Online High-Flow Peptide Immunoaffinity Enrichment and Nanoflow LC-MS/MS: Assay Development for Total Salivary Pepsin/Pepsinogen
Online High-Flow Peptide Immunoaffinity Enrichment and Nanoflow LC-MS/MS: Assay Development for Total Salivary Pepsin/Pepsinogen
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DOI：
10.1373/clinchem.2010.144576
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发表时间：
2010-09-01
影响因子：
9.3
通讯作者：
Muirhead, David
中科院分区：
文献类型：
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作者：
Neubert, Hendrik;Gale, Jeremy;Muirhead, David
背景技术背景：与测量低丰度蛋白质生物标志物相关的检测限挑战可以通过混合免疫亲和-质谱测定来解决，例如抗肽抗体捕获，然后进行液相色谱/串联质谱（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米流LC-MS/MS或在线耦合到常规LCMS/MS的高流量免疫亲和色谱。作为原理证明，我们描述了一种新型在线免疫亲和LC-MS/MS配置，其结合了高流量肽免疫亲和富集和纳米流LC-MS/MS。我们配置并验证了一种使用胃蛋白酶原标准品测量人唾液中总胃蛋白酶/胃蛋白酶原的测定方法。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-末端序列DRANNQVGLAPVA的抗肽抗体的在线免疫亲和富集与纳流液相色谱和选择反应监测质谱相关联。我们用该方法来测量胃蛋白酶/胃蛋白酶原浓度在人唾液中从假定健康volunteers.RESULTS：热灭活在100摄氏度为25分钟稳定的目标肽。在4.08-2980 pmol/L人胃蛋白酶原（0.165-120 μ g/L）范围内，最终测定具有< 15%的测定间相对误差和< 15%的测定间CV。低，但可定量的信号，观察到在一些样品从假定正常的健康志愿者范围从4.3至16.6 pmol/L（0.17-0.67 μ g/L）总唾液胃蛋白酶/胃蛋白酶gen.CONCLUSIONS：这种测定方法提供了一个高灵敏度的平台，在低皮摩尔范围内的蛋白质生物分析。它有可能提供关于胃蛋白酶/胃蛋白酶原唾液发生率的额外数据，置信度比以前更高。(C)2010年美国临床化学协会
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 LCMS/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 mu g/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 mu g/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. (C) 2010 American Association for Clinical Chemistry