肝癌化疗栓塞术前后血清蛋白质组学研究
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摘要
经导管动脉化疗栓塞术(TACE)是目前公认的非手术治疗肝癌和肝转移癌的首选方法。目前在TACE术后评判肝癌转移、复发的手段主要是综合运用影像学和血清学相结合的方法,要求肝癌患者TACE术后定期复查B超、CT、MR等,对AFP阳性的病人则定期复查血清AFP值。甲胎蛋白(AFP)多年来被作为初筛诊断肝癌的首选指标,但有20%～40%的肝癌患者AFP阴性或低值。
新兴的蛋白组学,是对基因组所编码的所有蛋白质进行研究的学科。通过蛋白质组学使我们对肿瘤的发生、发展有了更详尽的理解,并能够通过发现特异的分子标志物来预测患者预后和治疗效果。
ClinProt技术(液体芯片-飞行时间质谱技术)磁珠分离系统、质谱系统、分析软件和可选的体液样品自动处理系统组成,是蛋白质组学新兴运用的技术,已有众多的研究组利用该技术在患者体液中发现了能够早期诊断肿瘤的标志物。本课题研究目的就是希望在该技术的基础上寻找介入治疗预后与疗效判断相关的分子标记物,并对对有意义的差异蛋白进行鉴定。
第一部分ClinProt技术平台的建立及肝癌门静脉癌栓血清标记物的初步研究
目的建立稳定的ClinProt液体蛋白质芯片技术平台,选择最优化的条件,评价该平台的重复性,进而借此平台研究肝癌门静脉癌栓(PVTT)形成相关标志物并建立预测模型。
材料和方法选取健康体检者血清,采用磁珠分离系统纯化,基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)测定其血清多肽谱,摸索适合血清的最佳技术参数评估该技术的稳定性和可靠性。采取无PVTT和有PVTT肝癌患者血清各20例及体检健康者血清,采用该技术平台建立其血清多肽谱,通过神经网络算法(SNN)建立肝癌门静脉癌栓诊断模型并进行验证。
结果我们总结出最佳点样体积比例是样品：基质=1：5(u1)、温度和湿度控制在20℃-25℃和30%-50%时,结晶效果最佳,质谱出峰情况最好。该平台日内重复性和日间重复性均小于23%,达到蛋白质组学实验的标准。通过对无PVTT和有PVTT患者血清多肽谱的检测,发现蛋白质峰质荷比(M/Z)主要集中在2000~30000范围内,经数据对比分析找到39个有显著性差异的质荷比峰(P<0.05),通过神经网络算法(SNN)建立图谱诊断模型,筛选出建立模型选用的特征峰：1466.52 m/z、5823.1 m/z、8933.09 m/z、5353.95 m/z、4055.19 m/z、4420.43 m/z,5337.19 m/z,和4091.46m/z。模型训练集的判断准确性对于无门静脉癌栓组为92.86%,PVTT组为92.86%。模型测试集的判断准确性对门静脉癌栓组为83.3%,无PVTT组为100%。
结论磁珠分离结合MALDI-TOF-MS的ClinProt蛋白质组学研究平台有良好的可操作性和重复性、特异的判别模型,为肝癌基础研究提供了新的手段。诊断模型可能对预测门静脉癌栓的形成具有重要临床意义。
第二部分肝癌介入前后的血清差异蛋白组学初步研究
目的对一组肝癌患者介入治疗前后血清蛋白谱进行比对,寻找肝癌介入治疗相关的血清标记物。
材料和方法选取TNM分期为Ⅰ、Ⅱ期肝癌患者20例,分别在首次介入前一天(t0)、介入治疗后45天(t1)清晨于肘静脉采集空腹静脉血5ml,其中8例患者行第二次介入TACE术治疗,于第二次介入术后45天(t2)第三次采集静脉血。采用采用磁珠分离系统纯化,基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)测定其血清多肽谱,并进行前后比较,寻找蛋白峰差异。
结果本组20例患者共行TACE术62次,平均9.9个月行介入治疗3.1次,患者全部存活,3例行外科根治性切除治疗,1例行肝移植治疗,3例PD,11例SD,2例PR。1例患者出现肝内动门脉瘘,无患者出现门静脉癌栓和远处转移。血清蛋白质谱建立后(图),在质荷比(M/z)800Da-10000Da范围内,共检测出107个蛋白峰,其中有统计学意义(P<0.05)的共有7个蛋白峰3883、5634、6072、4645、8934、6091、6112m/z。8例患者在首次介入45天后行第二次介入,利用3883、5634m/z进一步验证,该蛋白峰有一定区分价值。
结论介入前后患者血清蛋白质谱对比,3883、5634、6072、4645、8934、6091、6112m/z蛋白峰差异显著,可能有其潜在的临床应用价值。我们拟在下一步实验中对3883Da峰进行鉴定。
第三部分介入治疗相关的差异蛋白鉴定
目的对所发现的3883Da的蛋白进行鉴定,并初步推测其在肝癌及肝癌介入治疗前后的临床意义。
材料和方法：选取上一部分研究中3883Da蛋白波峰值最明显者血清为待鉴定目标,选取3883蛋白峰高表达的10例肝癌患者血清,每个病人取100μl先进行磁珠富集,然后采用液相色谱-串联质谱法(liquid chromatography tandem mass spectrometry, LC-MS/MS进行氨基酸序列测定,然后进行数据库检索比对从而定性。使用数据分析软件BioworksBrowser 3.3.1 SP1进行SequestTM检索。检索数据库为International Protein Index (IPI human v3.45 fasta with 71983 entries)。母离子误差设定为50ppm,碎片离子误差设为1Da,酶切方式为非酶切,可变修饰为M(Methionine)甲硫氨酸氧化。
结果经过液相色谱-串联质谱法鉴定,其氨基酸序列为"R.SARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP.-",通过搜库证明其为SERPINA5. "Plasma serine protease inhibitor",即“丝氨酸蛋白酶抑制剂”、又称“蛋白C抑制剂”、“纤溶酶原激活物抑制剂3(PAI-3)”的肽片段。
结论
我们采用超高效液相色谱-串联质谱联用的方法成功鉴定前一部分介入治疗前后差异明显的3883Da蛋白峰为PAI-3(纤溶酶原激活物抑制剂3)的肽段。复习文献结合本研究推测PAI-3在肝癌的发生、发展中起一定作用,进一步的对PAI-3的功能的探讨和验证有助于深入肝癌TACE治疗的认识,指导提高TACE治疗的效果。
