Development of a liquid chromatography-tandem mass spectrometry method for quantification of the egg allergen ovalbumin in rat plasma
Development of a liquid chromatography-tandem mass spectrometry method for quantification of the egg allergen ovalbumin in rat plasma
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建立定量大鼠血浆中鸡蛋过敏原卵清蛋白的液相色谱-串联质谱法
DOI：
10.1016/j.alit.2018.12.005
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发表时间：
2019
影响因子：
6.8
通讯作者：
Matsuo H.
中科院分区：
文献类型：
--
作者：
Ogino R;Yokooji T;Omoto A;Taogoshi T;Morita E;Matsuo H.
在激发试验期间监测食物过敏原的血清浓度在诊断食物过敏时提供重要信息。1夹心ELISA已被用于测量食品2和生物样品中食物过敏原的浓度。1然而，ELISA中使用的抗体有时会与样品中的多种组分发生交叉反应和/或干扰，导致高估和/或低估。因此，ELISA的广泛使用由于特异性差而受到限制。最近，液相色谱串联质谱（LC-MS/MS）已被用于分析加工食品中的食物过敏原。2，3这些报告表明，使用质谱法（MS）检测加工食品中的食物过敏原有助于质量控制。此外，MS已用于定量生物样品中的生物来源肽4和基于蛋白质的生物药物5。然而，没有研究表明，血浆（或血清）浓度的食物过敏原的测定使用MS。相比于检测食品过敏原在加工食品中使用MS，过敏原在血浆中的检测是复杂的，因为血浆中的食物过敏原的浓度通常远低于那些在加工食品。为了检测生物样品中低浓度的靶蛋白，需要通过适当的方法如免疫捕获方法从样品中除去任何丰富的杂质。我们采用免疫沉淀鸡蛋过敏原OVA和nanoLC结合四极杆飞行时间串联质谱仪测定大鼠口服后血浆中的OVA浓度。图1显示了我们测定血浆中OVA浓度的示意性策略。在本研究中，我们使用Ekspert ™ nanoLC 425（AB sciex）与TripleTOF 5600+（AB Sciex）组合作为LCMS/MS系统。涉及动物的实验由广岛大学动物委员会批准（批准号A16 - 138）。所有细节见补充方法。为了确定用于OVA定量的特征肽离子，我们首先确认水中的OVA浓度与由胰蛋白酶消化的OVA产生的产物离子的峰面积之间的相关性。当使用nanoLC-MS/MS和Skyline软件分析来自胰蛋白酶消化的OVA的肽时，获得了11个前体离子峰（补充表1）。在这些前体离子中，5种OVA特异性前体离子表现出OVA浓度与产物离子的峰面积之间的相关性（补充表2）。特别是，在50 μ g/mL和500 μ g/mL范围内，GGLEPINESTAADQAR [M +2H] 2+在测量OVA浓度时显示出最高的强度和再现性。GGLEPINTAADQAR肽已被鉴定为可用于定量加工食品中的OVA含量的标记物。3此外，该肽含有OVA的部分IgE结合表位序列（RGGLEPINFQ），用于对鸡蛋过敏的I型患者。6因此，我们确定GGLEPINTAADQAR为特征肽，并合成了其稳定的同位素标记（SIL）肽。接着，我们尝试检测大鼠血浆中的OVA，并确认了大鼠血浆中的标准OVA浓度与OVA与SIL-肽的产物离子峰面积比之间的相关性。为了消除大量生物蛋白质的定量干扰，我们使用多克隆抗卵蛋白抗体偶联的磁珠通过免疫沉淀法浓缩血浆样品中的卵蛋白。在.之后
Monitoring of serum concentrations of food allergens during challenge tests provides important information when diagnosing food allergies. 1 Sandwich ELISA has been used to measure the concentrations of food allergens in foodstuffs 2 and in biological samples. 1 However, the antibodies used in ELISA sometimes show cross-reactivity and/or interference with multiple components in samples, resulting in overestimation and/or underestimation. Thus, the widespread use of ELISA is limited because of poor specificity. Recently, liquid chromatography tandem mass spectrometry (LC-MS/MS) has been used for the analysis of food allergens in processed foods. 2, 3 These reports suggest that detection of food allergens in processed foods using mass spectrometry (MS) is helpful for quality control. Furthermore, MS has been used for quantification of biological origin peptides 4 and protein-based biopharmaceuticals 5 in biological samples. However, there are no studies that the plasma (or serum) concentrations of food allergens were measured using MS. Compared to the detection of food allergens in processed foods using MS, detection of allergen in plasma is complicated since concentrations of food allergen in plasma are generally much lower than those in processed foods. To detect a target protein at low concentrations in biological samples, it is necessary to remove any abundant impurities from the samples by appropriate methods such as immunocapture methods. We applied immunoprecipitation of egg allergen OVA and nanoLC combined with quadrupole time of flight tandem mass spectrometer to measure the concentration of OVA in rat plasma after oral ingestion. Figure 1 shows our schematic strategy to determine the concentrations of OVA in plasma. In this study, we used Ekspert™ nanoLC 425 (AB sciex) combined with a TripleTOF 5600+(AB Sciex) as a LCMS/MS system. Experiments involving animals were approved by the animal committee of Hiroshima University (Approval No. A16-138). All details are shown in Supplementary Methods. To determine the signature peptide ions for OVA quantification, we first confirmed the correlation between the OVA concentrations in water and peak areas of the product ions generated from tryptic digested OVA. When the peptides from tryptic digested OVA were analyzed using nanoLC-MS/MS and Skyline software, 11 precursor ion peaks were obtained (Supplementary Table 1). Among these precursor ions, five OVA-specific precursor ions exhibited correlations between the OVA concentrations and peak areas of the product ions(Supplementary Table 2). In particular, GGLEPINFQTAADQAR [M+ 2H] 2+ showed the highest intensity and reproducibility for measuring the OVA concentration in the range of 50e500 μg/mL. GGLEPINFQTAADQAR peptide has been identified as a marker that can be used to quantify the OVA contents in processed foods. 3 In addition, this peptide contains a partial IgE-binding epitope sequence of OVA (RGGLEPINFQ) for patients with type I hypersensitivity to eggs. 6 Thus, we determined GGLEPINFQTAADQAR as a signature peptide and synthesized its stable isotopelabeled (SIL) peptide. SIL-peptide was used as internal standard in OVA quantification.Next, we tried to detect OVA in rat plasma and confirmed correlation between the standard OVA concentration in rat plasma and peak area ratios of product ions from OVA to SIL-peptide. To eliminate the quantitative interferences by large amounts of biological proteins, we concentrated OVA from plasma samples by the immunoprecipitation method using polyclonal anti-OVA antibody-coupled magnetic beads. After …