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颖的 血清蛋白质谱定量比较的蛋白质组学策略 能够检测到低至ng/ml水平的蛋白质。这大约是100- 比直接分析血清的灵敏度高1000倍 范围或窄范围的2-D凝胶，它代表 大多数已知肿瘤血清标志物的浓度范围。这么高 敏感性蛋白质组策略将开发并使用嵌合体进行测试 含有肿瘤生长所分泌的人类蛋白的小鼠血清 皮下注射人黑色素瘤或胰腺癌细胞 台词。用新方法对人体分泌蛋白质进行系统分析 在这个项目中开发的应该确定多个新的潜在诊断 癌症筛查的目标。这一蛋白质组策略将整合几个 新颖的支护方法除了进一步优化外，还有一个非常 最新发明的蛋白质组分离装置和方法前景看好 实验室。“微尺度溶液等电聚焦”分级法 (Microsol-IEF)使用数量可变的串联小容量腔室 在特定pH下用含有固定剂的半透隔板隔开？S。 对几种类型的样品进行了原则性验证实验 包括小鼠血清，并证明该方法可以干净地分离 至少几毫克的粗提物分成一小部分。这个 得到的分辨率很好的分数可以在一系列轻微的 重叠的窄幅二维凝胶使用的蛋白质负载比 使用的是未分馏的样品。尽管这样做的可行性 预分馏方法已经证明，必须有更可靠的设备 必须优化显影和分离参数以最大限度地提高分辨率 而可靠性之前的这种方法可以成为一种可靠的稳健蛋白质组学 方法。整体蛋白质组分析策略的其他新特征包括 使用定制的略有重叠的窄pH范围梯度，以确保 蛋白质不会在凝胶边界丢失，并使用双重敏感染色来 增加检测光点的动态范围。似乎具有特异性的蛋白质 将对荷瘤小鼠进行鉴定，并确定它们的起源物种(小鼠= 宿主或人类=肿瘤)将使用LC-MS/MS方法确定。具体的 目的是：1)开发优化的Microsol-IEF装置和方法；2)开发 高样品容量MicroSol-IEF器件，3)开发高灵敏度桩 Microsol-IEF分析方法，以及4)系统地应用集成蛋白质组 含人黑色素瘤或胰腺癌血清标本的分析策略 癌症肿瘤。

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.