Transcatheter arterial chemoembolization (TACE) is widely recognized as the preferred method of non-surgical treatment of hepatic carcinoma and metastatic liver tumor. Evaluation of metastasis and recurrence after TACE for hepatic carcinoma currently is the integrated use of the means of imaging and serological methods such as periodic review of ultrasound, CT, MR, ECT, etc. serum AFP value is regularly reviewed for the AFP-positive patients. For many years Alpha-fetoprotein (AFP) has been a target of choice for screening diagnosis of hepatic carcinoma, but 20% to 40% of liver cancer patients with negative or low AFP.
Emerging proteomics, is a subject studying all the proteins encoded by the genome. Through the help of Proteomics we can have a more detailed understanding of occurrence and development of tumor and able to find specific molecular markers to predict prognosis and treatment result.
ClinProt technology (liquid-chip-time of flight mass spectrometry) is a promising new approach to proteome profiling of human serum based on magnetic bead separation and MALDI-TOF MS. It consists of bead separation system, MS system, analysis software and automatic processing system for fluid samples. It is a new application of proteomics technologies, a large number of groups have been using the technology in the study body fluids of patients and biomarkers for early diagnosis of cancer have been found. Purpose of this research is to search for molecular markers of the prognosis and efficacy of intervention therapy of hepatic carcinoma using the ClinProt technology and establish a model for diagnosis the disease and prediction of the efficacy, and identify the protein which has significant meaning for the therapy.
Part One
The establishment of the ClinProt platform and the preliminary study of serum markers for hepatic carcinoma with portal vein tumor thrombus
Purpose To establish a stable ClinProt liquid platform for protein chip technology, choose the most optimal conditions, to evaluate the repeatability of the platform, then set up a screen potential protein biomarkers associated with portal vein thrombi (PVTT) formation in hepatocellular carcinorma(HCC) and build a predictive model.
Materials and Methods Serum sample from healthy people was selected and purified by magnetic bead separation system, detected by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) system. Serum peptide spectrum was evaluated and explore the best technical parameters for assessment of serum sample and stability and reliability of the technology. The serum samples from 40 HCC patients (20 with PVTT and 20 free of PVTT) were enriched by the magnetic bead and analyze by MALDI-TOF-MS. Diagnostic model was established using genetic arithmetic (GA) combined with statistical neural networks (SNN).
