修回日期: 2007-03-15
接受日期: 2007-04-03
在线出版日期: 2007-05-18
胰腺癌是一种十分凶险的恶性肿瘤. 胰腺癌早期症状不明显, 临床确诊的患者多已处于中晚期, 大多失去手术治疗机会. 蛋白质组技术的快速发展为研究胰腺癌发病机制、发现早期诊断和预后的生物标志物提供了可能. 本文就蛋白质组学技术在胰腺癌细胞系、组织、血清和胰液等方面的应用作一综述.
引文著录: 刘辉, 张海婧, 陈国强, 吕丽艳, 李智立. 应用蛋白质组学技术筛选胰腺癌生物标志物的研究进展. 世界华人消化杂志 2007; 15(14): 1628-1633
Revised: March 15, 2007
Accepted: April 3, 2007
Published online: May 18, 2007
Pancreatic cancer is one kind of devastating diseases. Those patients without nonspecific symptoms at early stage had mostly lost the opporunity of surgical therapy when pancreatic cancer was detected at advanced stage. Rapid growth of proteomic technologies provides possibilities to study etiopathogenesis, and screen early diagnostic and prognosis biomarkers of pancreatic cancer. In this paper, the application of proteomic techniques in cell lines, tissues, serum and pancreatic juice from patients with pancreatic cancer is reviewed briefly.
- Citation: Liu H, Zhang HJ, Chen GQ, Lv LY, Li ZL. Research progress in screening biomarkers of pancreatic cancer by proteomic techniques. Shijie Huaren Xiaohua Zazhi 2007; 15(14): 1628-1633
- URL: https://www.wjgnet.com/1009-3079/full/v15/i14/1628.htm
- DOI: https://dx.doi.org/10.11569/wcjd.v15.i14.1628
胰腺癌是消化系统常见的恶性肿瘤之一. 在西方国家他是第四大恶性肿瘤, 5 a存活率不足1%[1]; 在中国是第六大恶性肿瘤, 5 a存活率1%-3%[2]. 近年来发病率呈现明显上升趋势. 虽然现阶段诊断胰腺癌的标志物CA19-9[3-5], CA50, CA125, CA242, K-ras[6-8], p53[9-11], CD44v6和端粒酶[12-16]等已在临床中应用, 但无论是利用单个或是组合标志物进行胰腺癌早期诊断, 其检测灵敏性和准确性都不高. 蛋白质组学技术[17-21]的迅速发展, 为攻克胰腺癌等重大疾病提供了可能. 本文就蛋白质组学技术在胰腺癌方面的研究结果作一综述.
蛋白质组(proteome)一词最早由澳大利亚学者Wilkins et al[22]于1994年提出, 并首次出现在1995年的Biotechnology and Genetic Engineering Reviews杂志上, 指的是由一个基因组(genome)或一个细胞、组织表达的所有蛋白质. 蛋白质组学(proteomics)是在蛋白质水平上定量、动态、整体性地研究生物体. 他旨在阐明生物体全部蛋白质的表达模式及功能模式, 其内容包括蛋白质的定性鉴定、定量检测、细胞内定位、相互作用研究等, 最终揭示蛋白质功能, 是基因组DNA序列与基因功能之间的桥梁. 当前蛋白质组学的研究策略主要有: "bottom-up"策略[23-24], 他是指从细胞、组织、体液中提取出的蛋白质混合物酶切后得到更为复杂的多肽混合物, 经多维高效液相色谱分离后进行串联质谱鉴定获得肽段序列信息, 再搜索蛋白数据库和人工释图确定最后的鉴定结果[25-28], 虽然此方法相对简单, 但是也有不足: (1)由于蛋白酶解产生大量的肽段, 致使原始样品的复杂性显著增高; (2)不同实验室之间很难获得高重复性; (3)蛋白质的鉴定依赖于对少数肽段的分析, 可能导致蛋白质修饰信息的丢失. "bottom-up"适合于研究mRNA是单顺反子的微生物[29]. "top-down" 策略, 即对蛋白质混合物分离后在蛋白水平上直接分析[30-32], 当前有几种因素限制这种策略的广泛应用: (1)目前的分离技术还不能把较复杂的蛋白混合物分离成单个蛋白质组分后进行质谱鉴定; (2)现阶段质谱直接鉴定的蛋白质分子质量最大能到229 kDa[33]; (3)自动化程度低; (4)费用高、耗费时间. 鉴于以上几点的限制, 当前一种常见折中办法是对蛋白质混合物预先分离后, 每组组分酶切后在多肽水平分析. 此方法适合于研究存在复杂的剪接体变化和翻译后修饰的真核生物蛋白质组[29].
