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磷酸化是一种常见的可逆的翻译后修饰，在细胞信号传导等众多的生物过程中发挥调节作用，因此在肿瘤等许多疾病的研究中具有重要的意义。对磷酸化的认识有助于我们了解疾病的发生过程。磷酸蛋白和磷酸肽通常浓度极低，且电离程度差，因此很难通过质谱（MS）进行检测。因此，当前迫切需要能特异富集磷酸化肽、且与质谱分析兼容的富集技术。
二氧化钛具有富集磷酸丝氨酸（pSer）、磷酸苏氨酸（pThr）和磷酸酪氨酸（pTyr）残基的选择性亲和力。TiO₂磁珠是一种专有的磁性材料微粒载体，能在复杂生物样品的蛋白消化物中简单、方便、高效、高特异、高重复性富集磷酸化肽。磁珠表面的TiO₂纳米粒子对于单磷酸化肽和多磷酸化肽没有明显的偏好，因而非常适合单步富集磷酸化肽用于基于质谱的蛋白质组学分析。另外，TiO₂磁珠还可以通过结合磷脂双层膜分离外泌体等细胞外囊泡（EVs）。TiO₂通过和EVs双层磷脂的磷酸基形成双齿结构捕获sEVs，磁性内核能进一步将磁珠-sEVs复合物进行分离。磁珠和EVs的结合是可逆的，通过碱性溶液清洗，便能洗脱和收集所捕获的sEVs。
阿拉丁提供两款二氧化钛磁珠：二氧化钛磁珠（薄壳层）和二氧化钛磁珠（厚壳层）。二氧化钛磁珠（薄壳层）为尺寸均一的单分散微米级磁珠，表面呈现纳米级粗糙度岛状结构，具有较高的比表面积，较强的饱和磁化强度，快速的磁响应时间等优点。二氧化钛磁珠（厚壳层）表面包覆较厚二氧化钛壳层，这种结构更加稳定，能够用于更加剧烈的化学环境。二氧化钛磁珠集合了磁性材料快速外磁场响应和金属氧化物稳定性的优点，具有很高的比表面积，简化了磷酸化多肽富集过程，提高了富集通量。
| 货号 | T1520785 | T1520786 |
| 产品名称 | 1 μm 二氧化钛磁珠（薄壳层） | 1 μm 二氧化钛磁珠（厚壳层） |
| 尺寸 | 1.2 ± 0.2 μm | 1.45 ± 0.2 μm |
| 水动力尺寸 | 1000±200 nm | 1000±200 nm |
| Zeta电位 | ≤ -20 mV | ≤ -20 mV |
| 磁性物质占比（氧化铁） | ≥ 10% | ≥ 10% |
| 溶剂 | 纯水 | 纯水 |
| 浓度 | 25 mg/mL | 25 mg/mL |
1. 产品形貌尺寸
二氧化钛磁珠（薄壳层）平均尺寸为1.20 μm（左图），二氧化钛磁珠（厚壳层）平均尺寸为1.45 μm（右图）。
图1：二氧化钛磁珠扫描电镜照片
2. 动态光散射（DLS）水动力尺寸
1 μm 二氧化钛磁珠（薄壳层）的 DLS 水动力尺寸为 1058 nm，多分散系数 PDI 为 0.348（上）； 1 μm 二氧化钛磁珠（厚壳层）的 DLS 水动力尺寸为 1185 nm，多分散系数 PDI 为 0.373（下）。
图2：1 μm 二氧化钛磁珠的DLS 水动力尺寸分布图
3. 表面电荷 Zeta 电位表征
二氧化钛磁珠（薄壳层）的 Zeta 电位为-35.0 mV（上）；二氧化钛磁珠（厚壳层）的 Zeta 电位为-35.7 mV（下图）。
图3：1 μm 二氧化钛磁珠表面Zeta电位图
产品特点
对磷酸化肽及外泌体具有高特异性；
对单磷酸化肽和多磷酸化肽无明显的偏好；
小于30 s的快速磁响应性，减少样品损失，更适合自动化操作；
抗氧化特性，降低样品被污染风险。
外泌体提取
洗脱缓冲液：含有10%氨水的PBS溶液（10 mM PBS用25%的氨水进行调节至pH为11.1，PBS中氨水的含量为10%）
TiO₂磁珠以25 mg/mL浓度保存于纯水中。使用前应对磁珠进行洗涤和平衡（恢复至室温），可根据实际需要放大和缩小磁珠用量。
1. 涡旋混合TiO₂磁珠以确保均匀分散。
2. 转移200 μL（5 mg）TiO₂磁珠到2 mL离心管。
3. 将离心管放置磁分离器上，放置30秒，移弃上清。
4. 用200 μL的10 mM PBS温和洗涤微粒（例如间或涡旋混合）5分钟。
5. 将离心管放在磁分离器上，放置30秒，移弃上清。
6. 重复步骤4和5。
7. 将100 μL样品（含有外泌体的血清）加入到磁珠中，在4°C共孵育5-10分钟。
8. 将离心管放置磁分离器上，放置30秒，磁分离去除上清，用10 mM PBS（pH=7.4）清洗2-3次。
9. 移去磁场，在磁珠-外泌体复合物中加入含有10%氨水的PBS溶液，在4°C共孵育5-10分钟，使外泌体从磁珠上释放出来。
10. 将离心管放在磁分离器上，放置30秒，取上清外泌体悬浮液，进一步可通过超滤调节外泌体重悬液pH。
注意事项
Phosphorylation is a common reversible post-translational modification that plays a regulatory role in numerous biological processes such as cell signaling, and therefore holds significant importance in research on many diseases, including tumors. Understanding phosphorylation aids in comprehending disease pathogenesis. Phosphoproteins and phosphopeptides are typically present in very low concentrations and exhibit poor ionization efficiency, making them difficult to detect by mass spectrometry (MS). Consequently, there is an urgent need for enrichment technologies that can specifically enrich phosphopeptides and are compatible with mass spectrometry analysis.
