Roche Diagnostics GmbH, Nonnenwald 2, 82377 Penzberg, Germany.
Roche Diagnostics GmbH, Nonnenwald 2, 82377 Penzberg, Germany.
Clin Chim Acta. 2018 May;480:1-8. doi: 10.1016/j.cca.2018.01.040. Epub 2018 Feb 20.
Recently, site-specific fucosylation of glycoproteins has attracted attention as it can be associated with several types of cancers including prostate cancer. However, individual glycoproteins, which might serve as potential cancer markers, often are very low-concentrated in complex serum matrices and distinct glycan structures are hard to detect by immunoassays. Here, we present a mass spectrometry-based strategy for the simultaneous analysis of core-fucosylated and total prostate-specific antigen (PSA) in human serum in the low ng/ml concentration range. Sample preparation comprised an immunoaffinity capture step to enrich total PSA from human serum using anti-PSA antibody coated magnetic beads followed by consecutive two-step on-bead partial deglycosylation with endoglycosidase F3 and tryptic digestion prior to LC-MS/MS analysis. The method was shown to be linear from 0.5 to 60 ng/ml total PSA concentrations and allows the simultaneous quantification of core-fucosylated PSA down to 1 ng/ml and total PSA lower than 0.5 ng/ml. The imprecision of the method over two days ranged from 9.7-23.2% for core-fucosylated PSA and 10.3-18.3% for total PSA depending on the PSA level. The feasibility of the method in native sera was shown using three human specimens. To our knowledge, this is the first MS-based method for quantification of core-fucosylated PSA in the low ng/ml concentration range in human serum. This method could be used in large patient cohorts as core-fucosylated PSA may be a diagnostic biomarker for the differentiation of prostate cancer and other prostatic diseases, such as benign prostatic hyperplasia (BPH). Furthermore, the described strategy could be used to monitor potential changes in site-specific core-fucosylation of other low-concentrated glycoproteins, which could serve as more specific markers ("marker refinement") in cancer research.
最近，糖蛋白的位点特异性岩藻糖基化引起了关注，因为它可能与包括前列腺癌在内的几种类型的癌症有关。然而，作为潜在癌症标志物的个体糖蛋白在复杂的血清基质中通常浓度非常低，并且免疫测定法难以检测到独特的聚糖结构。在这里，我们提出了一种基于质谱的策略，用于同时分析人血清中核心岩藻糖基化和总前列腺特异性抗原（PSA），浓度范围在低 ng/ml。样品制备包括免疫亲和捕获步骤，使用抗 PSA 抗体包被的磁性珠从人血清中富集总 PSA，然后进行两步连续的珠上部分糖基化，使用内切糖苷酶 F3 和胰蛋白酶消化，然后进行 LC-MS/MS 分析。该方法在总 PSA 浓度为 0.5 至 60ng/ml 范围内呈线性，允许同时定量核心岩藻糖基化 PSA 低至 1ng/ml 和总 PSA 低于 0.5ng/ml。该方法在两天内的精密度取决于 PSA 水平，核心岩藻糖基化 PSA 的范围为 9.7-23.2%，总 PSA 的范围为 10.3-18.3%。使用三个人类标本证明了该方法在天然血清中的可行性。据我们所知，这是首次在人血清中建立基于 MS 的方法，用于定量核心岩藻糖基化 PSA 在低 ng/ml 浓度范围内的方法。该方法可用于大型患者队列，因为核心岩藻糖基化 PSA 可能是区分前列腺癌和其他前列腺疾病（如良性前列腺增生（BPH））的诊断生物标志物。此外，所描述的策略可用于监测其他低浓度糖蛋白的位点特异性核心岩藻糖基化的潜在变化，这可能作为癌症研究中更特异的标志物（“标志物细化”）。
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