Protein Microarrays for the Humoral Response in Cancer

描述（由申请人提供）：在拟议的研究中，我们计划开发检查对前列腺肿瘤抗原的体液反应的方法，作为疾病的诊断和预后血清生物标志物。该方法涉及使用由细胞裂解物的二维液相分级分离产生的蛋白质微阵列。在第一维中利用基于Pl的色谱分离，以在扩展的pH范围内将大量蛋白质分离成离散的pH级分。在第二维中，将使用每个pH级分的无孔（NH 4）反相（RP）HPLC分离纯化的蛋白质，将其从柱洗脱液中点样到硝酸纤维素载玻片上。结果将是由>2500个蛋白质点组成的蛋白质微阵列，其可以针对患者血清进行测试以检查所得的体液应答。前列腺癌蛋白微阵列将用于询问来自健康对照个体、良性前列腺增生（BPH）患者和活检证实的前列腺癌患者的血清。使用这种方法，我们希望能够描述前列腺癌患者的标志性体液反应。该方法将来自实际肿瘤细胞的蛋白质排列，使得患者血浆中的抗体可以与不一定由其他方法产生的蛋白质的修饰形式反应。可以通过质谱法直接从液体级分中分析引起对血清的体液应答的蛋白质，其中精确的MW值和通过CE-TOF-MS和MALDI-TOF-MS进行的酶促肽图谱分析可以用于鉴定和测定存在的修饰。修饰的蛋白质可以提供对血清中抗体的选择性应答，而未修饰的蛋白质可能不能提供。蛋白质在液相中的存在使得该方法适合于大量样品的自动化筛选。将探索抗体应答在筛查/诊断（即补充PSA检测）和预后（即，区分惰性和侵袭性前列腺癌）。此外，生物信息学方法将用于确定是否存在更好地区分临床结果的前列腺癌亚类。将开发一个预测模型，根据局部前列腺癌样本的体液表达谱对有临床意义的标志物进行分类;因此，可以鉴定候选肿瘤抗原用于进一步的实验研究。将使用组织微阵列进一步验证候选肿瘤抗原。

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.