Ovarian cancer is the most lethal gynecologic malignancy in women, and high-grade serous ovarian cancer (HGSOC) is the most common subtype. Currently, no clinical test has been approved by the FDA to screen the general population for ovarian cancer. This underscores the critical need for the development of a robust methodology combined with novel technology to detect diagnostic biomarkers for HGSOC in the sera of women. Targeted mass spectrometry (MS) can be used to identify and quantify specific peptides/proteins in complex biological samples with high accuracy, sensitivity, and reproducibility. In this study, we sought to develop and conduct analytical validation of a multiplexed Tier 2 targeted MS parallel reaction monitoring (PRM) assay for the relative quantification of 23 putative ovarian cancer protein biomarkers in sera. To develop a PRM method for our target peptides in sera, we followed nationally recognized consensus guidelines for validating fit-for-purpose Tier 2 targeted MS assays. The endogenous target peptide concentrations were calculated using the calibration curves in serum for each target peptide. Receiver operating characteristic (ROC) curves were analyzed to evaluate the diagnostic performance of the biomarker candidates. We describe an effort to develop and analytically validate a multiplexed Tier 2 targeted PRM MS assay to quantify candidate ovarian cancer protein biomarkers in sera. Among the 64 peptides corresponding to 23 proteins in our PRM assay, 24 peptides corresponding to 16 proteins passed the assay validation acceptability criteria. A total of 6 of these peptides from insulin-like growth factor-binding protein 2 (IBP2), sex hormone-binding globulin (SHBG), and TIMP metalloproteinase inhibitor 1 (TIMP1) were quantified in sera from a cohort of 69 patients with early-stage HGSOC, late-stage HGSOC, benign ovarian conditions, and healthy (non-cancer) controls. Confirming the results from previously published studies using orthogonal analytical approaches, IBP2 was identified as a diagnostic biomarker candidate based on its significantly increased abundance in the late-stage HGSOC patient sera compared to the healthy controls and patients with benign ovarian conditions. A multiplexed targeted PRM MS assay was applied to detect candidate diagnostic biomarkers in HGSOC sera. To evaluate the clinical utility of the IBP2 PRM assay for HGSOC detection, further studies need to be performed using a larger patient cohort.

中文翻译：

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使用多重靶向质谱分析对假定的卵巢癌血清蛋白生物标志物进行定量

卵巢癌是女性最致命的妇科恶性肿瘤，高级别浆液性卵巢癌（HGSOC）是最常见的亚型。目前，FDA 尚未批准任何临床测试来筛查一般人群是否患有卵巢癌。这强调了迫切需要开发一种与新技术相结合的稳健方法来检测女性血清中 HGSOC 的诊断生物标志物。靶向质谱 (MS) 可用于识别和定量复杂生物样品中的特定肽/蛋白质，具有高精度、灵敏度和重现性。在本研究中，我们试图开发并进行多重 2 级靶向 MS 平行反应监测 (PRM) 测定法的分析验证，以对血清中 23 种假定的卵巢癌蛋白质生物标志物进行相对定量。为了开发血清中目标肽的 PRM 方法，我们遵循国家认可的共识指南来验证适合目的的第 2 层靶向 MS 检测。使用每种目标肽的血清校准曲线计算内源性目标肽浓度。分析受试者工作特征（ROC）曲线以评估候选生物标志物的诊断性能。我们描述了开发和分析验证多重 Tier 2 靶向 PRM MS 测定的努力，以量化血清中候选卵巢癌蛋白质生物标志物。在我们的 PRM 测定中对应 23 种蛋白质的 64 种肽中，对应 16 种蛋白质的 24 种肽通过了测定验证可接受标准。在 69 名早期患有糖尿病的患者的血清中，对来自胰岛素样生长因子结合蛋白 2 (IBP2)、性激素结合球蛋白 (SHBG) 和 TIMP 金属蛋白酶抑制剂 1 (TIMP1) 的总共 6 种肽进行了定量。 -阶段 HGSOC、晚期 HGSOC、良性卵巢状况和健康（非癌症）对照。证实了之前发表的使用正交分析方法的研究结果，由于与健康对照和良性卵巢疾病患者相比，晚期 HGSOC 患者血清中 IBP2 的丰度显着增加，因此 IBP2 被确定为候选诊断生物标志物。应用多重靶向 PRM MS 测定来检测 HGSOC 血清中的候选诊断生物标志物。为了评估 IBP2 PRM 检测在 HGSOC 检测中的临床效用，需要使用更大的患者队列进行进一步研究。