Prince Henry's Institute, Monash Medical Centre, Clayton, Victoria, Australia, 3168.
J Proteome Res. 2013 Sep 6;12(9):4074-88. doi: 10.1021/pr400618v. Epub 2013 Aug 16.
Urine offers a number of attractive features as a sample type for biomarker discovery, including noninvasive sampling, quantity and availability, stability, and a narrow dynamic range. In this study we report the first application of isotope coded protein labeling (ICPL), coupled with in-solution isoelectric fractionation and LC-MALDI-TOF/TOF, to examine and prioritize urinary proteins from ovarian cancer patients. Following the definition of stringent exclusion criteria a total of 579 proteins were identified with 43% providing quantitation data. Protein abundance changes were validated for selected proteins by ESI-Qq-TOF MS, following which Western blot and immunohistochemical analysis by tissue microarray was used to explore the biological relevance of the proteins identified. Several established markers (e.g., HE4, osteopontin) were identified at increased levels in ovarian cancer patient urine, validating the approach used; we also identified a number of potential marker candidates (e.g., phosphatidylethanolamine binding protein 1, cell-adhesion molecule 1) previously unreported in the context of ovarian cancer. We conclude that the ICPL strategy for identification and relative quantitation of urine proteins is an appropriate tool for biomarker discovery studies, and can be applied for the selection of potential biomarker candidates for further characterization.
尿液作为生物标志物发现的样本类型具有许多吸引人的特点，包括非侵入性采样、数量和可用性、稳定性以及狭窄的动态范围。在这项研究中，我们报告了同位素编码蛋白质标记 (ICPL) 的首次应用，该方法与溶液等电聚焦和 LC-MALDI-TOF/TOF 相结合，用于检查和优先考虑卵巢癌患者的尿液蛋白。在定义严格的排除标准后，共鉴定出 579 种蛋白质，其中 43%提供定量数据。通过 ESI-Qq-TOF MS 对选定蛋白质的丰度变化进行了验证，随后使用组织微阵列进行 Western blot 和免疫组织化学分析，以探索鉴定的蛋白质的生物学相关性。几种已建立的标志物（例如 HE4、骨桥蛋白）在卵巢癌患者尿液中水平升高，验证了所采用的方法；我们还鉴定了一些以前未在卵巢癌背景下报道的潜在标志物候选物（例如，磷脂酰乙醇胺结合蛋白 1、细胞黏附分子 1）。我们得出结论，用于鉴定和相对定量尿液蛋白质的 ICPL 策略是生物标志物发现研究的合适工具，并且可以用于选择进一步表征的潜在生物标志物候选物。