双向凝胶电泳(two-dimensional gel electrophoresis, 2-DE)[34-36]、质谱(mass spectrometry, MS)[37]技术是当前分离鉴定蛋白质的两大支柱技术, 在很多文献中已有详细介绍本文不做赘述. 随着科学技术的不断进步, 出现了许多新的蛋白质组学技术, 如表面增强激光解吸飞行时间质谱(surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, SELDI-TOF MS)[38]技术; 同位素标记相对和绝对定量(isobaric tag for relative and absolute quantitation, iTRAQ)[39]技术是近年来新开发的一种蛋白质组学定量研究技术, 可对复杂样本进行相对和绝对定量研究, 具有较好的定量效果和较高的重复性, 适合差异蛋白质组学研究. 这些新技术的出现有力的推动了蛋白质组学技术在临床医学研究中的广泛应用. 多维色谱(multidimensional chromatography)[40-42]分离技术的概念由Giddings提出, 指把不同分离机理的色谱技术有机结合在一起的分离模式, 与一维色谱分离模式相比其优点是大大地提高了峰容量, 适用于样品复杂程度高的蛋白质组学研究. 多维色谱是有不同分离原理的色谱组合而成[43]: 如(1)离子交换色谱/反相液相色谱; (2)体积排阻色谱/反相液相色谱; (3)亲和液相色谱/反相液相色谱等.
Moller et al[44]在不同浓度柔红霉素条件下孵育细胞系(EPP85-181P)72 h. 细胞暴露在这种药物环境中导致17种蛋白上调表达, 其中12种蛋白被鉴定. 3种蛋白显示出强烈的浓度依赖调节, 细胞角蛋白7(cytokeratin 7)、细胞角蛋白19(cytokeratin19)和分化相关基因产物1(differentiation related gene product 1). Cecconi et al[45-46]使用酶抑制剂曲古抑菌素-A(trichostatin-A)和5-氮-2'-脱氧胞嘧啶核苷(5-aza-2'-deosycytidine)进行胰腺癌细胞培养, 发现与细胞凋亡相关的新蛋白. 其结果与观测的trichostatin-A和5-aza-2'-deosycytidine通过影响细胞周期停滞和细胞凋亡抑制细胞生长的结果相吻合. Tseng et al[47]建立了一株耐5-氟尿嘧啶的细胞系, 通过2-DE的方法观察到差异蛋白, 鉴定了其中的一些蛋白, 他们属于膜联蛋白家族. Imamura et al[48]建立了一株敲除Smad4基因的胰腺癌细胞系, 通过2-DE的方法研究了经TGF-β处理与未经TGF-β处理的胰腺癌细胞蛋白表达的差异, 并鉴定了13个表达量差异大于1.3倍的蛋白, 包括参与细胞骨架调节、细胞周期调节、细胞增殖的蛋白. 这种方法的成功应用使检测未知通路中新的靶分子成为可能.
Hu et al[49]使用2-DE的方法建立了人的胰腺蛋白质数据库, 他包括302个蛋白质. 在这些蛋白中30%属于酶, 具有各种催化活性, 这与胰腺的生理作用一致. 这个研究小组还对胰腺癌和癌旁附近的正常组织进行了2-DE的比较分析[50]. 结果发现在癌组织中有70个蛋白表达上调, 而在正常组织中有41个蛋白表达上调, 其中大部分是胰腺外分泌产生的支持胰腺正常功能的蛋白质, 如淀粉酶、脂肪酶、肽酶等. 在胰腺癌中表达上调的蛋白反映了他们在恶性肿瘤中的作用, 如增殖活性、黏连性消失、获得活动性和转移性. 癌组织中表达上调的组织蛋白酶D和fascin经免疫组化方法得到确认. Shen et al[51]通过应用2-DE的方法分析了胰腺癌与邻近正常组织以及慢性胰腺炎患者胰腺组织与非胰腺疾病患者的胰腺组织. 鉴定了在胰腺癌中有差异表达的40个蛋白, 包括一组抗氧化蛋白、伴侣蛋白、钙依赖结合蛋白、细胞外基质蛋白. 其中7个调节蛋白经蛋白印记法或免疫组化法得到确认. 以上两个研究使用了完整的胰腺组织. Shekouh et al[52]利用激光捕获显微切割(laser capture microdissection)技术富集非恶性和恶性胰腺导管上皮细胞后进行2-DE, 获得了4个差异表达的蛋白(annexinⅢ, lactate dehydrogenase, trypsin和S100A6). S100A6的差异表达得到免疫组化方法确认. 在正常胰腺细胞中S100A6不表达或弱表达, 在80%低分化和中等分化肿瘤细胞中S100A6表达. 该蛋白属于S100蛋白家族, S100蛋白家族中某些蛋白与各种疾病相关, 包括癌症的发展和转移. Chen et al[53]使用ICAT(isotope-coded affinity tag)标记技术[54]对胰腺癌组织和正常组织进行定量蛋白谱研究, 定量分析了656个特异性蛋白, 其中151个蛋白差异表达量相差2倍以上. 其中大部分调节蛋白在细胞生理活动、上皮和肿瘤细胞的细胞外基质相互作用的通讯系统中发挥作用.
