗脱将糖链从酰肼磁珠上释放出来，用于后续的质谱法分析。
产品信息
基质 | 磁性微球 |
粒径 | 约 1 μm |
磁珠浓度 | 10 mg/mL |
官能团载量 | ~170 μmol/g 磁珠 |
保存条件 | 密封，2-8°C/24 个月，禁止冷冻，使用前请充分混匀 |
使用说明
注意：以下所述的方案是将糖蛋白与酰肼磁珠 (1 μm)进行结合的示例，需要根据实际情况进行磁珠用量相应的调整。应避免使用 Tris 或其他含有伯胺或糖类的缓冲液，因为这些物质会与预期的偶联反应发生竞争。
方法 1：酰肼磁珠对糖蛋白进行分离（高碘酸钠氧化法）
偶联缓冲液：0.1 M 乙酸钠缓冲液（pH 5.6）
氧化剂：高碘酸钠（NaIO₄）
洗涤缓冲液：1 M 氯化钠
（1）糖蛋白的氧化
注意：该反应对光敏感，应在黑暗中进行。
1）在 1 mL 偶联缓冲液中溶解或稀释 0.5~10 mg 糖蛋白。（注意：如果蛋白质已经悬浮在其他缓冲液中，请通过透析脱盐柱进行缓冲液交换）。
2）将蛋白质溶液（1 mL）添加到含有 2 mg 高碘酸钠（终浓度应保持在 10 mM 左右）的琥珀色小瓶中。轻轻旋转以溶解氧化剂。
3）将样品在室温避光条件下静置 30 分钟，轻轻旋转以保持均匀。
4）终止反应，并通过 Sephadex G25 色谱柱进行脱盐和缓冲液交换，除去未反应的高碘酸钠。用偶联缓冲液对 5 毫升 Sephadex G-25 色谱柱进行预平衡。将氧化后的样品上样至色谱柱，使其进入凝胶床。应用 0.5 mL 的偶联缓冲液冲洗，并使其也进入凝胶床。最后加入 2 mL 偶联缓冲液并收集洗脱液。
（2）氧化的糖蛋白与酰肼磁珠偶联
1）将磁珠分散均匀，取 1.5 mL 磁珠（10 mg/mL）于离心管中。
2）磁分离，去除上清，并用 1.5 mL 偶联缓冲液重悬磁珠。
3）重复步骤2)， 2~3 次。
4）从分离器中取出离心管，并用 750 µL 偶联缓冲液重悬磁珠。
5）将磁珠与 250 µL 氧化蛋白溶液混合，并在室温下孵育至少 6 小时。
注意：偶联效率取决于目标糖蛋白的结构和大小。用户应根据实际情况优化蛋白浓度。对于 10 mg 磁珠，我们建议从 100~250 µg 开始。
6）用 1 mL 洗涤缓冲液洗涤磁珠 3 次。
7）将结合糖蛋白的磁珠重悬于 PBS 缓冲液（含 0.1％ Proclin 300（v/v））中至所需浓度，并保存在 4°C 直至使用。不要冻结。
（3）分离糖肽
1）将结合糖蛋白的磁珠分散于 50 mmol/L NH₄HCO₃ 缓冲液中，加入 20 µg 胰蛋白酶，在 37°C 下恒温振荡消化 4 小时，再加入 20 µg 胰蛋白酶，继续消化过夜。
2）分别用 2 mol/L NaCl 溶液、100%甲醇和 50 mmol/L NH₄HCO₃ 缓冲液充分洗涤磁珠来去除胰蛋白酶释放的肽段。将磁珠重新悬浮于 50 mmol/L NH₄HCO₃ 缓冲液中，通过在 37°C 下与 2 µL PNGase F 去糖基化酶共同孵育 4 小时来释放 N-糖肽。将释放的去糖基化肽段加载到预处理过的柱子中，并用 0.1%三氟乙酸中的 80%乙腈洗脱。洗脱液通过质谱进行分析。
方法 2：通用的亲和层析纯化流程结合糖蛋白
该方法是一种通用的亲和纯化流程。但由于没有两种蛋白质是完全相同的，所以不可能设计出适用于所有蛋白质纯化的通用方案。为了获得最佳效果，每位用户都必须确定针对特定目标蛋白质进行纯化的最佳操作条件。
偶联缓冲液：0.1 M 醋酸钠缓冲液，pH 5.6
储存缓冲液：15 mM PBS，pH 7.4，含 0.02% Proclin 300
（1）聚糖/多肽的制备
将 5 µL 的血清溶解在 200 µL 的 0.4 mmol NH₄HCO₃ 缓冲液（pH 8.0 - 8.3）中，然后在 100°C 下加热 5 分钟进行变性。将样品冷却至室温后，向血清溶液中加入 500 µL 1 mg/µL 的胰蛋白酶，并在 37°C 下孵育 16 小时。消化后的样品在 100°C 下加热 5 分钟以使胰蛋白酶失活，然后加入 10 µL 的去糖基化酶 PNGase F（500 单位/µL）。然后将样品在 37°C 下孵育 16 小时，并在 HPLC 水中重悬。在分析肽之前，从消化后的溶液中提取出糖类物质后，该样本通过层析柱进行纯化，以去除盐分和其他小分子物质。
（2）酰肼磁珠结合聚糖：
将酰肼磁珠用甲醇-醋酸缓冲液（85：15，v/v）清洗 2 次，去上清。向 100 µL 醋酸盐缓冲液和甲醇的磁珠中加入 5 µL 聚糖和肽混合物。加入 10 mM 苯胺作为催化剂，使聚糖还原端与肼基磁珠发生结合反应。将样品和磁珠混合，微波炉 50%条件下反应 20 min，为避免样品-磁珠过热，每 5 分钟微波照射，1-2 分钟间隔。
（3）聚糖的释放：
用 1 mL 50 mmol/L NH₄HCO₃ （pH 值为 8.0-8.3）冲洗结合了聚糖的酰肼磁珠。冲洗五次以完全清除非结合的样品（包括肽类）。随后，向清洗后的磁珠中加入 200 µL 10%甲酸，将其置于 60°C 的烘箱中孵育 30分钟，冷却至室温后，进行磁分离收集上清，并用 200 µL 5%乙酸溶液再清洗磁珠两次。上清液再通过过柱子进行浓缩，以进行进一步的分析。
注意事项
1. 磁珠取用前应充分混匀，防止取用改变磁珠浓度，避免长时间超声对磁珠表面破坏；
2. 磁珠使用和保存过程中应避免冻融。
Glycosylation is one of the most complex post-translational modifications of proteins, and more than 50% of proteins in mammals are predicted to be glycosylated. Current methods for the enrichment and purification of glycoproteins mainly include lectin affinity, boronate affinity, hydrazide chemistry, and hydrophilic interaction chromatography. Among these methods, hydrazide chemistry provides strong enrichment capability and high selectivity for glycoproteins. Aladdin provides uniform superparamagnetic beads with a particle size of 1 μm and hydrazide functional groups on the surface. These beads are widely used for the covalent coupling of aldehyde-containing ligands, such as antibodies, lectins, glycoproteins, and carbohydrates, through the formation of stable hydrazone bonds. This coupling method provides an efficient approach for protein immobilization while preserving key active sites.
