种子中蛋白质结合氨基酸的高通量绝对水平定量

Q1 Agricultural and Biological Sciences

Current protocols in plant biology Pub Date : 2018-11-08 DOI:10.1002/cppb.20084

Abou Yobi, Ruthie Angelovici

{"title":"种子中蛋白质结合氨基酸的高通量绝对水平定量","authors":"Abou Yobi, Ruthie Angelovici","doi":"10.1002/cppb.20084","DOIUrl":null,"url":null,"abstract":"<p>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.</p>","PeriodicalId":10932,"journal":{"name":"Current protocols in plant biology","volume":"3 4","pages":""},"PeriodicalIF":0.0000,"publicationDate":"2018-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cppb.20084","citationCount":"12","resultStr":"{\"title\":\"A High-Throughput Absolute-Level Quantification of Protein-Bound Amino Acids in Seeds\",\"authors\":\"Abou Yobi, Ruthie Angelovici\",\"doi\":\"10.1002/cppb.20084\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>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.</p>\",\"PeriodicalId\":10932,\"journal\":{\"name\":\"Current protocols in plant biology\",\"volume\":\"3 4\",\"pages\":\"\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2018-11-08\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://sci-hub-pdf.com/10.1002/cppb.20084\",\"citationCount\":\"12\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Current protocols in plant biology\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://onlinelibrary.wiley.com/doi/10.1002/cppb.20084\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"Agricultural and Biological Sciences\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current protocols in plant biology","FirstCategoryId":"1085","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cppb.20084","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"Agricultural and Biological Sciences","Score":null,"Total":0}

引用次数: 12

摘要

在本单元中，我们描述了16种蛋白质结合氨基酸(PBAAs)的高通量绝对定量方案，该方案结合了微尺度蛋白质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孔板萃取装置，避免了添加剂的使用，每周可以分析几百个样品。重要的是，该方法每个样品仅使用~ 3mg的组织，并包括12个重氨基酸内标，以高精度、准确性和可重复性对PBAAs的绝对水平进行定量。本文所述的方案已针对种子样品进行了优化，但适用于其他植物组织。©2018 by John Wiley &儿子,Inc。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

查看原文本刊更多论文

A High-Throughput Absolute-Level Quantification of Protein-Bound Amino Acids in Seeds

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.

求助全文

通过发布文献求助，成功后即可免费获取论文全文。去求助

来源期刊

Current protocols in plant biology Agricultural and Biological Sciences-Plant Science

自引率

0.00%

发文量

期刊介绍： Sound and reproducible laboratory methods are the foundation of scientific discovery. Yet nuances that are critical for an experiment''s success are not captured in the primary literature but exist only as part of a lab''s oral tradition. Current Protocols in Plant Biology provides reproducible step-by-step instructions for protocols relevant to plant research. Furthermore, Current Protocols content is thoughtfully organized by topic for optimal usage and to maximize contextual knowledge. Quarterly issues allow Current Protocols in Plant Biology to constantly evolve to keep pace with the newest discoveries and developments. Current Protocols in Plant Biology is the comprehensive source for protocols in the multidisciplinary field of plant biology, providing an extensive range of protocols from basic to cutting edge. Coverage includes: Extraction and analysis of DNA, RNA, proteins Chromosome analysis Transcriptional analysis Protein expression Metabolites Plant enzymology Epigenetics Plant genetic transformation Mutagenesis Arabidopsis, Maize, Poplar, Rice, and Soybean, and more.