Wandschneider et al[55]通过分析不同条件下胰液的2-DE图谱, 研究不同胰酶抑制剂、不同浓度以及不同的pH范围对胰液分析结果的影响. 研究结果表明: (1)在pH4以下的蛋白质很少, 大部分蛋白存在于pH4-7之间, pH10以上的蛋白也很少; (2)未加入胰酶抑制剂的2-DE图与加入胰酶抑制剂的2-DE图中的差异蛋白被后继试验证实为蛋白降解产物; (3)加入不同胰酶抑制剂的胰液2-DE图表明PMSF抑制剂的效果较好. Gronborg et al[56]采用1-DE和LC-MS/MS(liquid chromatography tandem mass spectrometry)方法对取自3例胰腺癌患者的未加任何胰酶抑制剂的胰液进行分析, 分别鉴定出76, 63, 115个蛋白. 由3例样本确定170个蛋白质, 胰液中正常组分如胰淀粉酶和胰脂肪酶等, 异常组分中过量表达的癌相关蛋白如脂皮质素1(lipocortin I)和CEACAM5等, 以及在胰液中首次报道的azurocidin和防卫素R-3(defensin R-3). 值得一提的是鉴定出一个与肝癌-小肠-胰腺/胰腺炎(hepatocarcinoma-intestine-pancreas/pancreatitis-associated-protein HIP/PAP)相关的新蛋白PAP-2. PAP-2和HIP/PAP的相关性为85%, HIP/PAP在胰腺癌患者的血清和胰液中都显著上调, 并且胰液中的含量比血清中高1000倍, 可能为胰腺癌标志物的候选物. Rosty et al[57]使用SELDI技术分析了22例胰液标本, 其中15例为胰腺癌, 7例为其他胰腺疾病, 发现有一个分子质量为16.5 kDa的蛋白在67%(10/15)胰腺癌患者的胰液标本中存在, 而对照组胰液标本中的阳性率仅为17%(1/7). 进一步鉴定认为这种蛋白是肝癌-小肠-胰腺/胰腺炎-相关蛋白1(hepatocarcinoma-intestine-pancreas/pancreatitis-associated-protein 1, HIP/PAP-1). 使用ELISA方法定量检测了HIP/PAP-1在43例患者(包括28例胰腺癌和15例胰腺其他疾病)胰液标本中的含量, 发现HIP/PAP-1在胰腺癌患者胰液中的含量(143.75-235.52 mg/L)显著高于对照组患者胰液中的含量(6.04±7.59 mg/L), 研究结果表明检测胰液中HIP/PAP-1的含量将可能有助于胰腺癌的诊断. Chen et al[53]使用ICAT的方法综合研究了胰液蛋白质谱, 并且定量鉴定了胰腺癌差异表达的蛋白. 通过对胰腺癌患者的胰液与正常人的胰液A组, 胰腺炎患者的胰液与正常人的胰液B组以及正常人的胰液与正常人的胰液C组进行分析, 共鉴定出136个蛋白, 其中20个蛋白在3组样品中存在, 40个蛋白在胰腺炎和胰腺癌的样品中存在, 34个蛋白在正常和胰腺癌的样品中存在. 最终鉴定了在胰腺癌胰液中差异表达2倍以上的21个蛋白. 需要注意的是在一对正常胰液的比较中发现有15个差异表达在2倍以上蛋白, 这种变化在进一步的分子标志物的评估中需要认真考虑. 在他们的另一研究中[58], 依然使用ICAT的方法定量研究胰液, 通过对1例胰腺癌胰液与11例正常胰液配对: 1例正常胰液与10例混合的正常胰液、1例胰腺癌胰液与10例混合的正常胰液比较研究, 总共鉴定105个蛋白, 在胰腺癌胰液与10例混合的正常胰液比较中显示30个蛋白定量差异在2倍以上, 其中有15个蛋白是首次发现与胰腺癌相关, IGFBP-2(insulin-like growth factor binding protein-2)是其中之一并使用蛋白质印迹方法验证.
