Department of Medical Biophysics, University of Toronto, Toronto, Canada.
Mol Cell Proteomics. 2012 Dec;11(12):1870-84. doi: 10.1074/mcp.M112.017889. Epub 2012 Sep 17.
Current protocols for the screening of prostate cancer cannot accurately discriminate clinically indolent tumors from more aggressive ones. One reliable indicator of outcome has been the determination of organ-confined versus nonorgan-confined disease but even this determination is often only made following prostatectomy. This underscores the need to explore alternate avenues to enhance outcome prediction of prostate cancer patients. Fluids that are proximal to the prostate, such as expressed prostatic secretions (EPS), are attractive sources of potential prostate cancer biomarkers as these fluids likely bathe the tumor. Direct-EPS samples from 16 individuals with extracapsular (n = 8) or organ-confined (n = 8) prostate cancer were used as a discovery cohort, and were analyzed in duplicate by a nine-step MudPIT on a LTQ-Orbitrap XL mass spectrometer. A total of 624 unique proteins were identified by at least two unique peptides with a 0.2% false discovery rate. A semiquantitative spectral counting algorithm identified 133 significantly differentially expressed proteins in the discovery cohort. Integrative data mining prioritized 14 candidates, including two known prostate cancer biomarkers: prostate-specific antigen and prostatic acid phosphatase, which were significantly elevated in the direct-EPS from the organ-confined cancer group. These and five other candidates (SFN, MME, PARK7, TIMP1, and TGM4) were verified by Western blotting in an independent set of direct-EPS from patients with biochemically recurrent disease (n = 5) versus patients with no evidence of recurrence upon follow-up (n = 10). Lastly, we performed proof-of-concept SRM-MS-based relative quantification of the five candidates using unpurified heavy isotope-labeled synthetic peptides spiked into pools of EPS-urines from men with extracapsular and organ-confined prostate tumors. This study represents the first efforts to define the direct-EPS proteome from two major subclasses of prostate cancer using shotgun proteomics and verification in EPS-urine by SRM-MS.
目前用于前列腺癌筛查的方案无法准确区分临床惰性肿瘤和侵袭性更强的肿瘤。一种可靠的预后指标是判断肿瘤是否局限于前列腺内（器官内）或已经侵犯前列腺外（器官外），但即使是这种判断也往往只能在前列腺切除术后做出。这凸显了探索其他途径以增强前列腺癌患者预后预测的必要性。与前列腺相邻的液体，如前列腺分泌液（EPS），是潜在前列腺癌生物标志物的有吸引力的来源，因为这些液体可能会浸润肿瘤。来自 16 名患有囊外（n=8）或器官内（n=8）前列腺癌患者的直接-EPS 样本被用作发现队列，并在 LTQ-Orbitrap XL 质谱仪上通过九步 MudPIT 进行重复分析。至少有两个独特肽段鉴定到 624 种独特蛋白质，假发现率为 0.2%。半定量光谱计数算法在发现队列中鉴定出 133 种差异表达蛋白。综合数据挖掘将 14 个候选物列为优先级，包括两种已知的前列腺癌生物标志物：前列腺特异性抗原和前列腺酸性磷酸酶，它们在器官内癌症组的直接-EPS 中显著升高。这些候选物以及另外五个候选物（SFN、MME、PARK7、TIMP1 和 TGM4）在一组具有生化复发疾病（n=5）的患者和未出现复发的患者（n=10）的独立直接-EPS 样本中通过 Western 印迹进行了验证。最后，我们使用经过重同位素标记的合成肽段，对 5 个候选物进行了基于 SRM-MS 的相对定量，这些肽段被添加到患有囊外和器官内前列腺肿瘤的男性的 EPS-尿液混合物中。这项研究代表了使用 shotgun 蛋白质组学定义两种主要前列腺癌亚型的直接-EPS 蛋白质组学的首次尝试，并通过 SRM-MS 在 EPS-尿液中进行了验证。
