Center for Systems and Computational Biology and Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA.
J Proteomics. 2013 Aug 26;89:165-78. doi: 10.1016/j.jprot.2013.06.016. Epub 2013 Jun 21.
New serological biomarkers for early detection and clinical management of ovarian cancer are urgently needed, and many candidates have been reported. A major challenge frequently encountered when validating candidates in patients is establishing quantitative assays that distinguish between highly homologous proteins. The current study tested whether multiple members of two recently discovered ovarian cancer biomarker protein families, chloride intracellular channel (CLIC) proteins and tropomyosins (TPM), were detectable in ovarian cancer patient sera. A multiplexed, label-free multiple reaction monitoring (MRM) assay was established to target peptides specific to all detected CLIC and TPM family members, and their serum levels were quantitated for ovarian cancer patients and non-cancer controls. In addition to CLIC1 and TPM1, which were the proteins initially discovered in a xenograft mouse model, CLIC4, TPM2, TPM3, and TPM4 were present in ovarian cancer patient sera at significantly elevated levels compared with controls. Some of the additional biomarkers identified in this homolog-centric verification and validation approach may be superior to the previously identified biomarkers at discriminating between ovarian cancer and non-cancer patients. This demonstrates the importance of considering all potential protein homologs and using quantitative assays for cancer biomarker validation with well-defined isoform specificity.
This manuscript addresses the importance of distinguishing between protein homologs and isoforms when identifying and validating cancer biomarkers in plasma or serum. Specifically, it describes the use of targeted in-depth LC-MS/MS analysis to determine the members of two protein families, chloride intracellular channel (CLIC) and tropomyosin (TPM) proteins that are detectable in sera of ovarian cancer patients. It then establishes a multiplexed isoform- and homology-specific MRM assay to quantify all observed gene products in these two protein families as well as many of the closely related tropomyosin isoforms. Using this assay, levels of all detected CLICs and TPMs were quantified in ovarian cancer patient and control subject sera. These results demonstrate that in addition to the previously known CLIC1, multiple tropomyosins and CLIC4 are promising new ovarian cancer biomarkers. Based on these initial validation studies, these new ovarian cancer biomarkers appear to be superior to most previously known ovarian cancer biomarkers.
迫切需要新的血清生物标志物来用于卵巢癌的早期检测和临床管理，并且已经报道了许多候选标志物。在患者中验证候选标志物时，经常遇到的一个主要挑战是建立能够区分高度同源蛋白的定量检测方法。本研究测试了最近发现的两个卵巢癌生物标志物蛋白家族（氯离子通道蛋白 CLIC 和原肌球蛋白 TPM）的多个成员是否可在卵巢癌患者血清中检测到。建立了一种多重、无标记的多重反应监测（MRM）检测方法，以靶向针对所有检测到的 CLIC 和 TPM 家族成员的特异性肽，并对卵巢癌患者和非癌症对照者的血清水平进行定量分析。除了最初在异种移植小鼠模型中发现的 CLIC1 和 TPM1 外，CLIC4、TPM2、TPM3 和 TPM4 也在卵巢癌患者血清中以明显高于对照组的水平存在。在这种同源验证和验证方法中鉴定的一些其他生物标志物在区分卵巢癌患者和非癌症患者方面可能优于先前鉴定的生物标志物。这证明了在鉴定和验证用于血浆或血清的癌症生物标志物时，考虑所有潜在蛋白同源物并使用定量检测方法进行区分的重要性。
本文解决了在血浆或血清中鉴定和验证癌症生物标志物时区分蛋白同源物和同工型的重要性。具体来说，它描述了使用靶向深度 LC-MS/MS 分析来确定在卵巢癌患者血清中可检测到的两个蛋白家族（氯离子通道（CLIC）和原肌球蛋白（TPM）蛋白）的成员。然后，它建立了一种多重、同型和同源特异性的 MRM 检测方法，以定量这些两种蛋白家族以及许多密切相关的原肌球蛋白同工型的所有观察到的基因产物。使用该检测方法，定量了卵巢癌患者和对照受试者血清中所有检测到的 CLIC 和 TPM 的水平。这些结果表明，除了先前已知的 CLIC1 之外，多种原肌球蛋白和 CLIC4 是很有前途的新卵巢癌生物标志物。基于这些初步验证研究，这些新的卵巢癌生物标志物似乎优于大多数先前已知的卵巢癌生物标志物。