Koopmann et al[59]应用SELDI技术在两种不同的芯片上[(weak cation exchange, immobilized metal affinity capture coupled with copper (IMAC-Cu2+)]分析来自胰腺癌、非恶性胰腺疾病和健康对照的180个血清样本, 有两个具有显著差异的质谱峰, 其灵敏性和特异性分别达到78%和97%. 在另一个独立的研究中[60], 经阴离子交换分离后使用IMAC-Cu2+芯片分析了103个来自胰腺癌和非胰腺癌患者的血清, 其灵敏性100%, 特异性93.5%. 虽然在这两个研究中观察到的质谱峰有差异, 但是都存在一个共同的质谱峰, 其质荷比为m/z 3967, 他是否为同一个蛋白, 仍需其他方法进行验证. Hong et al[61]利用2-DE、蛋白质印迹和质谱技术结合的方法分析了102例血清样本, 其中36例胰腺癌、18例慢性胰腺炎、33例非胰腺肿瘤和15例健康对照, 在一特殊类型(a particular tumor type)胰腺癌中鉴定出可诱导体液免疫反应产生自身抗体的钙网织蛋白1和2. Honda et al[62]应用SELDI技术在两种不同的芯片[(weak hydrophobic (H50), cationic (CM10)]上分析了245例血清样本, 其中113例肿瘤样本、6例胰腺囊肿样本、5例慢性胰腺炎样本和121例正常对照样本. 在肿瘤与正常样本比较中有4个质谱峰m/z 8766, 17 272, 28 080和14 779差异在2倍以上. 这组质谱峰与CA19-9联合检测29例胰腺癌患者有100%的敏感性, 但是在检测39例正常对照时产生6例假阳性. 这一组蛋白是否可以用于临床筛查有待进一步检验.
白质组学已经成为生命科学领域研究热点之一, 并已形成临床蛋白质组学分支. 在蛋白质组学研究策略普遍被接受的同时, 也应该清醒地认识到蛋白质组学技术现阶段还不是一种完美的工具, 在当前技术条件的限制下, 还有一些问题等待解决. 例如在生物样品的制备与处理过程中, 由于生物样品极其复杂, 如血清含有上千种性质各异的物质如蛋白质、核酸、脂类和代谢产物等, 如何消除生物基质的干扰; 如何解决高丰度蛋白对低丰度蛋白的掩盖; 如何避免电喷雾过程中的离子抑制现象和电化学反应等问题显得非常重要[63-64]. 胰腺癌早期无典型临床表现, 上腹疼痛需要鉴别的疾病甚多, 易发生误诊误治, 故寻找高特异性和高敏感性的胰腺早期诊断的标志物具有重要的临床意义和社会意义. 胰腺癌具有极强的隐蔽性, 因此血液中的特殊生物分子将有可能成为胰腺癌早期诊断标志物, 但是由于血液成分极其复杂, 多种分析和分离技术的有机结合才有可能从血液样本中获得胰腺癌早期诊断标志物.
本文简述蛋白质组学相关技术及筛选胰腺癌生物标志物的研究进展.
目前蛋白质组学技术被广泛用于胰腺癌早期诊断标志物的筛选, 研究表明血清/血浆样本是获得肿瘤生物标志物最佳资源之一, 但由于血清/血浆的成分极其复杂, 利用目前技术方法获得高特异性和高敏感性的标志物仍面临较大挑战.
本文较为详细列出蛋白质组学技术的局限性和胰腺癌生物标志物筛选的样本选择.
