利用MALDI-TOF MS技术检测乳腺癌患者血清蛋白指纹图谱并建立乳腺癌诊断模型
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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。
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.
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。
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.
引文
1 Christine H. Chung, Shawn Levy, Pierre Chaurand. Genomics and proteomics:Emerging tech-nologies in clinical cancer research. Critical Reviews in Oncology/Hematology, 2007, 61(1): 1-15
2 Etzioni R, Urban N, Ramsey S, McIntosh M, Schwartz S, Reid B, et al.The case for early detection [J]. Nat Rev Cancer 2003, 3(5):243-252
3 Aldred S, Grant MM, Griffiths HR. The use of proteomics for the assessment of clinical samples in research [J]. Clin Biochem,2004, 8(37):943-952
4 Wasinger VC. Progress with gene-product mapping of the Mollicutes: My coplasma genitailium[J]. Electrophoresis,1995,16(7):1090
5 Blackstock WP, Weir MP. Proteomics: quantitative and physical mapping of cellular proteins. Trends Biotechnol, 1999,17(3): 121-127
6 Fields S. Proteomics in genomeland. Science, 2001,291(5507): 1221-1224.
7 O’Farrell. PH High resolution two-dimensional electrophoresis of proteins[J]. J Biol Chem,1975,250:4007
8 Arthur M Moseley. Current trends in differential expression proteomics:isotopically coded tags. Trends in biotechnology, 2001, 19(suppl): 10-16
9 lahm HW,langen H.Mass Spectrometry:a tool for the indentification of proteins separated by gels.[J]Electrophoresis,2000,21(2):2105-2114
10 karas M,Hillenkamp F.Laser desorption ionization of proteins with molecular masses exceeding 10000 daltons.[J] Anal Chem,1988,60(4):2299-2301
11 Fenn JB,Mann M,Meng CK, et al.Electrospray ionization for mass spectrometry of large biomolecules.[J]Sience,1989,246(6):64-71
12赵冰,沈学静。飞行时间质谱分析技术的发展[J]。现代科学仪器2006,4(3):30-33
13王胜林,王强斌,古宏晨,等.磁性微球的生物医学应用研究进展磁性微球简介[J].化学世界, 2001, 7(4): 384-387
14布和巴特尔。磁珠技术在生命科学领域的应用及其制备[J]。化学工程师,2004, 6(7):57-58
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16 Amy V. Rapkiewicz, Virginia Espina, Emanuel, et al. Biomarkers of ovarian tumours [J]. European Journal of cancer, 2004, 40(17):2604-2612
17 Timothy D. Veenstra, DaRue A. Prieto, Thomas P. Conrads. Proteomic patterns for early cancer detection[J]. Drug Discovery Today, 2004,9(20):889-897
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2 Etzioni R, Urban N, Ramsey S, McIntosh M, Schwartz S, Reid B, et al.The case for early detection [J]. Nat Rev Cancer 2003, 3(5):243-252
3 Aldred S, Grant MM, Griffiths HR. The use of proteomics for the assessment of clinical samples in research [J]. Clin Biochem,2004, 8(37):943-952
4 Wasinger VC. Progress with gene-product mapping of the Mollicutes: My coplasma genitailium[J]. Electrophoresis,1995,16(7):1090
5 Blackstock WP, Weir MP. Proteomics: quantitative and physical mapping of cellular proteins. Trends Biotechnol, 1999,17(3): 121-127
6 Fields S. Proteomics in genomeland. Science, 2001,291(5507): 1221-1224.
7 O’Farrell. PH High resolution two-dimensional electrophoresis of proteins[J]. J Biol Chem,1975,250:4007
8 Arthur M Moseley. Current trends in differential expression proteomics:isotopically coded tags. Trends in biotechnology, 2001, 19(suppl): 10-16
9 lahm HW,langen H.Mass Spectrometry:a tool for the indentification of proteins separated by gels.[J]Electrophoresis,2000,21(2):2105-2114
10 karas M,Hillenkamp F.Laser desorption ionization of proteins with molecular masses exceeding 10000 daltons.[J] Anal Chem,1988,60(4):2299-2301
11 Fenn JB,Mann M,Meng CK, et al.Electrospray ionization for mass spectrometry of large biomolecules.[J]Sience,1989,246(6):64-71
12赵冰,沈学静。飞行时间质谱分析技术的发展[J]。现代科学仪器2006,4(3):30-33
13王胜林,王强斌,古宏晨,等.磁性微球的生物医学应用研究进展磁性微球简介[J].化学世界, 2001, 7(4): 384-387
14布和巴特尔。磁珠技术在生命科学领域的应用及其制备[J]。化学工程师,2004, 6(7):57-58
15 Nicolas Chignard,Laura Beretta.Proteomics for hepatocellular carcinoma marker discovery [J]. Gastroenterology, 2004, 127(5):120-125
16 Amy V. Rapkiewicz, Virginia Espina, Emanuel, et al. Biomarkers of ovarian tumours [J]. European Journal of cancer, 2004, 40(17):2604-2612
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