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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：在线高流量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 °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 μg/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 μg/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：可以通过混合免疫亲和质谱分析法（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））解决与测量低丰度蛋白质生物标志物相关的检测极限挑战。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偶联的高流免疫亲和色谱。作为原理的证明，我们描述了一种新颖的在线免疫亲和LC-MS / MS配置，该配置结合了高流量肽免疫亲和富集和纳流LC-MS / MS。方法：我们配置并验证了用于测定总胃蛋白酶/胃蛋白酶原的测定方法来自使用胃蛋白酶原标准品的人唾液。将唾液热灭活以淬灭残余的酶活性，然后用内切蛋白酶AspN消化。使用针对胃蛋白酶C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°C加热灭活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（0.165–120μg/ L）范围内的测定间相对误差<15％，测定间CV <15％。从唾液中的胃蛋白酶/胃蛋白酶原总含量估计为4.3至16.6 pmol / L（0.17–0.67μg/ L）的正常健康志愿者的一些样品中观察到的信号很低，但可量化。在低皮摩尔范围内。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更多数据的潜力。

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