Discovery and validation of novel serological biomarkers of colon cancer

描述（由申请人提供）：本申请的总体目标是确定人类结肠癌的新型血清学生物标志物，并评估其预测疾病发生或临床结果的能力。为了实现这一目标，我们将使用我们实验室最近开发的一种强大的新型4-D蛋白质分析方法，该方法可以识别许多低丰度的血清蛋白。该方法采用三种串联正交蛋白分离方法，包括：主蛋白缺失、溶液IEF和一维SDS凝胶。每个凝胶通道被切成均匀的薄片，用胰蛋白酶消化。每个胰蛋白酶消化，仍然含有许多蛋白质，然后通过LC-MS/MS使用纳米毛细管反相柱耦合到高性能线性离子阱质谱仪进行分析。在携带人类肿瘤的SCID小鼠中，候选的人类生物标志物将通过基于物种序列差异区分人类和小鼠蛋白质来鉴定。候选生物标志物的验证将分两个阶段进行。最初，候选人类生物标志物将使用中通量定量质谱分析和Western blots来评估一小群结肠癌患者和匹配对照的血清。更高通量的夹心ELISA检测将用于最有希望的生物标志物，这些检测将用于系统地检测来自大量早期和晚期癌症患者以及匹配对照的血清。将评估单个生物标志物以及生物标志物组的水平，以评估其预测疾病发生和临床结果的能力。这个雄心勃勃但可行的五年项目包括以下具体目标：1)使用一种新的多维蛋白质分析方法在SCID小鼠xenograph模型系统中鉴定候选人类结肠癌生物标志物；2)验证患者候选血清生物标志物，并使用中通量测定控制血清；3)开发高通量定量免疫分析，从最初的患者筛选中获得最有希望的生物标志物；4)采用高通量免疫测定法比较患者血清和对照血清。与公共卫生有关。发现新的结肠癌蛋白质生物标志物，可以通过简单的血液测试来测量，这对减少与这种疾病相关的痛苦和生命损失具有巨大的潜力。最近开发的强大的基于质谱的方法为系统地发现结肠癌生物标志物群提供了独特的机会。一组生物标记物（生物标记物特征）很可能比单个生物标记物更有能力监测癌症。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to identify novel serological biomarkers of human colon cancer and evaluate their capacities to predict disease occurrence or clinical outcome. To achieve this goal, we will use a powerful new 4-D protein profiling method recently developed in our lab that can identify many low abundance serum proteins. This method utilizes three tandem orthogonal protein separations consisting of: major protein depletion, solution IEF and 1-D SDS gels. Each gel lane is cut into uniform slices, and digested with trypsin. Each tryptic digest, which still contains many proteins, is then analyzed by LC-MS/MS using a nanocapillary reverse phase column coupled to a high performance linear ion trap mass spectrometer. Candidate human biomarkers in SCID mice bearing human tumors will be identified by distinguishing human and mouse proteins based on species sequence differences. Validation of candidate biomarkers will be conducted in two stages. Initially, candidate human biomarkers will be tested using medium throughput quantitative mass spectrometer assays and Western blots to evaluate sera from a small group of colon cancer patients and matched controls. Higher throughput sandwich ELISA assays will then be developed for the most promising biomarkers, and these assays will be used to systematically test sera from a larger number of early and late stage cancer patients and matched controls. Levels of individual biomarkers as well as groups of biomarkers will be evaluated for their capacity to predict disease occurrence and clinical outcome. This ambitious but feasible five year project involves the following Specific Aims: 1) Identify candidate human colon cancer biomarkers in a SCID mouse xenograph model system using a novel multi-dimensional protein profiling method; 2) Validate candidate serum biomarkers in patient and control sera using medium throughput assays; 3) Develop high throughput quantitative immunoassays for the most promising biomarkers from initial patient screens; and 4) Compare patient and control serum using high throughput immunoassays. Relevance to public health. Discovery of novel protein biomarkers of colon cancer that can be measured using a simple blood test has great potential to decrease the suffering and loss of life associated with this disease. Recently developed, powerful mass spectrometry-based methods provide a unique opportunity to systematically discovery groups of colon cancer biomarkers. It is most likely that groups of biomarkers (biomarker signatures) will have greater power to monitor cancer than individual biomarkers.