Titanium dioxide possesses selective affinity for enriching phosphoserine (pSer), phosphothreonine (pThr), and phosphotyrosine (pTyr) residues. TiO₂ magnetic beads are a proprietary particulate carrier of magnetic material that enable simple, convenient, efficient, highly specific, and highly reproducible enrichment of phosphopeptides from protein digests of complex biological samples. The TiO₂ nanoparticles on the bead surface show no significant preference for monophosphorylated versus multiphosphorylated peptides, making them highly suitable for single-step phosphopeptide enrichment for mass spectrometry-based proteomics analysis. Additionally, TiO₂ magnetic beads can be used to isolate extracellular vesicles (EVs), such as exosomes, by binding to the phospholipid bilayer membrane. TiO₂ captures small EVs (sEVs) by forming a bidentate structure with the phosphate groups of the EV bilayer phospholipids, while the magnetic core enables further separation of the bead-sEV complexes. The binding between the beads and EVs is reversible; the captured sEVs can be eluted and collected by washing with an alkaline solution.
Aladdin offers two types of titanium dioxide magnetic beads: Titanium Dioxide Magnetic Beads (Thin Shell) and Titanium Dioxide Magnetic Beads (Thick Shell). The Titanium Dioxide Magnetic Beads (Thin Shell) are monodisperse micron-sized beads with uniform size, featuring a nanoscale rough island-like surface structure, high specific surface area, strong saturation magnetization, and fast magnetic response time. The Titanium Dioxide Magnetic Beads (Thick Shell) have a thicker titanium dioxide shell coating, providing enhanced stability suitable for use in more rigorous chemical environments. Titanium dioxide magnetic beads combine the advantages of rapid magnetic field response of magnetic materials and the stability of metal oxides, offering high specific surface area, simplifying the phosphopeptide enrichment process, and increasing enrichment throughput.