Result We summarize the best ratio of the sample injection volume: substrate= 1:5 (ul).When the temperature and humidity are controlled at 20℃-25℃and 30%-50% the crystallization effect is the best and the MS peaks are optimal. The replication of within-run and between-run tests were less than 23% and reaches the standard of proteomics experiments.39 peaks with mass charge ratio (m/z) were found (P<0.05). Among them, 8 protein peaks with them/z value of 1466.52,5823.1、8933.09、5353.95、4055.19、4420.43、5337.19 and 4091.46m/z were selected to establish predictive model of PVTT. Specificity and accuracy of this model and 92.86% and 92.86 in predicting PVTT. Negative predictive value is 100%, and positive predictive value is 83.3%.
Conclusion Serum proteomic profiling using the ClinProt system is a promising good approach for proteomics study, ClinProt system has good operability and repeatability, the specificity of the discriminant model provides a new means for liver cancer research. The eight protein peaks may be potential biomarkers of PVTT in HCC patients and the predictive model may have great clinical significance in predicting the formation of PVTT.
Part Two Preliminary study of serum markers for hepatic carcinoma patients before and after TACE
Purpose Compare the serum proteomic patterns of a group of patients of hepatocellular carcinoma before and after TACE treatment. To explore the serum markers related to interventional radiolody.
Materials and Methods Select 20 cases of hepatocellular carcinoma patients which the TNM stage wasⅠ,Ⅱ. Patients were all in the early morning fasting state, and we got 5ml sera from them before TACE(t0). Then we performed TACE by selectively introducing a microcatheter into the right or the left hepatic artery or a segmental branch of the hepatic artery and injecting a mixture of iodized oil (Lipiodol; Andre Guerbet, Aulnaysous-Bois,France) and epirubicin hydrochloride (30~50mg per body surface). This was followed by the introduction of a gelatin sponge (lmm*lmm*lmm). And after 45days (t1)we got another 5ml sera. And among them 8 patients got the third blood drawings 45 days after they were treated with TACE for the second time(t2). Clinical parameters (etiology, tumor grading, AFP levels) were recorded. The choice of TACE as treatment was based on the AASLD-EASL guidelines. The serum samples were purified using magnetic bead separation system and then tested with matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), and differences protein peak before and after the therapy were studied.
Result The total of 20 patients in this group had undergone a total of 62times of TACE, an average of 3.1 times interventional therapy in 9.9 months, all patients survived, and 3 patients went for radical surgical resection,1 patient accepted liver transplantation,3 cases of PD,11 cases SD,2 cases of PR. One patients had arterioportal fistula, no patients had portal vein tumor thrombus or metastasis. After the establishment of serum protein spectrum, During m/z 800Da-10000Da range, a total of 107 protein peaks were detected,7 of which were statistically significant (P<0.05,3883,5634,6072,4645,8934,6091,6112 m/z).8 patients had a second TACE, use of 3883,5634 m/z further verification, the protein peaks have value of a certain distinction.
Conclusion Comparing the serum proteomic patterns of patients of hepatocellular carcinoma before and after TACE treatment,3883, 5634,6072,4645,8934,6091,6112 m/z protein peaks were statistically significant and may have potential clinical value. We intend to identify the 3883Da peak in the next part.
Part Three Identification of different protein concerning TACE
Purpose To identify of the most significantly different protein peak(3883Da) in previous part of study and speculate the role it plays in liver cancer and its meaning before and after TACE.
Materials and Methods Identification of the most significantly different protein peak 3883Da in previous part of study. Select ten high 3883Da peak expression cases for protein enrichment. Serum proteins were enriched and purified from the mixture of the sera of above ten using Clinprot magnetic beads.The purified protein was analyzed by using a LC-MS/MS system. Use data analysis software BioworksBrowser 3.3.1 SP1 for Sequest TM search. Database searching was performed with the program IPI Human (3.45). Parent ion error is set to 50ppm, fragment ion error set 1Da, non-endonuclease digestion, variable modification for the methionine oxidation.
Result Through the identification of LC-MS/MS system, the amino acid sequence of 3883Da peak is "R.SARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP.-".Searching the database, we confirm it as the peptide fragment of SERPINA5 (Plasma serine protease inhibitor) also Protein C inhibitor or Plasminogen activator inhibitor 3.