胰腺癌具有极强的隐蔽性, 因此血液中的特殊生物分子将有可能成为胰腺癌早期诊断标志物, 但是由于血液成分极其复杂, 多种分析和分离技术的有机结合才有可能从血液样本中获得胰腺癌早期诊断标志物.
电编:张敏 编辑:王晓瑜
| 1. | Neoptolemos JP, Dunn JA, Stocken DD, Almond J, Link K, Beger H, Bassi C, Falconi M, Pederzoli P, Dervenis C. Adjuvant chemoradiotherapy and chemotherapy in resectable pancreatic cancer: a randomised controlled trial. Lancet. 2001;358:1576-1585. [PubMed] [DOI] |
| 2. | Guo X, Cui Z. Current diagnosis and treatment of pancreatic cancer in China. Pancreas. 2005;31:13-22. [PubMed] [DOI] |
| 3. | Chang CY, Huang SP, Chiu HM, Lee YC, Chen MF, Lin JT. Low efficacy of serum levels of CA 19-9 in prediction of malignant diseases in asymptomatic population in Taiwan. Hepatogastroenterology. 2006;53:1-4. [PubMed] |
| 4. | Lowe D, Lee J, Schade R, Chaudhary A. Patient with markedly elevated CA 19-9 not associated with malignancy. South Med J. 2006;99:306-308. [PubMed] [DOI] |
| 5. | Kilic M, Gocmen E, Tez M, Ertan T, Keskek M, Koc M. Value of preoperative serum CA 19-9 levels in predicting resectability for pancreatic cancer. Can J Surg. 2006;49:241-244. [PubMed] |
| 6. | Marchese R, Muleti A, Pasqualetti P, Bucci B, Stigliano A, Brunetti E, De Angelis M, Mazzoni G, Tocchi A, Brozzetti S. Low correspondence between K-ras mutations in pancreatic cancer tissue and detection of K-ras mutations in circulating DNA. Pancreas. 2006;32:171-177. [PubMed] [DOI] |
| 7. | Campman SC, Fajardo MA, Rippon MB, Kraegel SA, Ruebner BH. Adenosquamous carcinoma arising in a mucinous cystadenoma of the pancreas. J Surg Oncol. 1997;64:159-162. [PubMed] [DOI] |
| 8. | Wu X, Lu XH, Xu T, Qian JM, Zhao P, Guo XZ, Yang XO, Jiang WJ. Evaluation of the diagnostic value of serum tumor markers, and fecal k-ras and p53 gene mutations for pancreatic cancer. Chin J Dig Dis. 2006;7:170-174. [PubMed] [DOI] |
| 9. | Gerdes B, Ramaswamy A, Ziegler A, Lang SA, Kersting M, Baumann R, Wild A, Moll R, Rothmund M, Bartsch DK. p16INK4a is a prognostic marker in resected ductal pancreatic cancer: an analysis of p16INK4a, p53, MDM2, an Rb. Ann Surg. 2002;235:51-59. [PubMed] [DOI] |
| 10. | Kawahira H, Kobayashi S, Kaneko K, Asano T, Ochiai T. p53 protein expression in intraductal papillary mucinous tumors (IPMT) of the pancreas as an indicator of tumor malignancy. Hepatogastroenterology. 2000;47:973-977. [PubMed] |
| 11. | Bergan A, Gladhaug IP, Schjolberg A, Bergan AB, Clausen OP. p53 accumulation confers prognostic information in resectable adenocarcinomas with ductal but not with intestinal differentiation in the pancreatic head. Int J Oncol. 2000;17:921-926. [PubMed] [DOI] |
| 12. | Ohuchida K, Mizumoto K, Yamada D, Yamaguchi H, Konomi H, Nagai E, Yamaguchi K, Tsuneyoshi M, Tanaka M. Quantitative analysis of human telomerase reverse transcriptase in pancreatic cancer. Clin Cancer Res. 2006;12:2066-2069. [PubMed] [DOI] |
| 13. | Mishra G, Zhao Y, Sweeney J, Pineau BC, Case D, Ho C, Blackstock AW, Geisinger K, Howerton R, Levine E. Determination of qualitative telomerase activity as an adjunct to the diagnosis of pancreatic adenocarcinoma by EUS-guided fine-needle aspiration. Gastrointest Endosc. 2006;63:648-654. [PubMed] [DOI] |
| 14. | Murakami Y, Uemura K, Sasaki T, Hayashidani Y, Sudo T, Sueda T. Long-term survival of pancreatic cancer patient diagnosed by positive telomerase activity of pancreatic juice. Surgery. 2005;138:962-963. [PubMed] [DOI] |
