人类十二指肠液蛋白质组学研究
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摘要
研究背景:
胰腺癌是恶性程度最高的肿瘤之一,据2007年统计结果显示,在美国胰腺癌是肿瘤死因顺位中的第4位。早期发现及早期干预对于胰腺癌患者提高生存率是至关重要的。但遗憾的是,目前临床上应用的肿瘤标志物,其敏感性和特异性都较差。近年来应用蛋白质组学方法寻找肿瘤标志物成为医学基础研究的热点,但目前尚未见到有关于人类十二指肠液蛋白质组学研究的相关报道。
目的与方法:
本课题以人类十二指肠液标本作为研究对象,以1-DE与2-DE进行蛋白分离,以LC MS/MS与MALDI-TOF/TOF进行蛋白鉴定。摸索人类十二指肠液的预处理方法,探索十二指肠液蛋白质组学的研究方法,建立十二指肠液蛋白数据库,并初步探讨十二指肠液替代胰液寻找肿瘤标志物的可行性。
结果:
1)预处理采用超滤离心法,可以达到除盐与蛋白浓缩的目的;以纯水作为超滤置换液,可以保证十二指肠液标本的稳定性;2)行二维凝胶电泳时,十二指肠液一旦与重泡胀液混合,就会出现明显蛋白降解的现象,加入大剂量蛋白酶抑制剂或同时给予更为彻底的蛋白变性处理,有助于减少标本中蛋白的降解;3)通过1-DE对44例患者的标本进行蛋白分离,发现十二指肠液呈现出3种图谱类型,其中1种与胃液图谱相似,怀疑部分标本中有胃液混入;4)通过1-DE对消化系统良性疾病患者的十二指肠液标本进行蛋白分离,以MALDI-TOF/TOF质谱进行鉴定,共鉴定出33种独立的蛋白,绝大部分为胰液蛋白成分,其中有3种为胃蛋白酶类,怀疑部分标本中有胃液混入;5)通过对比发现,十二指肠液与胰液之间从1-DE图谱形态到蛋白鉴定的结果均存在很好的一致性。
结论:
1)超滤置换法是十二指肠液标本预处理过程中一种有效的除盐和浓缩蛋白的方法;超滤置换液选用纯水可保证标本的稳定性;2)行2-DE时,十二指肠液标本中蛋白的降解问题出现在一向等点聚焦过程中,其原因可能是由于重泡胀液提供的碱性环境,使得某些酶类或小分子物质得以激活;3)对十二指肠液1-DE图谱进行了分类与分析,共发现3种图谱类型,其中的Ⅱ型图谱可能是由于十二指肠液标本中混入了胃液;4)建立了消化系统良性疾病患者的十二指肠液蛋白数据库;5)十二指肠液可以起到辅助胰液蛋白质组学研究的作用。
Background:Pancreatic cancer is one of the most formidable malignant tumors worldwide.According to the cancer statistics report in 2007,pancreatic cancer is the fourth most common cause of cancer death in the United States.Early detection is the most feasible strategies to improve outcome.Unfortunately,the biomarkers currently available for pancreatic cancer lack adequate sensitivity and specificity.In recent years, there has been a substantial interest in applying proteomics technologies to identify protein biomarkers for early detection of cancer.There is no report on duodenal juice proteomics analysis up to date.
Objectives and Methods:In the present study,we collected human duodenal juice as our study samples.Duodenal juice was first fractionated by 1-DE or 2-DE and subsequently analyzed by LC-MS/MS or MALDI-TOF/TOF.Our objectives are as following:1) to explore an effective sample preparation method;2) to establish the duodenal juice proteomics study method;3) to establish the 1-DE gel protein database of duodenal juice;4) to evaluate the feasibility of identifying biomarker from duodenal juice,substituting for pancreatic juice.
Results:1) Ultra-centrifugation has great performance in salt removal and protein enrichment.When mixed with pure water,duodenal juice remained stable during the ultra-centrifugation process.2) Degradation will occur when duodenal juice is mixed with rehydration stock solution during first-dimension isoelectric focusing process. PIC is able to improve this condition.3) 1-DE images of 44 cases can be divided into three categories.4) Duodenal juice was fractionated by 1-DE and analyzed by MALDI-TOF/TOF.A total of 33 unique proteins were identified including 3 types of pepsin.5) 1-DE image and protein composition of both duodenal juice and pancreatic juice are in good concordance.
