Biomarkers for Prostate and Bladder Cancer

描述（由申请人提供）：一种基于质谱蛋白质组学的策略用于发现前列腺癌和膀胱癌的生物标志物。目标标记物是由癌细胞和/或癌症相关细胞分泌的蛋白质。肿瘤组织标本取自手术切除的器官。标本在无血清培养基中用胶原酶消化成单细胞。消化介质称为COL，通过离心使细胞游离，它含有细胞产生的蛋白质。一个匹配的非癌标本也同样处理以进行比较分析。癌症和非癌症COL对中差异表达的蛋白质物种通过称为糖肽捕获的方法进行筛选，然后进行串联质谱鉴定。由于几乎所有分泌的蛋白质都是通过添加碳水化合物基团进行翻译后修饰的，因此该方法选择性地分析了这些蛋白质。定量是通过多肽的同位素标记实现的，即癌症的轻同位素和非癌症的重同位素。候选标记物将通过COL样品的Western blotting和免疫组织化学（如果有特异性抗体）或原位核酸探针杂交进行验证。在我们最初的实验中发现的TIMP1说明了这一标记发现过程。将对尿样进行蛋白质组学分析以检测标记物的可行性进行研究。初步实验已经生成了239种蛋白质的人类尿蛋白质组。将从癌症患者和年龄相匹配的志愿者身上获取尿液样本。目标是开发一种检测癌症相关蛋白标志物的方法，可以在尿中检测出前列腺癌和膀胱癌的诊断。

DESCRIPTION (provided by applicant): A strategy based on mass spectrometry proteomics is used to discover biomarkers for prostate and bladder cancer. The markers targeted are proteins secreted by the cancer cells and/or cancer-associated cells. Tumor tissue specimens are obtained from surgically resected organs after pathology. The specimens are digested by collagenase into single cells in a serum-free media. The digestion media, termed COL, is made cell-free by centrifugation, and it contains proteins made by the cells. A matched non-cancer specimen is processed similarly for comparative analysis. Differentially expressed protein species in the cancer and non-cancer COL pairs are screened by a method termed glycopeptide capture followed by tandem mass spectrometry for identification. Since virtually all secreted proteins are post-translationally modified by the addition of carbohydrate groups this method selectively analyzes these proteins. Quantification is achieved by isotopic labeling of the peptides, i.e. light isotopes for cancer and heavy isotopes for non-cancer. Candidate markers will be validated by Western blotting of the COL samples and immunohistochemistry if specific antibodies are available, or by in situ nucleic acid probe hybridization. This marker discovery process is illustrated by TIMP1, found in our initial experiments. The feasibility of proteomic analysis of urine samples to detect markers will be done. Preliminary experiments have generated a human urinary proteome of 239 protein species. Urine samples will be obtained from cancer patients and age-matched volunteers. The goal is to develop an assay for cancer associated protein markers that can be detected in voided urine for the diagnosis of prostate and bladder cancer.