| 15. | Mizumoto K, Tanaka M. Detection of telomerase activity in patients with pancreatic cancer. Methods Mol Med. 2005;103:199-205. [PubMed] |
| 16. | Vezzosi D, Bouisson M, Escourrou G, Laurell H, Selves J, Seguin P, Pradayrol L, Caron P, Buscail L. Clinical utility of telomerase for the diagnosis of malignant well-differentiated endocrine tumours. Clin Endocrinol (. Oxf). 2006;64:63-67. [PubMed] [DOI] |
| 17. | Geisow MJ. Proteomics: one small step for a digital computer, one giant leap for humankind. Nat Biotechnol. 1998;16:206. [PubMed] [DOI] |
| 18. | Persidis A. Proteomics. Nat Biotechnol. 1998;16:393-394. [PubMed] [DOI] |
| 19. | Anderson NL, Anderson NG. Proteome and proteomics: new technologies, new concepts, and new words. Electrophoresis. 1998;19:1853-1861. [PubMed] [DOI] |
| 21. | DeWitt N. Proteomics get complex. Nat Biotechnol. 1999;17:626. [PubMed] [DOI] |
| 22. | Wilkins MR, Sanchez JC, Gooley AA, Appel RD, Humphery-Smith I, Hochstrasser DF, Williams KL. Progress with proteome projects: why all proteins expressed by a genome should be identified and how to do it. Biotechnol Genet Eng Rev. 1996;13:19-50. [PubMed] [DOI] |
| 23. | McDonald WH, Yates JR 3rd. Shotgun proteomics and biomarker discovery. Dis Markers. 2002;18:99-105. [PubMed] [DOI] |
| 24. | Wolters DA, Washburn MP, Yates JR 3rd. An automated multidimensional protein identification technology for shotgun proteomics. Anal Chem. 2001;73:5683-5690. [PubMed] [DOI] |
| 25. | Leung KY, Wait R, Welson SY, Yan JX, Abraham DJ, Black CM, Pearson JD, Dunn MJ. A reference map of human lung MRC-5 fibroblast proteins using immobilized pH gradient-isoelectric focusing-based two-dimensional electrophoresis. Proteomics. 2001;1:787-794. [PubMed] [DOI] |
| 26. | Wait R, Gianazza E, Eberini I, Sironi L, Dunn MJ, Gemeiner M, Miller I. Proteins of rat serum, urine, and cerebrospinal fluid: VI. Further protein identifications and interstrain comparison. Electrophoresis. 2001;22:3043-3052. [PubMed] [DOI] |
| 27. | Davidsson P, Paulson L, Hesse C, Blennow K, Nilsson CL. Proteome studies of human cerebrospinal fluid and brain tissue using a preparative two-dimensional electrophoresis approach prior to mass spectrometry. Proteomics. 2001;1:444-452. [PubMed] [DOI] |
| 28. | Piubelli C, Galvani M, Hamdan M, Domenici E, Righetti PG. Proteome analysis of rat polymorphonuclear leukocytes: a two-dimensional electrophoresis/mass spectrometry approach. Electrophoresis. 2002;23:298-310. [PubMed] [DOI] |
| 29. | Guerrera IC, Kleiner O. Application of mass spectrometry in proteomics. Biosci Rep. 2005;25:71-93. [PubMed] [DOI] |
| 30. | Champion KM, Arnott D, Henzel WJ, Hermes S, Weikert S, Stults J, Vanderlaan M, Krummen L. A two-dimensional protein map of Chinese hamster ovary cells. Electrophoresis. 1999;20:994-1000. [PubMed] [DOI] |
| 31. | Poutanen M, Salusjarvi L, Ruohonen L, Penttila M, Kalkkinen N. Use of matrix-assisted laser desorption/ionization time-of-flight mass mapping and nanospray liquid chromatography/electrospray ionization tandem mass spectrometry sequence tag analysis for high sensitivity identification of yeast proteins separated by two-dimensional gel electrophoresis. Rapid Commun Mass Spectrom. 2001;15:1685-1692. [PubMed] [DOI] |
| 32. | Shaw AC, Gevaert K, Demol H, Hoorelbeke B, Vandekerckhove J, Larsen MR, Roepstorff P, Holm A, Christiansen G, Birkelund S. Comparative proteome analysis of Chlamydia trachomatis serovar A, D and L2. Proteomics. 2002;2:164-186. [PubMed] [DOI] |
