原发性肝细胞癌血清蛋白质组双向凝胶电泳—飞行时间质谱分析
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摘要
目的：
运用蛋白质组学双向凝胶电泳和质谱技术，研究原发性肝细胞癌患者与正常健康个体血清蛋白质组表达图谱的差异，寻找和确定原发性肝细胞癌特征性血清标志物。为肝癌的临床早期诊断与治疗提供可靠的、准确灵敏的实验室参考指标。
方法：
原发性肝细胞癌患者与正常人血清标本获得后，双向凝胶电泳的第一向电泳采用固相pH梯度电泳，第二向采用SDS-聚丙烯酰胺凝胶电泳分析血清蛋白质组成分。电泳后经银染或考马斯亮蓝染色，ImageMaster双向凝胶电泳软件分析，将有意义的差异蛋白质点胶内胰蛋白酶解，经过基质辅助激光解吸电离飞行时间质谱(MALDI-TOF)鉴定。本项研究还对血清白蛋白和IgG的去除、蛋白上样量、等电聚焦参数、平衡条件、两向之间的转移、SDS-聚丙烯酰胺凝胶的浓度等血清蛋白质组双向凝胶电泳的关键步骤进行了改进和优化。
结果：
1．对血清双向凝胶电泳的关键步骤改进和优化后获得了较好的肝细胞癌患者及正常人血清蛋白质组双向电泳图谱。
2．原发性肝细胞癌患者血清与正常健康个体血清相比较，经双向凝胶电泳软件分析，共找到33个差异表达的蛋白质，其中6个蛋白质在原发性肝细胞癌患者血清中含量降低，5个蛋白质含量增加，另有17个蛋白质在原发性肝细胞癌患者血清中缺失，5个蛋白质点为原发性肝细胞癌患者血清所特有。
天津医科大学硕士学位论文
肝细胞癌患者血清中缺失，5个蛋白质点为原发性肝细胞癌患者血清所特
有。
.选取在原发性肝细胞癌中缺失的3个蛋白质点(Spotl、2、3)，在原发性
肝细胞癌中含量减低的2个蛋白质点(Spot 16、17)和在原发性肝细胞癌中
含量增加的2个蛋白质点(Spot18、32)共7个蛋白质点进行MALDI一TOF
质谱鉴定，成功得到了7张肤质量指纹图谱，经数据库检索鉴定分析，确
定在原发性肝细胞癌患者血清中缺失的Spotl、2、3分别为血清白蛋白、
血清转铁蛋白、CDS抗原类蛋白;在原发性肝细胞癌患者血清中含量减低
的蛋白质点SPot16、17也是血清白蛋白;在原发性肝细胞癌患者血清中含
量增加的蛋白质点Spot 18、Spot32分别为锌一。2糖蛋白和免疫球蛋白Y-
1链C区。
结论:
1.根据蛋白质不同的分子量和等电点，通过双向凝胶电泳技术可将人血清中
蛋白质成分进行分离，并可利用分析软件，找到不同疾病状态下血清中的
差异蛋白质。
2.原发性肝细胞癌与正常人相比，在血清蛋白质组成分中存在差异。
.找到的这些差异蛋白质为进一步完善原发性肝细胞癌血清学诊断指标提供
了参考依据。
Analyzing the serum proteome of the patients suffering from primary hepatocellular carcinoma (PHCC ) and healthy donors, to search tumor markers of hepatocellular carcinoma for diagnosis.
Methods
The serum proteome of the patients from primary hepatocellular carcinoma and healthy donors were separated and identified using immobilized pH gradient (IPG). Isoelectric focusing (IEF) electrophoresis was run as the first dimensional electrophoresis, and then horizontal SDS-PAGE as the second electrophoresis. After silver staining or coomassie blue staining, images were captured by scanner, and then the images were edited and matched using Imagemaster2DE analysis software. The differentially expressed proteins were analyzed by peptide mass fingerprint based on matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF-MS) and SWISS-PROT or BLAST nr database searching.
Results
1. By removing albumin and IgG in serum, trying different loding quantity, procedure of isoelectric focusing electrophoresis, time of equilibrium, density of SDS-PAGE gel and other important procedures of two-dimensional electrophoresis, the images were analyzed successfully and good reproducibility were obtained.
