An Expedited Qualitative Profiling of Free Amino Acids in Plant Tissues Using Liquid Chromatography-Mass Spectrometry (LC–MS) in Conjunction With MS-DIAL

IF 1.9 3区 化学 Q3 BIOCHEMICAL RESEARCH METHODS
Anish Kaachra, Anish Tamang, Vipin Hallan
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引用次数: 0

Abstract

The estimation of relative levels of amino acids is crucial for understanding various biological processes in plants, including photosynthesis, stress tolerance, and the uptake and translocation of nutrients. A wide range of liquid chromatography (LC; HPLC/UHPLC)-based methods is available for measuring the quantity of amino acids in plants. Additionally, the coupling of LC with mass spectrometry (MS) significantly enhanced the robustness of existing chromatographic methods used for amino acid quantification. However, accurate annotation and integration of mass peaks can be challenging for plant biologists with limited experience in analyzing MS data, especially in studies involving large datasets with multiple treatments and/or replicates. Further, there are instances when the experiment demands an overall view of the amino acids profile rather than focusing on absolute quantification. The present protocol provides a detailed LC–MS method for obtaining a qualitative amino acids profile using MS-DIAL, a versatile and user-friendly program for processing MS data. Free amino acids were extracted from the leaves of control and Tomato leaf curl Palampur virus (ToLCPalV)-infected Nicotiana benthamiana plants. Extracted amino acids were derivatized and separated using UHPLC-QTOF, with each amino acid subsequently identified by aligning mass data with a custom text library created in MS-DIAL. Further, MS-DIAL was employed for internal standard-based normalization to obtain a qualitative profile of 15 amino acids in control and virus-infected plants. The outlined method aims to simplify the processing of MS data to quickly assess any modulation in amino acid levels in plants with a higher degree of confidence.

利用液相色谱-质谱(LC-MS)联用 MS-DIAL 对植物组织中的游离氨基酸进行快速定性分析
估算氨基酸的相对含量对于了解植物的各种生物过程(包括光合作用、抗逆性以及养分的吸收和转运)至关重要。目前有多种基于液相色谱(LC;HPLC/UHPLC)的方法可用于测量植物中氨基酸的数量。此外,液相色谱与质谱(MS)的结合大大提高了现有氨基酸定量色谱方法的稳定性。然而,对于分析质谱数据经验有限的植物生物学家来说,准确标注和整合质谱峰可能是一项挑战,尤其是在涉及多个处理和/或重复的大型数据集的研究中。此外,在某些情况下,实验需要对氨基酸谱进行整体观察,而不是关注绝对定量。本方案提供了一种详细的 LC-MS 方法,利用 MS-DIAL(一种处理 MS 数据的多功能且用户友好的程序)获得氨基酸的定性图谱。从对照和受番茄卷叶病毒(ToLCPalV)感染的烟草植物叶片中提取游离氨基酸。提取的氨基酸通过 UHPLC-QTOF 进行衍生和分离,然后将质量数据与 MS-DIAL 中创建的自定义文本库进行比对,从而鉴定出每种氨基酸。此外,还利用 MS-DIAL 进行基于内标的归一化,以获得对照植物和病毒感染植物中 15 种氨基酸的定性曲线。所概述的方法旨在简化 MS 数据的处理过程,从而以更高的可信度快速评估植物中氨基酸水平的任何变化。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Journal of Mass Spectrometry
Journal of Mass Spectrometry 化学-光谱学
CiteScore
5.10
自引率
0.00%
发文量
84
审稿时长
1.5 months
期刊介绍: The Journal of Mass Spectrometry publishes papers on a broad range of topics of interest to scientists working in both fundamental and applied areas involving the study of gaseous ions. The aim of JMS is to serve the scientific community with information provided and arranged to help senior investigators to better stay abreast of new discoveries and studies in their own field, to make them aware of events and developments in associated fields, and to provide students and newcomers the basic tools with which to learn fundamental and applied aspects of mass spectrometry.
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