Glycoproteins or glycans can be coupled to hydrazide magnetic beads by oxidizing cis-diol groups in the carbohydrate moieties with sodium periodate. The resulting aldehyde groups spontaneously react with hydrazide groups on the bead surface to form stable covalent bonds. Alternatively, the reducing ends of glycans released from glycoproteins can react with the hydrazide groups on the bead surface, thereby covalently immobilizing the glycans onto the magnetic beads. The glycans can subsequently be released from the hydrazide magnetic beads by reversible acid elution for downstream mass spectrometric analysis.
Product Information
Matrix | Magnetic microspheres |
Particle Size | Approx. 1 μm |
Magnetic Bead Concentration | 10 mg/mL |
Functional Group Loading | ~170 μmol/g magnetic beads |
Storage Conditions | Sealed, 2–8°C/24 months, do not freeze, mix thoroughly before use |
Instructions for Use
Note: The following protocol is an example for coupling glycoproteins with Hydrazide Magnetic Beads (1 μm). The amount of magnetic beads should be adjusted according to actual conditions. Tris or other buffers containing primary amines or carbohydrates should be avoided, as these substances may compete with the intended coupling reaction.
Method 1: Separation of Glycoproteins Using Hydrazide Magnetic Beads (Sodium Periodate Oxidation Method)
Coupling Buffer: 0.1 M sodium acetate buffer (pH 5.6)
Oxidizing Agent: Sodium periodate (NaIO₄)
Wash Buffer: 1 M sodium chloride
(1) Oxidation of Glycoproteins
Note: This reaction is light-sensitive and should be performed in the dark.
1) Dissolve or dilute 0.5~10 mg of glycoprotein in 1 mL of coupling buffer. (Note: If the protein is already suspended in another buffer, perform buffer exchange using a dialysis desalting column).
2) Add the protein solution (1 mL) to an amber vial containing 2 mg of sodium periodate (the final concentration should be maintained at approximately 10 mM). Gently rotate to dissolve the oxidizing agent.
3) Incubate the sample at room temperature in the dark for 30 minutes, with gentle rotation to maintain homogeneity.
4) Terminate the reaction and remove unreacted sodium periodate by desalting and buffer exchange using a Sephadex G25 chromatography column. Pre-equilibrate a 5 mL Sephadex G-25 chromatography column with coupling buffer. Load the oxidized sample onto the column and allow it to enter the gel bed. Rinse with 0.5 mL of coupling buffer and allow it to enter the gel bed as well. Finally, add 2 mL of coupling buffer and collect the eluate.