2. Analyzing by Imagemaster2DE software, thirty-three proteins were found differentially expressed in sera from hepatocellular carcinoma patients and healthy donors. Six proteins were down-regulated in HCC, five proteins were up-regulated in HCC, seventeen proteins were found especially in healthy controls and five proteins were found especially in HCC.
3. After analyzing by MALDI-TOF-MS, five differentially expressed proteins were identified. Albumin , Serotransferrin, CD5 antigen-like precursor ( IgM -associated peptide) were down-regulated in HCC, and Zinc-alpha-2-glycoprotein and Ig gamma-1 chain C region were up-regulated in HCC.
Conclusion
1. Using immobilized pH gradient two-dimensional polyacrylamide gel electrophoresis (2-DE), good reproducibility and images could be obtained to separate and identify the proteome in serum.
2. Thirty-three proteins which were found differentially expressed in HCC and healthy donors provided useful information for screening diagnostic tumor markers of human HCC.
运用蛋白质组学双向凝胶电泳和质谱技术，研究原发性肝细胞癌患者与正常健康个体血清蛋白质组表达图谱的差异，寻找和确定原发性肝细胞癌特征性血清标志物。为肝癌的临床早期诊断与治疗提供可靠的、准确灵敏的实验室参考指标。
方法：
原发性肝细胞癌患者与正常人血清标本获得后，双向凝胶电泳的第一向电泳采用固相pH梯度电泳，第二向采用SDS-聚丙烯酰胺凝胶电泳分析血清蛋白质组成分。电泳后经银染或考马斯亮蓝染色，ImageMaster双向凝胶电泳软件分析，将有意义的差异蛋白质点胶内胰蛋白酶解，经过基质辅助激光解吸电离飞行时间质谱(MALDI-TOF)鉴定。本项研究还对血清白蛋白和IgG的去除、蛋白上样量、等电聚焦参数、平衡条件、两向之间的转移、SDS-聚丙烯酰胺凝胶的浓度等血清蛋白质组双向凝胶电泳的关键步骤进行了改进和优化。
结果：
1．对血清双向凝胶电泳的关键步骤改进和优化后获得了较好的肝细胞癌患者及正常人血清蛋白质组双向电泳图谱。
2．原发性肝细胞癌患者血清与正常健康个体血清相比较，经双向凝胶电泳软件分析，共找到33个差异表达的蛋白质，其中6个蛋白质在原发性肝细胞癌患者血清中含量降低，5个蛋白质含量增加，另有17个蛋白质在原发性肝细胞癌患者血清中缺失，5个蛋白质点为原发性肝细胞癌患者血清所特有。
天津医科大学硕士学位论文
肝细胞癌患者血清中缺失，5个蛋白质点为原发性肝细胞癌患者血清所特
有。
.选取在原发性肝细胞癌中缺失的3个蛋白质点(Spotl、2、3)，在原发性
肝细胞癌中含量减低的2个蛋白质点(Spot 16、17)和在原发性肝细胞癌中
含量增加的2个蛋白质点(Spot18、32)共7个蛋白质点进行MALDI一TOF
质谱鉴定，成功得到了7张肤质量指纹图谱，经数据库检索鉴定分析，确
定在原发性肝细胞癌患者血清中缺失的Spotl、2、3分别为血清白蛋白、
血清转铁蛋白、CDS抗原类蛋白;在原发性肝细胞癌患者血清中含量减低
的蛋白质点SPot16、17也是血清白蛋白;在原发性肝细胞癌患者血清中含
量增加的蛋白质点Spot 18、Spot32分别为锌一。2糖蛋白和免疫球蛋白Y-
1链C区。
结论:
1.根据蛋白质不同的分子量和等电点，通过双向凝胶电泳技术可将人血清中
蛋白质成分进行分离，并可利用分析软件，找到不同疾病状态下血清中的
差异蛋白质。
2.原发性肝细胞癌与正常人相比，在血清蛋白质组成分中存在差异。
.找到的这些差异蛋白质为进一步完善原发性肝细胞癌血清学诊断指标提供
了参考依据。
Analyzing the serum proteome of the patients suffering from primary hepatocellular carcinoma (PHCC ) and healthy donors, to search tumor markers of hepatocellular carcinoma for diagnosis.