| 33. | Han X, Jin M, Breuker K, McLafferty FW. Extending top-down mass spectrometry to proteins with masses greater than 200 kilodaltons. Science. 2006;314:109-112. [PubMed] [DOI] |
| 34. | O'Farrell PH. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975;250:4007-4021. [PubMed] |
| 35. | Klose J. Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues. A novel approach to testing for induced point mutations in mammals. Humangenetik. 1975;26:231-243. [PubMed] |
| 36. | Scheele GA, Palade GE. Studies on the guinea pig pancreas. Parallel discharge of exocrine enzyme activities. J Biol Chem. 1975;250:2660-2670. [PubMed] |
| 37. | Aebersold R, Mann M. Mass spectrometry-based proteomics. Nature. 2003;422:198-207. [PubMed] [DOI] |
| 38. | Tang N, Tornatore P, Weinberger SR. Current developments in SELDI affinity technology. Mass Spectrom Rev. 2004;23:34-44. [PubMed] [DOI] |
| 39. | Ross PL, Huang YN, Marchese JN, Williamson B, Parker K, Hattan S, Khainovski N, Pillai S, Dey S, Daniels S. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics. 2004;3:1154-1169. [PubMed] [DOI] |
| 40. | Giddings JC. Two-dimensional separations: concept and promise. Anal Chem. 1984;56:1258A-1260A, 1262A, 1264A passim. [PubMed] |
| 41. | Davis JM, Giddings JC. Statistical theory of component overlap in multicomponent chromatograms. Anal Chem. 1983;55:418-424. [DOI] |
| 42. | Davis JM, Giddings JC. Statistical method for estimation of number of components from single complex chromatograms: theory, computer-based testing, and analysis of errors. Anal Chem. 1985;57:2168-2177. [PubMed] [DOI] |
| 43. | Issaq HJ, Chan KC, Janini GM, Conrads TP, Veenstra TD. Multidimensional separation of peptides for effective proteomic analysis. J Chromatogr B Analyt Technol Biomed Life Sci. 2005;817:35-47. [PubMed] [DOI] |
| 44. | Moller A, Malerczyk C, Volker U, Stoppler H, Maser E. Monitoring daunorubicin-induced alterations in protein expression in pancreas carcinoma cells by two-dimensional gel electrophoresis. Proteomics. 2002;2:697-705. [PubMed] [DOI] |
| 45. | Cecconi D, Astner H, Donadelli M, Palmieri M, Missiaglia E, Hamdan M, Scarpa A, Righetti PG. Proteomic analysis of pancreatic ductal carcinoma cells treated with 5-aza-2'-deoxycytidine. Electrophoresis. 2003;24:4291-4303. [PubMed] [DOI] |
| 46. | Cecconi D, Scarpa A, Donadelli M, Palmieri M, Hamdan M, Astner H, Righetti PG. Proteomic profiling of pancreatic ductal carcinoma cell lines treated with trichostatin-A. Electrophoresis. 2003;24:1871-1878. [PubMed] [DOI] |
| 47. | Tseng JF, Willett CG, Fernandez-del Castillo C, Ryan DP, Clark JW, Zhu AX, Rattner DW, Winkelmann JL, Warshaw AL. Patients undergoing treatment for pancreatic adenocarcinoma can mount an effective immune response to vaccinations. Pancreatology. 2005;5:67-74. [PubMed] [DOI] |
| 48. | Imamura T, Kanai F, Kawakami T, Amarsanaa J, Ijichi H, Hoshida Y, Tanaka Y, Ikenoue T, Tateishi K, Kawabe T. Proteomic analysis of the TGF-beta signaling pathway in pancreatic carcinoma cells using stable RNA interference to silence Smad4 expression. Biochem Biophys Res Commun. 2004;318:289-296. [PubMed] [DOI] |
| 49. | Hu L, Evers S, Lu ZH, Shen Y, Chen J. Two-dimensional protein database of human pancreas. Electrophoresis. 2004;25:512-518. [PubMed] [DOI] |