(2) Coupling of Oxidized Glycoproteins to Hydrazide Magnetic Beads
1) Thoroughly disperse the magnetic beads and transfer 1.5 mL of magnetic beads (10 mg/mL) into a centrifuge tube.
2) Perform magnetic separation, remove the supernatant, and resuspend the magnetic beads in 1.5 mL of coupling buffer.
3) Repeat Step 2), 2~3 times.
4) Remove the centrifuge tube from the magnetic separator and resuspend the magnetic beads in 750 µL of coupling buffer.
5) Mix the magnetic beads with 250 µL of oxidized protein solution and incubate at room temperature for at least 6 hours.
Note: Coupling efficiency depends on the structure and size of the target glycoprotein. Users should optimize the protein concentration according to actual conditions. For 10 mg of magnetic beads, we recommend starting with 100~250 µg.
6) Wash the magnetic beads 3 times with 1 mL of wash buffer.
7) Resuspend the glycoprotein-bound magnetic beads in PBS buffer (containing 0.1% Proclin 300 (v/v)) to the desired concentration and store at 4°C until use. Do not freeze.
(3) Isolation of Glycopeptides
1) Disperse the glycoprotein-bound magnetic beads in 50 mmol/L NH₄HCO₃ buffer, add 20 µg of trypsin, and digest with constant shaking at 37°C for 4 hours. Then add another 20 µg of trypsin and continue digestion overnight.
2) Thoroughly wash the magnetic beads with 2 mol/L NaCl solution, 100% methanol, and 50 mmol/L NH₄HCO₃ buffer, respectively, to remove peptides released by trypsin digestion. Resuspend the magnetic beads in 50 mmol/L NH₄HCO₃ buffer and incubate with 2 µL of PNGase F deglycosylation enzyme at 37°C for 4 hours to release N-glycopeptides. Load the released deglycosylated peptides onto a pretreated column and elute with 80% acetonitrile in 0.1% trifluoroacetic acid. Analyze the eluate by mass spectrometry.
Method 2: General Affinity Chromatography Purification Procedure for Glycoprotein Binding
This method is a general affinity purification procedure. However, because no two proteins are completely identical, it is impossible to design a universal protocol suitable for the purification of all proteins. To achieve optimal results, each user must determine the optimal operating conditions for purification of the specific target protein.
Coupling Buffer: 0.1 M sodium acetate buffer, pH 5.6
Storage Buffer: 15 mM PBS, pH 7.4, containing 0.02% Proclin 300
(1) Preparation of Glycans/Peptides
Dissolve 5 µL of serum in 200 µL of 0.4 mmol NH₄HCO₃ buffer (pH 8.0 - 8.3), and then heat at 100°C for 5 minutes for denaturation. After cooling the sample to room temperature, add 500 µL of 1 mg/µL trypsin to the serum solution and incubate at 37°C for 16 hours. Heat the digested sample at 100°C for 5 minutes to inactivate trypsin, and then add 10 µL of PNGase F deglycosylation enzyme (500 units/µL). Then incubate the sample at 37°C for 16 hours and resuspend it in HPLC water. Before peptide analysis, after extracting the carbohydrate components from the digested solution, purify the sample through a chromatography column to remove salts and other small-molecule substances.
(2) Binding of Glycans to Hydrazide Magnetic Beads:
Wash the hydrazide magnetic beads twice with methanol-acetic acid buffer (85:15, v/v) and remove the supernatant. Add 5 µL of the glycan and peptide mixture to the magnetic beads in 100 µL of acetate buffer and methanol. Add 10 mM aniline as a catalyst to promote the binding reaction between the reducing ends of glycans and the hydrazide magnetic beads. Mix the sample and magnetic beads and react under 50% microwave power for 20 min. To avoid overheating of the sample-bead mixture, apply microwave irradiation for 5 minutes at a time, with 1-2 minute intervals.
(3) Release of Glycans:
Wash the glycan-bound hydrazide magnetic beads with 1 mL of 50 mmol/L NH₄HCO₃ (pH 8.0-8.3). Wash five times to completely remove unbound samples (including peptides). Then add 200 µL of 10% formic acid to the washed magnetic beads and incubate in an oven at 60°C for 30 minutes. After cooling to room temperature, perform magnetic separation and collect the supernatant, and then wash the magnetic beads twice with 200 µL of 5% acetic acid solution. The supernatant is then concentrated through a column for further analysis.
Precautions
1. Thoroughly mix the magnetic beads before use to prevent changes in bead concentration during sampling, and avoid prolonged ultrasonication to prevent damage to the surface of the magnetic beads;
2. Avoid freeze-thaw cycles during use and storage of the magnetic beads.
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