Development of Methods for Cancer Biomarker Detection

描述（由申请人提供）： 这项为期三年的R33提案的总体目标是开发一种新的 血清蛋白质谱定量比较的蛋白质组学策略 能够检测低至ng/ml水平的蛋白质。这是大约100- 比直接分析血清灵敏1000倍， 范围或窄范围2-D凝胶，它代表了 大多数已知的肿瘤血清学标志物的浓度范围。这种高 敏感性蛋白质组策略将开发和测试使用嵌合 含有肿瘤生长分泌的人类蛋白质的小鼠血清 皮下注射人黑色素瘤或胰腺癌细胞 线使用新方法系统分析分泌的人类蛋白质 在这个项目中开发的技术应该能够识别出多种新的潜在的诊断方法， 癌症筛查的目标。这种蛋白质组策略将包括几个 新的支持方法，除了进一步优化一个非常 我们最近发明了有前途的蛋白质组分馏装置和方法 实验室分级方法，“微量溶液等电聚焦” （microsol-IEF）使用可变数量的串联小体积腔室 分离的半渗透性分区含有immobilines在特定的pH值？S. 已经在几种类型的样品上完成了原理验证实验 包括小鼠血清，并证明该方法可以干净地将 将至少几mg粗提取物分成少量级分。的 得到的良好分离的级分可以在一系列轻微的 重叠的窄范围2-D凝胶，使用比当 使用未分级的样品。尽管这样做的可行性 预分馏方法已经得到证实，必须使用更可靠的装置， 开发和分离参数必须优化，以最大限度地提高分辨率 和可靠性，才能使这种方法成为一种可靠的蛋白质组学方法 法整体蛋白质组分析策略的其他新特征包括 使用定制的略微重叠的窄pH范围梯度，以确保 蛋白质在凝胶边界处不会丢失， 增加了检测斑点的动态范围。蛋白质似乎是特定的 将鉴定其来源物种（小鼠= 宿主或人=肿瘤）的浓度将使用LC-MS/MS方法测定。具体 目的是：1）开发优化的微溶胶IEF装置和方法，2）开发 高采样容量的微溶胶-IEF装置，3）开发高灵敏度柱 microsol-IEF分析方法; 4）系统地应用整合蛋白质组 含有人黑素瘤或胰腺癌的血清样品的分析策略 恶性肿瘤

DESCRIPTION (provided by applicant): The overall goal of this three-year R33 proposal is to develop a novel proteomic strategy for quantitative comparisons of serum protein profiles capable of detecting proteins down to the low ng/ml level. This is about 100- to 1000-fold more sensitive than direct analysis of serum on either broad range or narrow range 2-D gels, and it represents the lower end of the concentration range for most known tumor serological markers. This high sensitivity proteome strategy will be developed and tested using chimeric mouse serum that contains human proteins secreted by tumors grown subcutaneously from injected human melanoma or pancreatic carcinoma cell lines. Systematic analyses of secreted human proteins using the new methods developed in this project should identify multiple new potential diagnostic targets for cancer screening. This proteome strategy will incorporate several novel supporting methods in addition to further optimization of a very promising proteome fractionation device and method recently invented in our laboratory. The fractionation method, "microscale solution isoelectrofocusing" (microsol-IEF) uses a variable number of tandem small volume chambers separated by semipermeable partitions containing immobilines at specific pH?s. Proof-of-principle experiments have been completed on several types of samples including mouse serum and demonstrate that this method can cleanly fractionate at least several mg of crude extracts into a small number of fractions. The resulting well-resolved fractions can be separated on a series of slightly overlapping narrow range 2-D gels using much higher protein loads than when unfractionated samples are used. Although the feasibility of this prefractionation method has been demonstrated, more reliable devices must be developed and separation parameters must be optimized to maximize resolution and reliability before this method can become a reliable robust proteomics method. Other novel features of the overall proteome analysis strategy include use of custom-made slightly overlapping narrow pH range gradients to ensure proteins are not lost at gel boundaries and use of dual sensitivity stains to increase dynamic range of detected spots. Proteins that appear to be specific to tumor-bearing mice will be identified and their species of origin (mouse = host or human = tumor) will be determined using LC-MS/MS methods. The Specific Aims are: 1) Develop an optimized microsol-IEF device and method, 2) Develop high sample capacity microsol-IEF devices, 3) Develop high sensitivity post microsol-IEF analysis methods, and 4) Systematically apply integrated proteome analysis strategy to serum samples containing human melanoma or pancreatic carcinoma tumors.