| 50. | Lu Z, Hu L, Evers S, Chen J, Shen Y. Differential expression profiling of human pancreatic adenocarcinoma and healthy pancreatic tissue. Proteomics. 2004;4:3975-3988. [PubMed] [DOI] |
| 51. | Shen J, Person MD, Zhu J, Abbruzzese JL, Li D. Protein expression profiles in pancreatic adenocarcinoma compared with normal pancreatic tissue and tissue affected by pancreatitis as detected by two-dimensional gel electrophoresis and mass spectrometry. Cancer Res. 2004;64:9018-9026. [PubMed] [DOI] |
| 52. | Shekouh AR, Thompson CC, Prime W, Campbell F, Hamlett J, Herrington CS, Lemoine NR, Crnogorac-Jurcevic T, Buechler MW, Friess H. Application of laser capture microdissection combined with two-dimensional electrophoresis for the discovery of differentially regulated proteins in pancreatic ductal adenocarcinoma. Proteomics. 2003;3:1988-2001. [PubMed] [DOI] |
| 53. | Chen R, Pan S, Brentnall TA, Aebersold R. Proteomic profiling of pancreatic cancer for biomarker discovery. Mol Cell Proteomics. 2005;4:523-533. [PubMed] [DOI] |
| 54. | Gygi SP, Rist B, Gerber SA, Turecek F, Gelb MH, Aebersold R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat Biotechnol. 1999;17:994-999. [PubMed] [DOI] |
| 55. | Wandschneider S, Fehring V, Jacobs-Emeis S, Thiesen HJ, Lohr M. Autoimmune pancreatic disease: preparation of pancreatic juice for proteome analysis. Electrophoresis. 2001;22:4383-4390. [PubMed] [DOI] |
| 56. | Gronborg M, Bunkenborg J, Kristiansen TZ, Jensen ON, Yeo CJ, Hruban RH, Maitra A, Goggins MG, Pandey A. Comprehensive proteomic analysis of human pancreatic juice. J Proteome Res. 2004;3:1042-1055. [PubMed] [DOI] |
| 57. | Rosty C, Christa L, Kuzdzal S, Baldwin WM, Zahurak ML, Carnot F, Chan DW, Canto M, Lillemoe KD, Cameron JL. Identification of hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein I as a biomarker for pancreatic ductal adenocarcinoma by protein biochip technology. Cancer Res. 2002;62:1868-1875. [PubMed] |
| 58. | Chen R, Pan S, Yi EC, Donohoe S, Bronner MP, Potter JD, Goodlett DR, Aebersold R, Brentnall TA. Quantitative proteomic profiling of pancreatic cancer juice. Proteomics. 2006;6:3871-3879. [PubMed] [DOI] |
| 59. | Koopmann J, Zhang Z, White N, Rosenzweig J, Fedarko N, Jagannath S, Canto MI, Yeo CJ, Chan DW, Goggins M. Serum diagnosis of pancreatic adenocarcinoma using surface-enhanced laser desorption and ionization mass spectrometry. Clin Cancer Res. 2004;10:860-868. [PubMed] [DOI] |
| 60. | Bhattacharyya S, Siegel ER, Petersen GM, Chari ST, Suva LJ, Haun RS. Diagnosis of pancreatic cancer using serum proteomic profiling. Neoplasia. 2004;6:674-686. [PubMed] [DOI] |
| 61. | Hong SH, Misek DE, Wang H, Puravs E, Giordano TJ, Greenson JK, Brenner DE, Simeone DM, Logsdon CD, Hanash SM. An autoantibody-mediated immune response to calreticulin isoforms in pancreatic cancer. Cancer Res. 2004;64:5504-5510. [PubMed] [DOI] |
| 62. | Honda K, Hayashida Y, Umaki T, Okusaka T, Kosuge T, Kikuchi S, Endo M, Tsuchida A, Aoki T, Itoi T. Possible detection of pancreatic cancer by plasma protein profiling. Cancer Res. 2005;65:10613-10622. [PubMed] [DOI] |
| 63. | McLuckey SA, Stephenson JL Jr. Ion/ion chemistry of high-mass multiply charged ions. Mass Spectrom Rev. 1998;17:369-407. [PubMed] [DOI] |
| 64. | Mora JF, Van Berkel GJ, Enke CG, Cole RB, Martinez-Sanchez M, Fenn JB. Electrochemical processes in electrospray ionization mass spectrometry. J Mass Spectrom. 2000;35:939-952. [PubMed] [DOI] |