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首页> 外文期刊>Molecular & cellular proteomics: MCP >Use of an immunoaffinity-mass spectrometry-based approach for the quantification of protein biomarkers from serum samples of lung cancer patients.
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Use of an immunoaffinity-mass spectrometry-based approach for the quantification of protein biomarkers from serum samples of lung cancer patients.

机译：基于免疫亲和质谱的方法用于量化肺癌患者血清样品中蛋白质生物标志物的用途。

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It is a challenging task to verify and quantify potential biomarkers expressed at elevated levels in sera from cancer patients. An immunoaffinity-mass spectrometry-based approach has been developed using antibodies to enrich proteins of interest from sera followed by mass spectrometry-based quantification. Antibodies specific to the protein of interest were immobilized to hydrazide resin via the carbohydrate moiety on the Fc region of the antibody. Captured proteins were eluted, reduced, alkylated, and digested with trypsin. Peptides were analyzed by LC coupled with multiple reaction monitoring approach, and quantification was achieved by the addition of stable isotope-labeled (heavy) standard peptides. Using this methodology, we were able to achieve a linear response from 15 to 250 ng/ml for carcinoembryonic antigen (CEA), a known tumor biomarker. Moreover we observed elevated levels of CEA in sera samples from lung cancer patients that to our knowledge is the first time that circulating CEA has been detected by mass spectrometry-based analysis. This approach was further applied to potential protein biomarkers discovered from tumor cell lines and tumor tissues. A linear response was obtained from a multiplex spiking experiment in normal human sera for secretory leukocyte peptidase inhibitor (4-500 ng/ml), tissue factor pathway inhibitor (TFPI) (42-1000 ng/ml), tissue factor pathway inhibitor 2 (TFPI2) (2-250 ng/ml), and metalloproteinase inhibitor 1 (TIMP1) (430-1000 ng/ml). A replicate experiment for a single concentration value yielded a relative coefficient of variation better than 11% for TFPI, secretory leukocyte peptidase inhibitor, and TFPI2. The expression level of the proteins in lung cancer patient sera was assayed by an immunoaffinity-multiple reaction monitoring method, and the results were comparable with those obtained from ELISA. This immunoaffinity-mass spectrometry-based quantification approach thus provides a specific and accurate assay for verifying the expression of potential biomarkers in patient serum samples especially for those proteins for which the necessary reagents for ELISA development are unavailable.
机译：验证和量化癌症患者血清中水平升高表达的潜在生物标志物是一项艰巨的任务。已开发出一种基于免疫亲和质谱的方法，该方法使用抗体从血清中富集目标蛋白，然后进行基于质谱的定量。通过抗体的Fc区上的碳水化合物部分将对目标蛋白具有特异性的抗体固定在酰肼树脂上。将捕获的蛋白洗脱，还原，烷基化并用胰蛋白酶消化。通过LC结合多种反应监测方法对肽进行分析，并通过添加稳定的同位素标记（重）标准肽进行定量。使用这种方法，我们能够对癌胚抗原（CEA）（一种已知的肿瘤生物标志物）实现15到250 ng / ml的线性响应。此外，我们观察到肺癌患者血清样品中CEA的水平升高，据我们所知，这是首次通过基于质谱的分析检测到循环中CEA。该方法进一步应用于从肿瘤细胞系和肿瘤组织中发现的潜在蛋白质生物标志物。从正常人血清中的多重加标实验获得线性应答，其针对分泌型白细胞肽酶抑制剂（4-500 ng / ml），组织因子途径抑制剂（TFPI）（42-1000 ng / ml），组织因子途径抑制剂2（ TFPI2）（2-250 ng / ml）和金属蛋白酶抑制剂1（TIMP1）（430-1000 ng / ml）。单一浓度值的重复实验得出的TFPI，分泌性白细胞肽酶抑制剂和TFPI2的相对变异系数优于11％。通过免疫亲和多重反应监测方法检测肺癌患者血清中蛋白质的表达水平，其结果与ELISA相当。因此，这种基于免疫亲和质谱的定量方法提供了一种特定而准确的测定方法，用于验证患者血清样品中潜在生物标记物的表达，尤其是对于那些无法获得用于ELISA开发的必需试剂的蛋白质。

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