Methods
The serum proteome of the patients from primary hepatocellular carcinoma and healthy donors were separated and identified using immobilized pH gradient (IPG). Isoelectric focusing (IEF) electrophoresis was run as the first dimensional electrophoresis, and then horizontal SDS-PAGE as the second electrophoresis. After silver staining or coomassie blue staining, images were captured by scanner, and then the images were edited and matched using Imagemaster2DE analysis software. The differentially expressed proteins were analyzed by peptide mass fingerprint based on matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF-MS) and SWISS-PROT or BLAST nr database searching.
Results
1. By removing albumin and IgG in serum, trying different loding quantity, procedure of isoelectric focusing electrophoresis, time of equilibrium, density of SDS-PAGE gel and other important procedures of two-dimensional electrophoresis, the images were analyzed successfully and good reproducibility were obtained.
2. Analyzing by Imagemaster2DE software, thirty-three proteins were found differentially expressed in sera from hepatocellular carcinoma patients and healthy donors. Six proteins were down-regulated in HCC, five proteins were up-regulated in HCC, seventeen proteins were found especially in healthy controls and five proteins were found especially in HCC.
3. After analyzing by MALDI-TOF-MS, five differentially expressed proteins were identified. Albumin , Serotransferrin, CD5 antigen-like precursor ( IgM -associated peptide) were down-regulated in HCC, and Zinc-alpha-2-glycoprotein and Ig gamma-1 chain C region were up-regulated in HCC.
Conclusion
1. Using immobilized pH gradient two-dimensional polyacrylamide gel electrophoresis (2-DE), good reproducibility and images could be obtained to separate and identify the proteome in serum.
2. Thirty-three proteins which were found differentially expressed in HCC and healthy donors provided useful information for screening diagnostic tumor markers of human HCC.
引文
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[2] Shigetoshi F, Motohiko T, Seishi M, et al. Tumor markers in early diagnosis, follow-up and management of patients with hepatocellular carcinoma. Oncology, 2002, 62 (S1): 57-63.
[3] Tissot JD, Sanchez JC, Vuadens F, et al. IgM are associated to Sp alpha (CD5 antigen-like). Electrophoresis. 2002 Apr; 23(7-8): 1203-1206.
[4] Pers JO, Berthou C, Porakishvili N, et al. CD5-induced apoptosis of B cells in some patients with chronic lymphocytic leukemia. Leukemia. 2002 Jan; 16(1): 44-52.
[5] Lopez-Boado YS, Diez-Itza I, Tolivia J, et al. Glucocorticoids and androgens up-regulate the Zn-alpha 2-glycoprotein messenger RNA in human breast cancer cells. Breast Cancer Res Treat. 1994; 29(3): 247-258.
[6] Brysk MM, Lei G, Selvanayagam P, et al. Modulation by interferon-gamma of zinc-alpha 2-glycoprotein gene expression in human epithelial cell lines. Anticancer Res. 1997 Sep-Oct; 17(5A): 3387-3391.
[7] Ghafouri B, Tagesson C, Lindahl M. gapping of proteins in human saliva using two-dimensional gel electrophoresis and peptide mass fingerprinting. Proteomics. 2003 Jun; 3(6): 1003-1015.
[8] BingC, Bao Y, Jenkins J, et al. Zinc-alpha2-glycoprotein, a lipid nobilizing factor, is expressed in adipocytes and is up-regulated in mice with cancer cachexia. Proc Natl Acad Sci U S A. 2004 Feb 24; 101(8): 2500-2505.
[9] Hale LP, Price DT, Sanchez LM, et al. Zinc alpha-2-glycoprotein is expressed
by malignant prostatic epithelium and may serve asa potential serum marker for prostate cancer. Clin Cancer Res. 2001 Apr; 7(4): 846-853.
[10] Perdew G, Schaup H, Selivonchick D. The use of a zwitterioncic detergent in two-dimensional eletrophoresis. Electrophoresis 1983, 19, 758-760.
[11] Marshall T, Williams K. Artifacts associated with 2-mercaptothanol upon high resolution two-dimensional electrophoresis. Anal Biochen, 1984, 139, 502-505.
[12] 张维铭主编．2003．现代分子生物学实验手册．北京：科学出版社．
[13] Kreczko S, Lipska A, Wysocka J. Alpha-fetoprotein: diagnostic value in hepatic disorders. Pol Merkuriusz Lek, 2000, 8(48): 420-423.
