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目的筛选较好的血清多肽富集方法与最优诊断模型,为开发替代性血清蛋白富集试剂盒奠定基础。方法建立日本血吸虫急性感染小鼠模型,利用分别包被有C3、C8、弱阳离子、螯合铜离子的磁珠富集纯化血清多肽,在m/z0.8~1.6ku区段应用基质辅助激光解吸电离时间飞行质谱(MALDI-TOF-MS)考察多肽峰数量与质量,结合ClinProTool建立诊断模型,盲测考察不同模型的检测效果。结果在m/z 0.8~16ku范围的多肽指纹图谱中,弱阳离子富集肽数量最多,组内均值为208个,螯合铜离子的多肽峰信噪比最佳达460.02,均显著高于C3与C8组(P<0.05);在实验条件下盲测,螯合铜离子富集的多肽峰应用于ClinProTool建立的诊断模型检测日本血吸虫感染的敏感性与准确率最高。结论 ClinProTool诊断准确性与多肽峰信噪比正相关;具有疏水作用的弱阳离子与螯合铜离子更易富集血清多肽,且血清肽谱峰质量较好,可为开发相应试剂提供参考。
Objective To screen better methods of serum peptide enrichment and the optimal diagnostic model to lay the foundation for the development of alternative serum protein enrichment kit. Methods Acute murine model of Schistosoma japonicum was established. Serum peptides were enriched using magnetic beads coated with C3, C8, weak cation and chelating copper ions, respectively. The basal-assisted laser was applied in the m / z 0.8 ~ Desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used to investigate the quantity and quality of the polypeptide peaks. ClinProTool was used to establish a diagnostic model, and blind detection was used to test the effect of different models. Results In the fingerprint of peptides with the range of 0.8 to 16 ku, the number of weak cation - rich peptides was the highest in the group with a mean of 208, and the peak signal - to - noise ratio of chelated copper ions reached 460.02, which was significantly higher than that of C3 (P <0.05). Under the experimental conditions, the peptide peak detected by chelation and chelation of copper ions was the most sensitive and accurate for the detection of Schistosoma japonicum infection in the diagnostic model established by ClinProTool. Conclusion ClinProTool diagnostic accuracy is positively correlated with peak signal-to-noise ratio of polypeptide. Weak cation with hydrophobic interaction is more likely to be enriched in serum peptides than chelated copper ions, and the peak of serum peptide peaks is better, which may provide a reference for the development of corresponding reagents.