| Cat. No. | T1520785 | T1520786 |
| Product Name | 1 μm Titanium Dioxide Magnetic Beads (Thin Shell) | 1 μm Titanium Dioxide Magnetic Beads (Thick Shell) |
| Size | 1.2 ± 0.2 μm | 1.45 ± 0.2 μm |
| Hydrodynamic Size | 1000±200 nm | 1000±200 nm |
| Zeta Potential | ≤ -20 mV | ≤ -20 mV |
| Magnetic Content (Iron Oxide) | ≥ 10% | ≥ 10% |
| Solvent | Pure Water | Pure Water |
| Concentration | 25 mg/mL | 25 mg/mL |
1. Product Morphology and Size
The average size of Titanium Dioxide Magnetic Beads (Thin Shell) is 1.20 μm (left image), and the average size of Titanium Dioxide Magnetic Beads (Thick Shell) is 1.45 μm (right image).
Figure 1: Scanning Electron Microscopy Images of Titanium Dioxide Magnetic Beads
2. Hydrodynamic Size by Dynamic Light Scattering (DLS)
The DLS hydrodynamic size of the 1 μm Titanium Dioxide Magnetic Beads (Thin Shell) is 1058 nm, with a polydispersity index (PDI) of 0.348 (top); The DLS hydrodynamic size of the 1 μm Titanium Dioxide Magnetic Beads (Thick Shell) is 1185 nm, with a polydispersity index (PDI) of 0.373 (bottom).
Figure 2: DLS Hydrodynamic Size Distribution Plots of 1 μm Titanium Dioxide Magnetic Beads
3. Surface Charge Zeta Potential Characterization
The Zeta potential of Titanium Dioxide Magnetic Beads (Thin Shell) is -35.0 mV (top); The Zeta potential of Titanium Dioxide Magnetic Beads (Thick Shell) is -35.7 mV (bottom).
Figure 3: Surface Zeta Potential Plots of 1 μm Titanium Dioxide Magnetic Beads
Product Features
High specificity for phosphopeptides and exosomes;
No significant preference for monophosphorylated versus multiphosphorylated peptides;
Fast magnetic responsiveness in less than 30 seconds, reducing sample loss and more suitable for automation;
Antioxidant properties, reducing the risk of sample contamination.
Exosome Extraction
Loading Buffer: 10 mM PBS (pH=7.4)
Elution Buffer: PBS solution containing 10% ammonia water (10 mM PBS adjusted to pH 11.1 with 25% ammonia water, with a final ammonia concentration of 10% in PBS)
TiO₂ magnetic beads are stored in pure water at a concentration of 25 mg/mL. The beads should be washed and equilibrated (allowed to reach room temperature) before use. The amount of beads can be scaled up or down according to actual needs.
Vortex mix the TiO₂ magnetic beads to ensure uniform dispersion.
Transfer 200 μL (5 mg) of TiO₂ magnetic beads to a 2 mL centrifuge tube.
Place the centrifuge tube on a magnetic separator for 30 seconds, then discard the supernatant.
Gently wash the microparticles with 200 μL of 10 mM PBS (e.g., by intermittent vortex mixing) for 5 minutes.
Place the centrifuge tube on the magnetic separator for 30 seconds, then discard the supernatant.
Repeat steps 4 and 5.
Add 100 μL of the sample (serum containing exosomes) to the beads and incubate together at 4°C for 5-10 minutes.
Place the centrifuge tube on the magnetic separator for 30 seconds, magnetically separate and remove the supernatant, then wash 2-3 times with 10 mM PBS (pH=7.4).
Remove from the magnetic field, add PBS solution containing 10% ammonia water to the bead-exosome complex, and incubate together at 4°C for 5-10 minutes to release the exosomes from the beads.
Place the centrifuge tube on the magnetic separator for 30 seconds, collect the supernatant exosome suspension. The pH of the exosome resuspension solution can be further adjusted by ultrafiltration.
Note: Ammonia water is corrosive. Please refer to the safety instructions for operation.
Precautions
Titanium dioxide magnetic beads will settle upon prolonged storage. Please mix thoroughly by stirring or vortexing before use.
Keep sealed and store in the refrigerator at 2°C-8°C, avoid drying into a solid mass.
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0434432 |
分析证书 | T1520785 |
危险品化学品经营许可证（带存储）