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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。
阿拉丁的5 μm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。
图1. 5 μm二氧化钛磁珠（薄壳层）扫描电镜照片
| 货号 | T1520787 |
| 产品名称 | 5 μm 二氧化钛磁珠（薄壳层） |
| 尺寸 | 5± 0.2 μm |
| 表面电位 | -20 mV 左右 |
| 磁含量 | 大约 10%-15% |
| 溶剂 | 纯化水 |
| 浓度 | 25 mg/mL |
产品特点
对磷酸化肽具有高特异性；
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 many biological processes, such as cell signaling, and is therefore of great importance in the study of many diseases, including cancer. Understanding phosphorylation helps us to understand disease pathogenesis. Phosphoproteins and phosphopeptides are typically present at very low concentrations and are poorly ionized, making them difficult to detect by mass spectrometry (MS). Therefore, enrichment techniques that can specifically enrich phosphopeptides and are compatible with MS analysis are urgently needed.
Titanium dioxide (TiO₂) has selective affinity for enriching phosphoserine (pSer), phosphothreonine (pThr), and phosphotyrosine (pTyr) residues. TiO₂ magnetic beads are proprietary magnetic microparticle carriers 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 obvious preference for mono‑phosphopeptides versus multi‑phosphopeptides, making them very suitable for single‑step enrichment of phosphopeptides for MS‑based proteomics analysis. In addition, TiO₂ magnetic beads can also be used to isolate extracellular vesicles (EVs) such as exosomes by binding to phospholipid bilayers. TiO₂ captures small EVs (sEVs) by forming a bidentate structure with the phosphate groups of the EV bilayer phospholipids, and the magnetic core further separates the magnetic bead‑sEV complexes. The binding between beads and EVs is reversible; captured sEVs can be eluted and collected by washing with an alkaline solution.
Aladdin's 5 μm Titanium Dioxide Magnetic Beads (thin shell layer) are monodisperse, micron‑sized magnetic beads with uniform size. The surface has a nanoscale rough island‑like structure, offering advantages such as high specific surface area, strong saturation magnetization, and fast magnetic response time. TiO₂ magnetic beads combine the rapid external magnetic field response of magnetic materials with the stability of metal oxides, feature a very high specific surface area, simplify the phosphopeptide enrichment process, and increase enrichment throughput.
Figure 1. SEM image of 5 μm titanium dioxide magnetic beads (thin shell layer)
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Product Features
High specificity for phosphopeptides
No obvious preference for mono‑phosphopeptides vs. multi‑phosphopeptides
Fast magnetic response (<30 seconds) reduces sample loss and is more suitable for automation
Antioxidant properties reduce the risk of sample contamination
Exosome Extraction
Loading buffer: 10 mM PBS (pH 7.4)
Elution buffer: PBS containing 10% ammonia water (adjust 10 mM PBS to pH 11.1 with 25% ammonia water; final ammonia concentration in PBS is 10%)
TiO₂ magnetic beads are stored in purified water at 25 mg/mL. Before use, the beads should be washed and equilibrated (allow to return to room temperature). The amount of beads can be scaled up or down as needed.
Transfer 200 μL (5 mg) of TiO₂ magnetic beads to a 2 mL centrifuge tube.
Place the tube on a magnetic separator for 30 seconds, then remove the supernatant.
Gently wash the beads with 200 μL of 10 mM PBS (e.g., intermittent vortex mixing) for 5 minutes.
Place the tube on the magnetic separator for 30 seconds and remove the supernatant.
Repeat steps 4 and 5.
Add 100 μL of sample (serum containing exosomes) to the beads and co‑incubate at 4 °C for 5–10 minutes.
Place the tube on the magnetic separator for 30 seconds, remove the supernatant by magnetic separation, and wash 2–3 times with 10 mM PBS (pH 7.4).
Remove the magnetic field, add PBS containing 10% ammonia water to the bead‑exosome complex, and co‑incubate at 4 °C for 5–10 minutes to release exosomes from the beads.
Place the tube on the magnetic separator for 30 seconds, collect the supernatant (exosome suspension). Further adjust the pH of the exosome resuspension by ultrafiltration if needed.
Note: Ammonia water is corrosive. Please follow the safety instructions for use.
Notes
Titanium dioxide magnetic beads will settle upon prolonged standing. Use after thorough stirring or vortexing to disperse evenly.
Store sealed at 2 °C–8 °C in a refrigerator. Avoid drying into a solid mass.
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0838159 |
分析证书 | T1520787 |
| ZJ26F0434434 |
分析证书 | T1520787 |
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