Conclusions:1) Ultra-centrifugation is an effective sample preparation method in removing salt and enriching protein.When mixed with pure water,duodenal juice remains stable during this process.2) Degradation occurs during first-dimension isoelectric focusing process.3) Duodenal juice 1-DE images are divided into three categories.Contamination of gastric juice may be the cause of TypeⅡimage.4) Establish the 1-DE gel protein database of duodenal juice.5) Duodenal juice can assist the pancreatic juice proteomics analysis study.
胰腺癌是恶性程度最高的肿瘤之一,据2007年统计结果显示,在美国胰腺癌是肿瘤死因顺位中的第4位。早期发现及早期干预对于胰腺癌患者提高生存率是至关重要的。但遗憾的是,目前临床上应用的肿瘤标志物,其敏感性和特异性都较差。近年来应用蛋白质组学方法寻找肿瘤标志物成为医学基础研究的热点,但目前尚未见到有关于人类十二指肠液蛋白质组学研究的相关报道。
目的与方法:
本课题以人类十二指肠液标本作为研究对象,以1-DE与2-DE进行蛋白分离,以LC MS/MS与MALDI-TOF/TOF进行蛋白鉴定。摸索人类十二指肠液的预处理方法,探索十二指肠液蛋白质组学的研究方法,建立十二指肠液蛋白数据库,并初步探讨十二指肠液替代胰液寻找肿瘤标志物的可行性。
结果:
1)预处理采用超滤离心法,可以达到除盐与蛋白浓缩的目的;以纯水作为超滤置换液,可以保证十二指肠液标本的稳定性;2)行二维凝胶电泳时,十二指肠液一旦与重泡胀液混合,就会出现明显蛋白降解的现象,加入大剂量蛋白酶抑制剂或同时给予更为彻底的蛋白变性处理,有助于减少标本中蛋白的降解;3)通过1-DE对44例患者的标本进行蛋白分离,发现十二指肠液呈现出3种图谱类型,其中1种与胃液图谱相似,怀疑部分标本中有胃液混入;4)通过1-DE对消化系统良性疾病患者的十二指肠液标本进行蛋白分离,以MALDI-TOF/TOF质谱进行鉴定,共鉴定出33种独立的蛋白,绝大部分为胰液蛋白成分,其中有3种为胃蛋白酶类,怀疑部分标本中有胃液混入;5)通过对比发现,十二指肠液与胰液之间从1-DE图谱形态到蛋白鉴定的结果均存在很好的一致性。
结论:
1)超滤置换法是十二指肠液标本预处理过程中一种有效的除盐和浓缩蛋白的方法;超滤置换液选用纯水可保证标本的稳定性;2)行2-DE时,十二指肠液标本中蛋白的降解问题出现在一向等点聚焦过程中,其原因可能是由于重泡胀液提供的碱性环境,使得某些酶类或小分子物质得以激活;3)对十二指肠液1-DE图谱进行了分类与分析,共发现3种图谱类型,其中的Ⅱ型图谱可能是由于十二指肠液标本中混入了胃液;4)建立了消化系统良性疾病患者的十二指肠液蛋白数据库;5)十二指肠液可以起到辅助胰液蛋白质组学研究的作用。
Background:Pancreatic cancer is one of the most formidable malignant tumors worldwide.According to the cancer statistics report in 2007,pancreatic cancer is the fourth most common cause of cancer death in the United States.Early detection is the most feasible strategies to improve outcome.Unfortunately,the biomarkers currently available for pancreatic cancer lack adequate sensitivity and specificity.In recent years, there has been a substantial interest in applying proteomics technologies to identify protein biomarkers for early detection of cancer.There is no report on duodenal juice proteomics analysis up to date.
Objectives and Methods:In the present study,we collected human duodenal juice as our study samples.Duodenal juice was first fractionated by 1-DE or 2-DE and subsequently analyzed by LC-MS/MS or MALDI-TOF/TOF.Our objectives are as following:1) to explore an effective sample preparation method;2) to establish the duodenal juice proteomics study method;3) to establish the 1-DE gel protein database of duodenal juice;4) to evaluate the feasibility of identifying biomarker from duodenal juice,substituting for pancreatic juice.
