【作者】 赵洪源；

【导师】 艾军； 单保恩；

【作者基本信息】 河北医科大学 ， 肿瘤学， 2010， 硕士

【摘要】 目的:利用基质辅助激光解吸离子化飞行时间质谱(matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF MS)技术检测血清样本蛋白多肽表达图谱,分析clinprot实验方法的重复性及血清不同的冻融次数对实验结果的影响,以便建立MALDI-TOF MS检测方法用于后续试验;同时比较分析弱阳离子交换磁珠(MB-WCX)、铜离子螯合磁珠(MB-IMAC Cu)、疏水C8磁珠(MB-HIC C8)进行血清蛋白多肽分离富集后图谱采集的差异,选取分离蛋白质能力强的MB-WCX对乳腺癌及健康对照组的血清样本进行检测,通过高通量分析软件进行数据处理,筛选出乳腺癌和健康对照组之间的差异蛋白多肽质荷比峰,并建立乳腺癌诊断模型,进行模型验证确定诊断模型具有高度灵敏度和特异度,为临床寻找乳腺癌血清肿瘤标志物提供理论依据。方法:1采用MB-WCX分别对冻融1、3、5次后的血清样本分离提纯,经MALDI-TOF MS检测后比较其蛋白质图谱,以此判断冻融次数对实验结果的影响。2采用MB-WCX进行血清样本蛋白分离提纯,经MALDI-TOF MS检测后,选择不同分子量范围的9个蛋白重复测定,比较组间和组内变异系数以考察重复性。3比较MB-WCX、MB-IMAC Cu、MB-HIC C8的平均出峰量、平均峰面积和平均峰强度等参数,从而选取分离蛋白质能力较好的一种磁珠用于大量血清样本的提纯。4选择分离蛋白质能力较好的MB-WCX对乳腺癌及健康对照组的血清样本进行检测,然后用MALDI-TOF MS检测不同组患者蛋白质谱图并加以对比分析。研究过程中仪器操作、数据分析和图像采集分别用flexControlMS3.0、ClinProToolsTM2.1和flexAnalysis 3.0软件。对所发现的差异蛋白计算识别率、预测能力和验证准确率,或差异蛋白组合后诊断的效果,以指导临床应用。结果:1研究发现,样品的冻融次数在3次以内不影响质谱峰的采集,没有显著性差异(P>0.05),冻融次数越多,对小分子量蛋白或多肽的影响越大。2重复性研究表明,标准品变异系数范围在10.67%~29.59%,混合样品变异系数范围在9.88%~30.07%,重复性很好。3相同样本利用MB-WCX处理后的平均出峰量和平均峰面积均明显优于MB-IMAC-Cu、MB-HIC C8(P<0.05)。4应用MB-WCX与MALDI-TOF MS检测到15个明显差异表达的蛋白质峰,分子量分别为4964.52Da、7765.28Da、3261.95Da、1621.2Da、3192.55Da、7008.09Da、2288.99Da、1741.74Da、3225.04Da、4363.01Da、6910.35Da、7631.46Da、3935.34Da、8140.66Da、4054.5Da。5分子量分别为4964.52Da和7765.28Da的2个蛋白质峰的差异最显著,在乳腺癌患者中的表达上调,有可能成为潜在的乳腺癌早期诊断标志物。6利用差异蛋白质峰建立乳腺癌诊断模型后应用QC(快速分类算法)、GA(遗传算法)和SNN(神经网络算法)对模型进行验证,得到该模型区分乳腺癌患者和健康者的灵敏度和特异度均高于80%。结论:1 MALDI-TOF MS作为一种高灵敏度的研究平台,严格操作规程减少冻融次数能够减少变异,提高检测结果的重复性和可信性。2 MB-WCX的蛋白分离效果优于MB-IMAC-Cu和MB-HIC C8;3利用MALDI-TOF MS技术可以建立高度敏感性和特异性的乳腺癌诊断模型。更多还原

【Abstract】 Objective: Magnetic bead purification for the analysis of proteins in body blood serum facilitates the identification of potential new biomarkers with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The aim of our study was to establish a standard proteome fractionation technique for proteomic pattern analysis. To screen a suitable magnetic bead from three kinds of magnetic beads IMAC-Cu, MB-WCX, MB-HIC C8, and use MB-WCX to search biomarkers and evaluate their diagnostic value by comparing different patients′serum protein. To provide a new method of finding biomarkers for breast cancer, while establish the diagnostic model of breast cancer and provide a simple way for the early diagnosis.Methods: 1 The influence of freeze-thaw cycles and the ratio of sample to matrix were evaluated by comparison of their mass spectrum.2 Serum sample′s protein spectrum was detected by MALDI-TOF MS after serum samples were purified by MB-WCX magnetic bead. In order to evaluate the reproducibility of MALDI-TOF MS at different spots, nine proteins in different mass ranges were selected and their CV% was calculated.3 Before large scale patients′serum testing, three kinds of magnetic bead (IMAC-Cu, MB-WCX, MB-HIC C8)were compared about their peak number, peak area and peak intensity of mass spectrum.4 The MB-WCX was selected and used to detect breast cancer patients′and healthy controls′blood serum. After separation and purification by magnetic bead MB-WCX, their mass spectrums were detected by MALDI-TOF MS. During the process, flexControlMS3.0, ClinProToolsTM2.1 and flexAnalysis3.0 software were used in instrumentation control, data analysis and mass spectrum collection. Results: 1 More freeze-thaw cycles had more influence on mass spectrum, especially in small range proteins, so the samples should be operated within 3 cycles in order to get good results.2 Reproducibility of MALDI-TOF test in this study was quite satisfactory; its CV% was within 9.88%-30.07%.3 After comparison, MB-WCX was better than IMAC-Cu and MB-HIC C8 magnetic bead.4 There were 15 main protein peaks were detected whose molecular mass were 4964.52Da, 7765.28Da, 3261.95Da, 1621.2Da, 3192.55Da, 7008.09Da, 2288.99Da, 1741.74Da, 3225.04Da, 4363.01Da, 6910.35Da, 7631.46Da, 3935.34Da, 8140.66Da, 4054.5Da.5 Two protein peaks were of significant difference whose molecular mass were 4964.52Da and 7765.28Da. These two proteins were up-regulated in breast cancer patients, and could be seen as potential breast cancer biomarkers.6 Breast cancer serum diagnostic model were built up and correct rate validation were both more than 80% according to QC, GA and SNN methods.Conclusion: 1 As MALDI-TOF MS is a high-tech method in proteomics analysis, quality control of operating sequence and reduce freeze-thaw cycles in whole procedure is very important.2 MB-WCX was better than IMAC-Cu and MB-HIC C8 magnetic bead in protein purification.3 There was significant difference between the group of breast cancer patients and healthy controls.更多还原