Mast cells are essential in mediating the inflammatory processes. When activated, mast cells can rapidly release characteristic granules and various mediators into the interstitium. Tryptase (TPS) and beta-hexosaminidase (HEXB) are typical protease mediators stored in granules and released upon activation. They have been recognized as important biomarkers of anaphylaxis, and the released level is associated with the severity of allergic reactions. In this study, a sensitive, accurate, and selective liquid chromatography tandem mass spectrometry (LC-MS/MS) method for simultaneously quantifying the two biomarkers was developed and validated in LAD2 cell culture supernatant, P14R was used as internal standard. Good linearity was observed in the range of 50-2500 ng/mL for TPS and 10-2000 ng/mL for HEXB both with R2 ＞ 0.99. The matrix effect and recovery were both within acceptable limits. We quantified TPS and HEXB released from Laboratory of Allergic Disease 2 (LAD2) mast cells treated with several potential allergens, results demonstrate that the method can be used to investigate TPS and HEXB levels in LAD2 mast cell model during allergy research. We anticipate our approach to be a robust and sensitive assessment method for more biomarkers with similar kinetics characteristics, and to be a major tool of allergic drug assessment or anti-allergic drug development in research.

中文翻译：

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基于液相色谱串联质谱的无标记定量方法，用于评估变应原诱导的类过敏反应。

肥大细胞在介导炎症过程中至关重要。激活后，肥大细胞可以迅速将特有的颗粒和各种介体释放到间质中。类胰蛋白酶（TPS）和β-己糖胺酶（HEXB）是典型的蛋白酶介质，存储在颗粒中，并在激活后释放。它们被认为是过敏反应的重要生物标志物，其释放水平与过敏反应的严重程度有关。在这项研究中，建立了同时定量两种生物标志物的灵敏，准确，选择性液相色谱串联质谱法（LC-MS / MS），并在LAD2细胞培养上清液中进行了验证，P14R作为内标。对于TPS，在50-2500 ng / mL的范围内，对于HEXB，在10-2000 ng / mL的范围内，均观察到良好的线性，R2＞ 0.99。基质效应和回收率均在可接受的范围内。我们量化了用几种潜在过敏原处理过的变应性疾病2（LAD2）肥大细胞实验室释放的TPS和HEXB，结果表明该方法可用于在变态反应研究期间研究LAD2肥大细胞模型中的TPS和HEXB水平。我们预期我们的方法将成为具有相似动力学特征的更多生物标志物的可靠且灵敏的评估方法，并且将成为研究中过敏性药物评估或抗过敏性药物开发的主要工具。结果表明，该方法可用于过敏研究期间LAD2肥大细胞模型中TPS和HEXB水平的研究。我们预计我们的方法将成为具有相似动力学特征的更多生物标志物的可靠且灵敏的评估方法，并且将成为研究中过敏性药物评估或抗过敏性药物开发的主要工具。结果表明，该方法可用于过敏研究期间LAD2肥大细胞模型中TPS和HEXB水平的研究。我们预计我们的方法将成为具有相似动力学特征的更多生物标志物的可靠且灵敏的评估方法，并且将成为研究中过敏性药物评估或抗过敏性药物开发的主要工具。