Discovery and validation of novel serological biomarkers of colon cancer

描述（由申请方提供）：本提案的总体目标是鉴定人结肠癌的新型血清学生物标志物，并评价其预测疾病发生或临床结局的能力。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4-D蛋白质谱分析方法，该方法可以识别许多低丰度血清蛋白。该方法采用三种串联正交蛋白质分离，包括：主要蛋白质去除、溶液IEF和1-D SDS凝胶。将每个凝胶泳道切成均匀的切片，并用胰蛋白酶消化。然后，使用与高性能线性离子阱质谱仪偶联的纳米毛细管反相柱，通过LC-MS/MS分析仍然含有许多蛋白质的每个胰蛋白酶消化物。携带人肿瘤的SCID小鼠中的候选人生物标志物将通过基于物种序列差异区分人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，将使用中等通量定量质谱仪测定和蛋白质印迹法检测候选人生物标志物，以评价来自一小组结肠癌患者和匹配对照的血清。然后将开发更高通量的夹心ELISA测定法用于最有希望的生物标志物，并且这些测定法将用于系统地测试来自大量早期和晚期癌症患者和匹配对照的血清。将评价单个生物标志物以及生物标志物组的水平，以确定其预测疾病发生和临床结局的能力。这个雄心勃勃但可行的五年项目涉及以下具体目标：1）使用新的多维蛋白质谱分析方法在SCID小鼠异种移植模型系统中鉴定候选人结肠癌生物标志物; 2）使用中等通量测定法在患者和对照血清中鉴定候选血清生物标志物; 3）开发用于来自初始患者筛选的最有希望的生物标志物的高通量定量免疫测定法;和4）使用高通量免疫测定比较患者和对照血清。与公共卫生的相关性。发现可以使用简单的血液测试测量的结肠癌新蛋白质生物标志物具有很大的潜力，可以减少与这种疾病相关的痛苦和生命损失。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组提供了独特的机会。最有可能的是，生物标志物组（生物标志物签名）将比单个生物标志物具有更大的监测癌症的能力。

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.