The utility of isotope-coded protein labeling for prioritization of proteins found in ovarian cancer patient urine

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备诸多优良特性，包括采样无创、样本量充足且易于获取、性质稳定，以及较窄的动态范围。本研究首次将同位素编码蛋白质标记（Isotope Coded Protein Labeling, ICPL）技术，结合溶液内等电点分级分离与液相色谱-基质辅助激光解吸电离飞行时间串联质谱（LC-MALDI-TOF/TOF），用于分析并筛选卵巢癌患者尿液中的蛋白质。在制定严格的排除标准后，本研究共鉴定出579种蛋白质，其中43%可获得定量数据。研究通过电喷雾电离三重四极杆飞行时间质谱（ESI-Qq-TOF MS）对筛选出的蛋白质的丰度变化进行了验证，随后采用蛋白质印迹法结合组织微阵列进行免疫组织化学分析，以探究所鉴定蛋白质的生物学相关性。本研究在卵巢癌患者尿液中检测到多种已确立的标志物（如HE4、骨桥蛋白）的水平升高，验证了所采用的实验方法；同时还鉴定出若干此前未在卵巢癌相关研究中被报道的潜在候选标志物（如磷脂酰乙醇胺结合蛋白1、细胞黏附分子1）。本研究结论表明，用于尿液蛋白质鉴定与相对定量的ICPL策略，是适用于生物标志物发现研究的可靠工具，可用于筛选潜在的生物标志物候选物以开展后续表征分析。