[14] Yoskida S, Kurokohchi K, Arima K et al. Clinical significance of lens culinaris agglytimin-reactive fraction of serum alpha-fetoprotein in patients with hepatocellular carcinoma Int J Oncol. 2002, 20(2): 305-309.
[15] Takeda S, Nakao A. Genetic detection and clinical application in patients with hepatocellular carcinoma Nippon Geka Gakkai Zasshi, 2002, 103(6): 472-475.
[16] Nomura F, Ishijima M, Kuwa K, et al. Serum des-gamma-carboy prothrom-bin levels determined by a new generation of sensitive immunoassays in patients with small-sized hepatocellular carcinoma Am J Gastroenterol, 1999, 94(3): 651-654.
[17] Mori S, Anyagi Y, Yanagi M, et al. Serum N-acetylglucosaninyl transferase Ⅲ activities in hepatocellularcarcinoma. Journal of Gastroenterology, 1998, 13(6): 610-619.
[18] Huskic J, Kulenovic H, Kardum D, et al. Serum angiotensin converting enzyme in patients with primary liver carcinoma Medicinski Arhiv, 1999, 53(2): 61-63.
[19] Seow TK, Ong SE, Liang RC, et al. Two-dimensional electrophoresis map of the separated proteins by mass spectrouetry[J]. Electrophoresis, 2000,
21(9): 1787-1813.
[20] 俞利荣，王楠，吴高德等．人肝癌细胞系BEL-7404和正常肝癌细胞系L-02表达的蛋白质组双向凝胶电泳分析．科学通报，2000，45(2)：170-178．
[21] 王征，阮幼冰，官阳等．肝细胞癌患者血清蛋白组成的双向凝胶电泳-飞行时间质谱分析．中华病理学杂志，2003，32(4)：333-336．
[22] Poon TC, Johnson PJ. Proteome analysis and its impact on the discovery of serological tumor markers[J].Clin Chin Acta, 2001, 313(1-2): 231-239.
[23] Kennedy S. Proteomic profiling from humun sample: the body alternative[J].Toxicol Lett, 2001, 120(1-3): 379-384.
[24] Mckerrow JH, Bhargava V, Hansell E, et al. A fuctional proteomics screen of proteases in colorectal carcinoma[J].Mol Med, 2000, 6(5): 450-460.
[25] Seow TK, Liang RC, Leow CK, et al. Hepatocellular carcinoma: from bedside to proteomics[J].Protoomics, 2001, 1(10): 1249-1263.
[26] International Human Genome Sequencing Consortium. Initial sequencing and analysis of the human genome[J]. Nature, 2001, 409: 860-921.
[27] Reynolds T. For ptoteomics research, a new race has begun. J Natl Cancer Inst, 2002, 94: 552-554.
[28] Simpson RJ, Dorow DS. Cancer protomics: From signaling network to tumor markers[J]. Trends Biotechnol, 2001, 19(sup.): 40-47.
[29] Wu W, HU W, Kavanagh J. Proteomics in cancer research. Iht J Gynecol Cancer. 2002, 12: 409-423.
[30] Gavin AC, Bosche M, Krause R, et al. Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature, 2002, 15: 141-147.
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[2] Shigetoshi F, Motohiko T, Seishi M, et al. Tumor markers in early diagnosis, follow-up and management of patients with hepatocellular carcinoma. Oncology, 2002, 62 (S1): 57-63.
[3] Tissot JD, Sanchez JC, Vuadens F, et al. IgM are associated to Sp alpha (CD5 antigen-like). Electrophoresis. 2002 Apr; 23(7-8): 1203-1206.
[4] Pers JO, Berthou C, Porakishvili N, et al. CD5-induced apoptosis of B cells in some patients with chronic lymphocytic leukemia. Leukemia. 2002 Jan; 16(1): 44-52.
[5] Lopez-Boado YS, Diez-Itza I, Tolivia J, et al. Glucocorticoids and androgens up-regulate the Zn-alpha 2-glycoprotein messenger RNA in human breast cancer cells. Breast Cancer Res Treat. 1994; 29(3): 247-258.
[6] Brysk MM, Lei G, Selvanayagam P, et al. Modulation by interferon-gamma of zinc-alpha 2-glycoprotein gene expression in human epithelial cell lines. Anticancer Res. 1997 Sep-Oct; 17(5A): 3387-3391.