Conclusion We identify the most significantly different protein peak (3883Da) using LC-MS/MS system successfully. The protein peak is Plasminogen activator inhibitor 3(PAI-3). Review related articles and concern with our study, we speculate PAI-3 plays important role in the occurrence and development liver cancer. Further investigation and validation the PAI-3 may help to in-depth the understanding of TACE for liver cancer will help to improve the effect of TACE treatment.
新兴的蛋白组学,是对基因组所编码的所有蛋白质进行研究的学科。通过蛋白质组学使我们对肿瘤的发生、发展有了更详尽的理解,并能够通过发现特异的分子标志物来预测患者预后和治疗效果。
ClinProt技术(液体芯片-飞行时间质谱技术)磁珠分离系统、质谱系统、分析软件和可选的体液样品自动处理系统组成,是蛋白质组学新兴运用的技术,已有众多的研究组利用该技术在患者体液中发现了能够早期诊断肿瘤的标志物。本课题研究目的就是希望在该技术的基础上寻找介入治疗预后与疗效判断相关的分子标记物,并对对有意义的差异蛋白进行鉴定。
第一部分ClinProt技术平台的建立及肝癌门静脉癌栓血清标记物的初步研究
目的建立稳定的ClinProt液体蛋白质芯片技术平台,选择最优化的条件,评价该平台的重复性,进而借此平台研究肝癌门静脉癌栓(PVTT)形成相关标志物并建立预测模型。
材料和方法选取健康体检者血清,采用磁珠分离系统纯化,基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)测定其血清多肽谱,摸索适合血清的最佳技术参数评估该技术的稳定性和可靠性。采取无PVTT和有PVTT肝癌患者血清各20例及体检健康者血清,采用该技术平台建立其血清多肽谱,通过神经网络算法(SNN)建立肝癌门静脉癌栓诊断模型并进行验证。
结果我们总结出最佳点样体积比例是样品：基质=1：5(u1)、温度和湿度控制在20℃-25℃和30%-50%时,结晶效果最佳,质谱出峰情况最好。该平台日内重复性和日间重复性均小于23%,达到蛋白质组学实验的标准。通过对无PVTT和有PVTT患者血清多肽谱的检测,发现蛋白质峰质荷比(M/Z)主要集中在2000~30000范围内,经数据对比分析找到39个有显著性差异的质荷比峰(P<0.05),通过神经网络算法(SNN)建立图谱诊断模型,筛选出建立模型选用的特征峰：1466.52 m/z、5823.1 m/z、8933.09 m/z、5353.95 m/z、4055.19 m/z、4420.43 m/z,5337.19 m/z,和4091.46m/z。模型训练集的判断准确性对于无门静脉癌栓组为92.86%,PVTT组为92.86%。模型测试集的判断准确性对门静脉癌栓组为83.3%,无PVTT组为100%。
结论磁珠分离结合MALDI-TOF-MS的ClinProt蛋白质组学研究平台有良好的可操作性和重复性、特异的判别模型,为肝癌基础研究提供了新的手段。诊断模型可能对预测门静脉癌栓的形成具有重要临床意义。
第二部分肝癌介入前后的血清差异蛋白组学初步研究
目的对一组肝癌患者介入治疗前后血清蛋白谱进行比对,寻找肝癌介入治疗相关的血清标记物。
材料和方法选取TNM分期为Ⅰ、Ⅱ期肝癌患者20例,分别在首次介入前一天(t0)、介入治疗后45天(t1)清晨于肘静脉采集空腹静脉血5ml,其中8例患者行第二次介入TACE术治疗,于第二次介入术后45天(t2)第三次采集静脉血。采用采用磁珠分离系统纯化,基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)测定其血清多肽谱,并进行前后比较,寻找蛋白峰差异。
结果本组20例患者共行TACE术62次,平均9.9个月行介入治疗3.1次,患者全部存活,3例行外科根治性切除治疗,1例行肝移植治疗,3例PD,11例SD,2例PR。1例患者出现肝内动门脉瘘,无患者出现门静脉癌栓和远处转移。血清蛋白质谱建立后(图),在质荷比(M/z)800Da-10000Da范围内,共检测出107个蛋白峰,其中有统计学意义(P<0.05)的共有7个蛋白峰3883、5634、6072、4645、8934、6091、6112m/z。8例患者在首次介入45天后行第二次介入,利用3883、5634m/z进一步验证,该蛋白峰有一定区分价值。
结论介入前后患者血清蛋白质谱对比,3883、5634、6072、4645、8934、6091、6112m/z蛋白峰差异显著,可能有其潜在的临床应用价值。我们拟在下一步实验中对3883Da峰进行鉴定。
第三部分介入治疗相关的差异蛋白鉴定
目的对所发现的3883Da的蛋白进行鉴定,并初步推测其在肝癌及肝癌介入治疗前后的临床意义。
材料和方法：选取上一部分研究中3883Da蛋白波峰值最明显者血清为待鉴定目标,选取3883蛋白峰高表达的10例肝癌患者血清,每个病人取100μl先进行磁珠富集,然后采用液相色谱-串联质谱法(liquid chromatography tandem mass spectrometry, LC-MS/MS进行氨基酸序列测定,然后进行数据库检索比对从而定性。使用数据分析软件BioworksBrowser 3.3.1 SP1进行SequestTM检索。检索数据库为International Protein Index (IPI human v3.45 fasta with 71983 entries)。母离子误差设定为50ppm,碎片离子误差设为1Da,酶切方式为非酶切,可变修饰为M(Methionine)甲硫氨酸氧化。
结果经过液相色谱-串联质谱法鉴定,其氨基酸序列为"R.SARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP.-",通过搜库证明其为SERPINA5. "Plasma serine protease inhibitor",即“丝氨酸蛋白酶抑制剂”、又称“蛋白C抑制剂”、“纤溶酶原激活物抑制剂3(PAI-3)”的肽片段。
结论
