Protein Microarrays for the Humoral Response in Cancer

描述(由申请人提供)：在拟议的研究中，我们计划开发检测体液对前列腺癌抗原的反应作为疾病诊断和预后血清生物标记物的方法。该方法涉及使用由细胞裂解产物的二维液相分离产生的蛋白质微阵列。基于PL的层析分离在第一维被用来在扩展的pH范围内将大量蛋白质分离成离散的pH组分。在第二个维度中，每个pH组分的无孔(NPS)反相(RP)高效液相色谱(RP)将被用来从柱洗脱液中分离出要从硝酸纤维玻片上分离的纯化蛋白质。其结果将是一个由2500个蛋白质点组成的蛋白质微阵列，可以用患者血清进行测试，以检查由此产生的体液反应。前列腺癌蛋白芯片将用于询问健康对照组、良性前列腺增生症(BPH)患者和经活检证实的前列腺癌患者的血清。通过这种方法，我们希望描述前列腺癌患者的标志性体液反应。该方法从实际的肿瘤细胞中排列蛋白质，以便患者血浆中的抗体可以对不一定由其他方法产生的修饰形式的蛋白质产生反应。对血清产生体液反应的蛋白质可以直接从液体组分中通过质谱分析进行分析，其中准确的MW值以及CE-TOF-MS和MALDI-TOF-MS的酶促肽图分析可以用于鉴定和确定当前的修饰。修饰的蛋白质可以对血清中的抗体产生选择性反应，而未修饰的蛋白质可能无法提供这种反应。蛋白质在液体中的存在使该方法适用于大量样品的自动筛选。将探索抗体反应在筛查/诊断(即补充PSA检测)和预后(即区分惰性前列腺癌和侵袭性前列腺癌)中的作用。此外，生物信息学方法将被用来确定是否存在更好地区分临床结果的前列腺癌亚类。根据前列腺癌局部样本的体液表达谱，将建立一个预测模型，对临床有意义的标志物进行分类，从而为进一步的实验研究识别候选的肿瘤抗原。候选肿瘤抗原将使用组织微阵列进一步验证。

DESCRIPTION (provided by applicant): In the proposed research, we plan to develop methods that examine the humoral response to prostate tumor antigens as a diagnostic and prognostic serum biomarker of disease. The method involves the use of protein microarrays produced by the two-dimensional liquid phase fractionation of cell lysates. A pl-based chromatographic separation is utilized in the first dimension to fractionate large numbers of proteins into discrete pH fractions over an extended pH range. In the second dimension, nonporous (NPS) reversed phase (RP) HPLC of each pH fraction will be used to separate out purified proteins to be spotted from the column eluent onto nitrocellulose slides. The result will be a protein microarray consisting of >2500 protein spots that can be tested against patient sera to examine resulting humoral response. The prostate cancer protein microarrays will be used to interrogate serum from healthy control individuals, patients with benign prostatic hyperplasia (BPH), and those with biopsy-proven prostate cancer. Using this approach, we hope to characterize a signature humoral response for patients with prostate cancer. The method arrays proteins from actual tumor cells so that antibodies in patient plasma may react to modified forms of a protein not necessarily produced by other methods. Proteins eliciting a humoral response to serum can be analyzed directly from liquid fractions by mass spectrometry where an accurate MW value and analysis of enzymatic peptide maps by CE-TOF-MS and MALDI-TOF-MS can be used for identification and determination of present modifications. Modified proteins may provide selective response to antibodies in sera that unmodified proteins may not provide. The presence of proteins in the liquid phase makes this method amenable to automated screening of large numbers of samples. The antibody response will be explored for its utility in screening/diagnosis (i.e. to supplement PSA testing) and prognosis (i.e., distinguish between indolent and aggressive prostate cancer). In addition, bioinformatic methods will be used to determine whether subclasses of prostate cancer exist that better discriminate among clinical outcomes. A predictive model will be developed cataloguing clinically meaningful markers based on humoral expression profiles from localized prostate cancer samples; thus, candidate tumor antigens can be identified for further experimental study. Candidate tumor antigens will be further validated using tissue microarrays.