[7] Ghafouri B, Tagesson C, Lindahl M. gapping of proteins in human saliva using two-dimensional gel electrophoresis and peptide mass fingerprinting. Proteomics. 2003 Jun; 3(6): 1003-1015.
[8] BingC, Bao Y, Jenkins J, et al. Zinc-alpha2-glycoprotein, a lipid nobilizing factor, is expressed in adipocytes and is up-regulated in mice with cancer cachexia. Proc Natl Acad Sci U S A. 2004 Feb 24; 101(8): 2500-2505.
[9] Hale LP, Price DT, Sanchez LM, et al. Zinc alpha-2-glycoprotein is expressed
by malignant prostatic epithelium and may serve asa potential serum marker for prostate cancer. Clin Cancer Res. 2001 Apr; 7(4): 846-853.
[10] Perdew G, Schaup H, Selivonchick D. The use of a zwitterioncic detergent in two-dimensional eletrophoresis. Electrophoresis 1983, 19, 758-760.
[11] Marshall T, Williams K. Artifacts associated with 2-mercaptothanol upon high resolution two-dimensional electrophoresis. Anal Biochen, 1984, 139, 502-505.
[12] 张维铭主编．2003．现代分子生物学实验手册．北京：科学出版社．
[13] Kreczko S, Lipska A, Wysocka J. Alpha-fetoprotein: diagnostic value in hepatic disorders. Pol Merkuriusz Lek, 2000, 8(48): 420-423.
[14] Yoskida S, Kurokohchi K, Arima K et al. Clinical significance of lens culinaris agglytimin-reactive fraction of serum alpha-fetoprotein in patients with hepatocellular carcinoma Int J Oncol. 2002, 20(2): 305-309.
[15] Takeda S, Nakao A. Genetic detection and clinical application in patients with hepatocellular carcinoma Nippon Geka Gakkai Zasshi, 2002, 103(6): 472-475.
[16] Nomura F, Ishijima M, Kuwa K, et al. Serum des-gamma-carboy prothrom-bin levels determined by a new generation of sensitive immunoassays in patients with small-sized hepatocellular carcinoma Am J Gastroenterol, 1999, 94(3): 651-654.
[17] Mori S, Anyagi Y, Yanagi M, et al. Serum N-acetylglucosaninyl transferase Ⅲ activities in hepatocellularcarcinoma. Journal of Gastroenterology, 1998, 13(6): 610-619.
[18] Huskic J, Kulenovic H, Kardum D, et al. Serum angiotensin converting enzyme in patients with primary liver carcinoma Medicinski Arhiv, 1999, 53(2): 61-63.
[19] Seow TK, Ong SE, Liang RC, et al. Two-dimensional electrophoresis map of the separated proteins by mass spectrouetry[J]. Electrophoresis, 2000,
21(9): 1787-1813.
[20] 俞利荣，王楠，吴高德等．人肝癌细胞系BEL-7404和正常肝癌细胞系L-02表达的蛋白质组双向凝胶电泳分析．科学通报，2000，45(2)：170-178．
[21] 王征，阮幼冰，官阳等．肝细胞癌患者血清蛋白组成的双向凝胶电泳-飞行时间质谱分析．中华病理学杂志，2003，32(4)：333-336．
[22] Poon TC, Johnson PJ. Proteome analysis and its impact on the discovery of serological tumor markers[J].Clin Chin Acta, 2001, 313(1-2): 231-239.
[23] Kennedy S. Proteomic profiling from humun sample: the body alternative[J].Toxicol Lett, 2001, 120(1-3): 379-384.
[24] Mckerrow JH, Bhargava V, Hansell E, et al. A fuctional proteomics screen of proteases in colorectal carcinoma[J].Mol Med, 2000, 6(5): 450-460.
[25] Seow TK, Liang RC, Leow CK, et al. Hepatocellular carcinoma: from bedside to proteomics[J].Protoomics, 2001, 1(10): 1249-1263.
[26] International Human Genome Sequencing Consortium. Initial sequencing and analysis of the human genome[J]. Nature, 2001, 409: 860-921.
[27] Reynolds T. For ptoteomics research, a new race has begun. J Natl Cancer Inst, 2002, 94: 552-554.
[28] Simpson RJ, Dorow DS. Cancer protomics: From signaling network to tumor markers[J]. Trends Biotechnol, 2001, 19(sup.): 40-47.
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