我们采用超高效液相色谱-串联质谱联用的方法成功鉴定前一部分介入治疗前后差异明显的3883Da蛋白峰为PAI-3(纤溶酶原激活物抑制剂3)的肽段。复习文献结合本研究推测PAI-3在肝癌的发生、发展中起一定作用,进一步的对PAI-3的功能的探讨和验证有助于深入肝癌TACE治疗的认识,指导提高TACE治疗的效果。
Transcatheter arterial chemoembolization (TACE) is widely recognized as the preferred method of non-surgical treatment of hepatic carcinoma and metastatic liver tumor. Evaluation of metastasis and recurrence after TACE for hepatic carcinoma currently is the integrated use of the means of imaging and serological methods such as periodic review of ultrasound, CT, MR, ECT, etc. serum AFP value is regularly reviewed for the AFP-positive patients. For many years Alpha-fetoprotein (AFP) has been a target of choice for screening diagnosis of hepatic carcinoma, but 20% to 40% of liver cancer patients with negative or low AFP.
Emerging proteomics, is a subject studying all the proteins encoded by the genome. Through the help of Proteomics we can have a more detailed understanding of occurrence and development of tumor and able to find specific molecular markers to predict prognosis and treatment result.
ClinProt technology (liquid-chip-time of flight mass spectrometry) is a promising new approach to proteome profiling of human serum based on magnetic bead separation and MALDI-TOF MS. It consists of bead separation system, MS system, analysis software and automatic processing system for fluid samples. It is a new application of proteomics technologies, a large number of groups have been using the technology in the study body fluids of patients and biomarkers for early diagnosis of cancer have been found. Purpose of this research is to search for molecular markers of the prognosis and efficacy of intervention therapy of hepatic carcinoma using the ClinProt technology and establish a model for diagnosis the disease and prediction of the efficacy, and identify the protein which has significant meaning for the therapy.
Part One
The establishment of the ClinProt platform and the preliminary study of serum markers for hepatic carcinoma with portal vein tumor thrombus
Purpose To establish a stable ClinProt liquid platform for protein chip technology, choose the most optimal conditions, to evaluate the repeatability of the platform, then set up a screen potential protein biomarkers associated with portal vein thrombi (PVTT) formation in hepatocellular carcinorma(HCC) and build a predictive model.
Materials and Methods Serum sample from healthy people was selected and purified by magnetic bead separation system, detected by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) system. Serum peptide spectrum was evaluated and explore the best technical parameters for assessment of serum sample and stability and reliability of the technology. The serum samples from 40 HCC patients (20 with PVTT and 20 free of PVTT) were enriched by the magnetic bead and analyze by MALDI-TOF-MS. Diagnostic model was established using genetic arithmetic (GA) combined with statistical neural networks (SNN).
