A High-Throughput Absolute-Level Quantification of Protein-Bound Amino Acids in Seeds
A High-Throughput Absolute-Level Quantification of Protein-Bound Amino Acids in Seeds
复制标题
种子中蛋白质结合氨基酸的高通量绝对水平定量
DOI：
10.1002/cppb.20084
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发表时间：
2018
影响因子：
--
通讯作者：
Angelovici, Ruthie
中科院分区：
文献类型：
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作者：
Yobi, Abou;Angelovici, Ruthie
在本单元中，我们描述了16种蛋白结合氨基酸（PBAA）的高通量绝对定量方案，该方案结合了微量蛋白水解步骤和使用基于多反应监测的液相色谱-串联质谱检测的绝对定量步骤。该方法通过使用96孔板提取装置和避免使用添加剂，每周可分析数百份样品。重要的是，该方法每个样本仅使用103 mg组织，并包括12个重氨基酸内标，以实现高精度，准确度和重现性的PBAA绝对水平的定量。本文所述的方案已经针对种子样品进行了优化，但也适用于其他植物组织。John Wiley & Sons，Inc.
In this unit, we describe a high‐throughput absolute quantification protocol for 16 protein‐bound amino acids (PBAAs) that combines a microscale protein hydrolysis step and an absolute quantification step using multiple reaction monitoring—based liquid chromatography–tandem mass spectrometry detection. The approach facilitates analysis of a few hundred samples per week by using a 96‐well‐plate extraction setup and avoiding use of additives. Importantly, the method uses only ∼3 mg of tissue per sample and includes 12 heavy‐amino‐acid internal standards to enable quantification of the absolute levels of PBAAs with high precision, accuracy, and reproducibility. The protocol described herein has been optimized for seed samples but is applicable to other plant tissues. © 2018 by John Wiley & Sons, Inc.