摘要
作为临床样本,血清因其易于获取等优势被广泛应用于疾病研究。血清中的外泌体不仅携带多种蛋白质、核酸等生物活性物质,还可以参与细胞之间的通讯,与多种疾病的发生发展相关联。作为关键的翻译后修饰之一,蛋白质磷酸化通过动态调控细胞信号网络参与疾病发生发展,其在外泌体中的特异性表达为疾病诊断提供了新视角。本论文设计制备了一种基于2-三氟甲基-4-氨基苯甲酸和Ti^(4+)的磁性双功能材料(MagphTi^(4+)),通过2-三氟甲基-4-氨基苯甲酸的疏水作用和Ti^(4+)的静电作用共同锚定外泌体的双层膜,通过基质的磁响应性质实现外泌体的分离提取。同时,利用材料表面的Ti^(4+)和磷酸根的螯合作用实现磷肽的富集。通过冷场发射扫描电子显微镜、热重分析、红外光谱、X射线光电子能谱和Zeta电位测定对MagphTi^(4+)进行了表征。通过冷场发射扫描电子显微镜、透射电子显微镜和十二烷基硫酸钠聚丙烯酰胺凝胶电泳对外泌体进行了表征,验证了MagphTi^(4+)对外泌体精准识别和快速分离的能力。以β-酪蛋白酶解物为标准磷肽模型,建立了磁性固相萃取(MSPE)-质谱检测技术平台,实现了MagphTi^(4+)对磷肽的高选择性(β-酪蛋白与牛血清白蛋白的质量比为1∶5000)和高灵敏度的检测(检出限低至0.4fmol/μL)。将该方法用于实际样品分析,实现了对血清外泌体的提取与低丰度磷肽的分离富集。本方法有望用于临床血清样本中外泌体的高通量磷酸化蛋白质组学分析和疾病标志物的筛查。
As a clinical sample,serum is widely used in disease research due to the minimally invasive nature of its collection and easy accessibility.Exosomes in serum not only carry various bioactive substances such as proteins and nucleic acids but also participate in intercellular communication,which is closely associated with the development of multiple diseases.As one of the most critical post-translational modifications,protein phosphorylation dynamically regulates cellular signaling networks and participates in disease pathogenesis.However,due to the low abundance of exosomes in serum and the fact that phosphoproteins constitute only 1%–2%of the total proteome,direct analysis of exosomal phosphoproteins from serum faces significant challenges.The specific expression of phosphoproteins in exosomes provides novel perspectives for disease diagnosis.In this study,magnetic bifunctional nanoparticles(MagphTi^(4+))based on 2-trifluoromethyl-4-aminobenzoic acid and Ti^(4+)were synthesized,which anchor the exosome bilayer membrane through the hydrophobic interactions of 2-trifluoromethyl-4-aminobenzoic acid and the electrostatic interactions of Ti^(4+),and the magnetic responsiveness ensures the isolation of exosomes.Simultaneously,the enrichment of phosphopeptides was achieved by the chelation between Ti^(4+)and phosphate groups.MagphTi^(4+)was characterized by cold field scanning electron microscopy,thermogravimetric analysis,Fourier transform infrared spectroscopy,X-ray photoelectron spectroscopy and zeta potential measurement,demonstrating the successful synthesis and functional characteristics of MagphTi^(4+).The precise recognition and rapid separation of exosomes were validated by cold field scanning electron microscopy,transmission electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis.Usingβ-casein as a standard phosphoprotein model,a magnetic solid-phase extraction(MSPE)-matrix-assisted laser desorption ionization-time of flight mass spectrometry platform was established,which demonstrated high selectivity(mass ratio ofβ-casein to bovine serum albumin was 1∶5000)and sensitivity(detection limit was 0.4 fmol/μL)for phosphopeptide detection.Subsequently,the method was applied to saliva and nonfat milk samples;11 phosphopeptides were identified in saliva and 20 were identified in nonfat milk,which demonstrates excellent enrichment capability for phosphopeptide detection in real samples.When applied to clinical serum samples,the method successfully achieved the extraction of exosomes in serum and enrichment of phosphopeptides with low abundance.This integrated approach provides a powerful tool for exosome capture and phosphopeptide enrichment from complex biological samples,showing significant potential for high-throughput phosphoproteomic analysis of exosomes from clinical serum and the screening of disease biomarkers.Further application of the method developed in this study holds promise for elucidating the mechanisms of exosome-mediated intercellular transmission of phosphorylation signaling,thereby identifying novel targets related to tumor metastasis and immune escape.
作者
王子瑞
郑海娇
贾琼
WANG Zirui;ZHENG Haijiao;JIA Qiong(College of Chemistry,Jilin University,Changchun 130012)
出处
《色谱》
北大核心
2026年第3期338-348,共11页
Chinese Journal of Chromatography
基金
吉林省教育厅科学技术研究项目(JJKH20241244KJ)。
关键词
血清外泌体
钛离子
磁性固相萃取
磷肽
质谱检测
serum exosomes
titanium ion
magnetic solid-phase extraction
phosphopeptides
mass spectrometric detection