Result We summarize the best ratio of the sample injection volume: substrate= 1:5 (ul).When the temperature and humidity are controlled at 20℃-25℃and 30%-50% the crystallization effect is the best and the MS peaks are optimal. The replication of within-run and between-run tests were less than 23% and reaches the standard of proteomics experiments.39 peaks with mass charge ratio (m/z) were found (P<0.05). Among them, 8 protein peaks with them/z value of 1466.52,5823.1、8933.09、5353.95、4055.19、4420.43、5337.19 and 4091.46m/z were selected to establish predictive model of PVTT. Specificity and accuracy of this model and 92.86% and 92.86 in predicting PVTT. Negative predictive value is 100%, and positive predictive value is 83.3%.
Conclusion Serum proteomic profiling using the ClinProt system is a promising good approach for proteomics study, ClinProt system has good operability and repeatability, the specificity of the discriminant model provides a new means for liver cancer research. The eight protein peaks may be potential biomarkers of PVTT in HCC patients and the predictive model may have great clinical significance in predicting the formation of PVTT.
Part Two Preliminary study of serum markers for hepatic carcinoma patients before and after TACE
Purpose Compare the serum proteomic patterns of a group of patients of hepatocellular carcinoma before and after TACE treatment. To explore the serum markers related to interventional radiolody.
Materials and Methods Select 20 cases of hepatocellular carcinoma patients which the TNM stage wasⅠ,Ⅱ. Patients were all in the early morning fasting state, and we got 5ml sera from them before TACE(t0). Then we performed TACE by selectively introducing a microcatheter into the right or the left hepatic artery or a segmental branch of the hepatic artery and injecting a mixture of iodized oil (Lipiodol; Andre Guerbet, Aulnaysous-Bois,France) and epirubicin hydrochloride (30~50mg per body surface). This was followed by the introduction of a gelatin sponge (lmm*lmm*lmm). And after 45days (t1)we got another 5ml sera. And among them 8 patients got the third blood drawings 45 days after they were treated with TACE for the second time(t2). Clinical parameters (etiology, tumor grading, AFP levels) were recorded. The choice of TACE as treatment was based on the AASLD-EASL guidelines. The serum samples were purified using magnetic bead separation system and then tested with matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), and differences protein peak before and after the therapy were studied.
Result The total of 20 patients in this group had undergone a total of 62times of TACE, an average of 3.1 times interventional therapy in 9.9 months, all patients survived, and 3 patients went for radical surgical resection,1 patient accepted liver transplantation,3 cases of PD,11 cases SD,2 cases of PR. One patients had arterioportal fistula, no patients had portal vein tumor thrombus or metastasis. After the establishment of serum protein spectrum, During m/z 800Da-10000Da range, a total of 107 protein peaks were detected,7 of which were statistically significant (P<0.05,3883,5634,6072,4645,8934,6091,6112 m/z).8 patients had a second TACE, use of 3883,5634 m/z further verification, the protein peaks have value of a certain distinction.
Conclusion Comparing the serum proteomic patterns of patients of hepatocellular carcinoma before and after TACE treatment,3883, 5634,6072,4645,8934,6091,6112 m/z protein peaks were statistically significant and may have potential clinical value. We intend to identify the 3883Da peak in the next part.
Part Three Identification of different protein concerning TACE
Purpose To identify of the most significantly different protein peak(3883Da) in previous part of study and speculate the role it plays in liver cancer and its meaning before and after TACE.
Materials and Methods Identification of the most significantly different protein peak 3883Da in previous part of study. Select ten high 3883Da peak expression cases for protein enrichment. Serum proteins were enriched and purified from the mixture of the sera of above ten using Clinprot magnetic beads.The purified protein was analyzed by using a LC-MS/MS system. Use data analysis software BioworksBrowser 3.3.1 SP1 for Sequest TM search. Database searching was performed with the program IPI Human (3.45). Parent ion error is set to 50ppm, fragment ion error set 1Da, non-endonuclease digestion, variable modification for the methionine oxidation.
Result Through the identification of LC-MS/MS system, the amino acid sequence of 3883Da peak is "R.SARLNSQRLVFNRPFLMFIVDNNILFLGKVNRP.-".Searching the database, we confirm it as the peptide fragment of SERPINA5 (Plasma serine protease inhibitor) also Protein C inhibitor or Plasminogen activator inhibitor 3.
Conclusion We identify the most significantly different protein peak (3883Da) using LC-MS/MS system successfully. The protein peak is Plasminogen activator inhibitor 3(PAI-3). Review related articles and concern with our study, we speculate PAI-3 plays important role in the occurrence and development liver cancer. Further investigation and validation the PAI-3 may help to in-depth the understanding of TACE for liver cancer will help to improve the effect of TACE treatment.
引文
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