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首页> 外文期刊>Clinical Chemistry: Journal of the American Association for Clinical Chemists >Online high-flow peptide immunoaffinity enrichment and nanoflow LC-MS/MS: assay development for total salivary pepsin/pepsinogen.
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Online high-flow peptide immunoaffinity enrichment and nanoflow LC-MS/MS: assay development for total salivary pepsin/pepsinogen.

机译：在线高流动肽免疫亲和富集和纳流LC-MS / MS：总唾液胃蛋白酶/胃蛋白酶原的测定开发。

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BACKGROUND: Detection limit challenges associated with measuring low-abundance protein biomarkers can be addressed with hybrid immunoaffinity-mass spectrometric assays, such as antipeptide antibody capture followed by liquid chromatography/tandem mass spectrometry (LC-MS/MS). Popular assay formats use magnetic bead-based immunoaffinity enrichment and nanoflow LC-MS/MS or high-flow immunoaffinity chromatography coupled online to conventional LC-MS/MS. As a proof of principle, we describe a novel online immunoaffinity LC-MS/MS configuration that combines high-flow peptide immunoaffinity enrichment and nanoflow LC-MS/MS. METHODS: We configured and validated an assay for the measurement of total pepsin/pepsinogen from human saliva that uses a pepsinogen standard. Saliva was heat-inactivated to quench residual enzymatic activity and then digested with endoproteinase AspN. Online immunoaffinity enrichment using an antipeptide antibody directed against the pepsin C-terminal sequence, DRANNQVGLAPVA, was linked to nanoflow liquid chromatography and selected reaction monitoring mass spectrometry. We used the assay to measure pepsin/pepsinogen concentrations in human saliva from presumed healthy volunteers. RESULTS: Heat inactivation at 100 degrees C for 25 min stabilized the target peptide. The final assay had <15% interassay relative error and <15% interassay CV across a range of 4.08-2980 pmol/L human pepsinogen (0.165-120 microg/L). Low but quantifiable signals were observed in some samples from presumed normal healthy volunteers ranging from 4.3 to 16.6 pmol/L (0.17-0.67 microg/L) total salivary pepsin/pepsinogen. CONCLUSIONS: This assay approach provides a high-sensitivity platform for protein bioanalysis in the low picomolar range. It bears the potential to deliver additional data on the salivary occurrence of pepsin/pepsinogen with greater confidence than previously.
机译：背景：与测量低丰度蛋白质生物标志物相关的检测极限挑战可通过混合免疫亲和质谱分析法解决，例如抗肽抗体捕获，然后进行液相色谱/串联质谱分析（LC-MS / MS）。流行的测定形式使用基于磁珠的免疫亲和富集和纳流LC-MS / MS或在线与常规LC-MS / MS偶联的高流量免疫亲和色谱。作为原理的证明，我们描述了一种结合了高流量肽免疫亲和富集和纳流LC-MS / MS的新型在线免疫亲和LC-MS / MS配置。方法：我们配置并验证了一种使用胃蛋白酶原标准物测定人唾液中总胃蛋白酶/胃蛋白酶原的方法。将唾液热灭活以淬灭残留的酶活性，然后用内蛋白酶AspN消化。使用针对胃蛋白酶C末端序列的抗肽抗体DRANNQVGLAPVA进行的在线免疫亲和富集与纳流液相色谱和选定的反应监测质谱联用。我们使用该测定法来测量假定健康志愿者在人唾液中的胃蛋白酶/胃蛋白酶原浓度。结果：在100℃下热失活25分钟稳定了目标肽。最终测定法在4.08-2980 pmol / L人胃蛋白酶原（0.165-120 microg / L）的范围内测定间相对误差<15％，测定间CV <15％。从正常健康志愿者的一些样本中观察到的信号很低但可量化，范围为唾液胃蛋白酶/胃蛋白酶原总量为4.3至16.6 pmol / L（0.17-0.67 microg / L）。结论：这种测定方法为低皮摩尔范围的蛋白质生物分析提供了一个高灵敏度的平台。它有可能以比以前更大的信心传递有关胃蛋白酶/胃蛋白酶原唾液出现的附加数据。

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