Discovery and validation of novel serological biomarkers of colon cancer

描述(由申请人提供)：这项提案的总体目标是确定人类结肠癌的新的血清生物标记物，并评估它们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的4-D蛋白质图谱方法，该方法可以识别许多低丰度的血清蛋白质。该方法利用三种串联的正交法分离蛋白质，包括：主蛋白质耗竭、溶液IEF和一维十二烷基硫酸钠凝胶。每个凝胶通道被切成均匀的切片，并用胰酶消化。每一种仍然含有许多蛋白质的胰酶消化液，然后用LC-MS/MS进行分析，使用与高性能线性离子陷阱质谱仪耦合的纳米毛细管反相柱。携带人类肿瘤的SCID小鼠中的候选人类生物标记物将通过基于物种序列差异区分人类和小鼠蛋白质来识别。候选生物标志物的验证将分两个阶段进行。最初，候选的人类生物标记物将使用中通量定量质谱仪分析和Western blots进行测试，以评估一小部分结肠癌患者和匹配对照的血清。然后将为最有前景的生物标记物开发更高通量的夹心ELISA检测方法，这些检测方法将用于系统地检测大量早期和晚期癌症患者和匹配对照的血清。将评估单个生物标记物以及生物标记物组的水平，以确定它们预测疾病发生和临床结果的能力。这项雄心勃勃但可行的五年计划涉及以下具体目标：1)使用新颖的多维蛋白质谱方法在SCID小鼠异种移植模型系统中识别候选的人类结肠癌生物标记物；2)使用中通量分析方法验证患者和对照血清中的候选血清生物标记物；3)从初始患者筛选出最有希望的生物标记物的高通量定量免疫分析方法；以及4)使用高通量免疫分析方法比较患者和对照血清。与公共卫生的相关性。新的结肠癌蛋白质生物标记物的发现，可以用简单的血液测试来测量，对于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学的方法为系统地发现结肠癌生物标记物提供了一个独特的机会。最有可能的是，一组生物标记物(生物标记物签名)将比单个生物标记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.