Results:1) Ultra-centrifugation has great performance in salt removal and protein enrichment.When mixed with pure water,duodenal juice remained stable during the ultra-centrifugation process.2) Degradation will occur when duodenal juice is mixed with rehydration stock solution during first-dimension isoelectric focusing process. PIC is able to improve this condition.3) 1-DE images of 44 cases can be divided into three categories.4) Duodenal juice was fractionated by 1-DE and analyzed by MALDI-TOF/TOF.A total of 33 unique proteins were identified including 3 types of pepsin.5) 1-DE image and protein composition of both duodenal juice and pancreatic juice are in good concordance.
Conclusions:1) Ultra-centrifugation is an effective sample preparation method in removing salt and enriching protein.When mixed with pure water,duodenal juice remains stable during this process.2) Degradation occurs during first-dimension isoelectric focusing process.3) Duodenal juice 1-DE images are divided into three categories.Contamination of gastric juice may be the cause of TypeⅡimage.4) Establish the 1-DE gel protein database of duodenal juice.5) Duodenal juice can assist the pancreatic juice proteomics analysis study.
引文
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[49]Zhou L,Lu Z,Yang A,Deng R,Mai C,Sang X,Faber KN,Lu X.Comparative proteomic analysis of human pancreatic juice:methodological study.Proteomics.2007 Apr;7(8):1345-55.
[2]Ferrone CR,Brennan MF,Gonen M,Coit DG,Fong Y,Chung S,Tang L,Klimstra D,Allen PJ.Pancreatic Adenocarcinoma:The Actual 5-Year Survivors.J Gastrointest Surg.2007 Nov 20.
[3]Steinberg,W.The clinical utility of the CA 19-9 tumor-associated antigen.Am.J.Gastroenterol.1990 Apr;85(4):350-5.
[4]Abbott A.And now for the proteome.Nature,2001,409:747.
[5]Fields S.Proteomics in genomeland.Science,2001,291:1221-1224.
[6]Hu S,Loo JA,Wong DT.Hman body fluid proteome analysis.Proteomics.2006Dec;6(23):6326-53.
[7]Thongboonkerd V,Malasit P.Proteomics 2005,5,1033-1042.
[8]Thongboonkerd V,McLeish KR,Arthur JM,Klein JB.Kidney Int.2002,62,1461-1469.
[9]Lafitte D,Dussol B,Andersen S,Vazi A.et al.Clin.Biochem.2002,35,581-589.
[10]Thongboonkerd V,Chutipongtanate S,Kanlaya R.J.Proteome Res.2006,5,183-191.
[11]Spahr CS,Davis MT,McGinley MD,Robinson JH.et al.Proteomics 2001,1,93-107.
[12]Rohlff C.Electrophoresis 2000,21,1227-1234.
[13]Turck CW,Maccarrone G,Sayan-Ayata E,Jacob AM.et al.J.Med.Invest.2005,52,231-235.
[14]Streckfus C,Bigler L,Dellinger T,Dai X.et al.Clin.Cancer Res.2000,6,2363-2370.
[15]Streckfus C,Bigler L,Tucci M,Thigpen JT.Cancer Invest.2000,18,101-109.
[16]Ben-Chetrit EFR,Rubinow A.Clin.Rheumatol.1993,12,471-474.
[17]Witte T.Ann.NYAcad.Sci.2005,1051,235-239.
[18]Ryu OH,Atkinson JC,Hoehn GT,Illei GG et al.Rheumatology(Oxford) 2006,45,1077-1086.
[19]Wattiez R,Falmagne P.J.Chromatogr.B 2005,815,169-178.
[20]Plymoth A,Lofdahl CG,Ekberg-Jansson A,Dahlback M.et al.Proteomics 2003,3,962-972.
[21]Yamada T et al,eds,Textbook of gastroenterology.Lippincott Williams & Wilkins, 2003,fourth edition,Vol 1,308-88.
[22]Gronborg M,Bunkenborg J,kristiansen TZ,Jensen ON,Yeo C J,Hruban RH,Maitra A,Goggins MG,Pandey A.Comprehensive proteomic analysis of human pancreatic juice.J Proteome Res.2004 Sep-Oct;3(5):1042-55.
[23]Zhou L,Lu Z,Yang A,Deng R,Mai C,Sang X,Faber KN,Lu X.Comparative proteomic analysis of human pancreatic juice:methodological study.Proteomics.2007 Apr;7(8):1345-55.
[24]龚晓明,陈元芳,陈原稼,等.胰腺癌患者胰液中K-ras基因突变的检测及其意义.中华内科杂志,1999,38:673-676.
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