High-throughput protein modification quantitation analysis using intact protein MRM and its application on hENGase inhibitor screening.
High-throughput protein modification quantitation analysis using intact protein MRM and its application on hENGase inhibitor screening.
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DOI：
10.1016/j.talanta.2021.122384
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发表时间：
2021-08-15
期刊：
影响因子：
6.1
通讯作者：
LeClair CA
中科院分区：
文献类型：
--
作者：
Tao D;Xu M;Farkhondeh A;Burns AP;Rodems S;Might M;Zheng W;LeClair CA
蛋白质被广泛用作许多疾病的药物靶点、酶底物和生物标志物。对蛋白质定量的新兴需求在多个领域不断增加。目前，利用无标记方法在完整蛋白质水平上进行高通量蛋白质定量仍存在很大差距。本文选择核糖核酸酶B（RNB）作为模型，它是人内切-β-N-乙酰氨基葡萄糖苷酶（hENGase）的底物，而hENGase是治疗N-聚糖酶缺乏症的一个很有前途的药物靶点。最初开发并优化了完整蛋白水平多反应监测（MRM）方法来定量RNB和去糖基化RNB（RNB-deg），与传统的全MS扫描方法相比，S/N比提高了近20倍。为了进一步提高通量，使hENGase抑制剂筛选成为可能，将蛋白质MRM方法引入RapidFire-MS/MS系统，实现了至少12倍的通量提高。将该测定进一步优化为用于化合物筛选的384孔板形式，S/B比>37倍且Z'因子>0.7，其适合于化合物集合的高通量筛选，速度为每384孔板2小时，并且能够以单一浓度剂量每天筛选超过3000种化合物。这种基于384孔板的自动SPE-MS/MS测定对于化合物筛选是有效和稳健的，并且测定形式对于其他疾病模型的蛋白质靶标具有广泛的适用性。
Proteins are widely used as drug targets, enzyme substrates, and biomarkers for numerous diseases. The emerging demand for proteins quantitation has been increasing in multiple fields. Currently, there is still a big gap for high-throughput protein quantitation at intact protein level using label-free method. Here we choose ribonuclease B (RNB) as a model, which is the substrate for human endo-β-N-acetylglucosaminidase (hENGase), a promising drug target for the treatment of N-Glycanase deficiency. Intact proteinlevel multiple reaction monitoring (MRM) methods were initally developed and optimized to quantify RNB and deglycosylated RNB (RNB-deg), with the S/N ratio improved by nearly 20-fold compared to the traditional full MS scan methods. To further increase the throughput making it possible for hENGase inhibitors screen, the protein MRM methods were introduced to the RapidFire-MS/MS system, achieving at least 12-fold throughput improvement. This assay was further optimized into 384-well plate format for compound screening with S/B ratio >37-fold and Z’ factor >0.7 that is suitable for high-throughput screening of compound collections with a speed of 2 h per 384-well plate and an ability to screen over 3000 compounds per day at a single concentration dose. This 384-well plate based automated SPE-MS/MS assay is efficient and robust for compound screening and the assay format has a wide applicability to protein targets for other disease models.
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