Highly accurate and precise quantification strategy using stable isotope dimethyl labeling coupled with GeLC-MS/MS
Highly accurate and precise quantification strategy using stable isotope dimethyl labeling coupled with GeLC-MS/MS
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DOI：
10.1016/j.bbrc.2021.02.101
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发表时间：
2021-03-05
影响因子：
3.1
通讯作者：
Kodera, Yoshio
中科院分区：
文献类型：
--
作者：
Konno, Ryo;Matsui, Takashi;Kodera, Yoshio
鸟枪蛋白质组学是一种全面鉴定和定量胰蛋白酶多肽的有力方法，但对蛋白水解事件的分析比较困难。一维凝胶和液相色谱-串联质谱法（GeLC-MS/MS）能够使用SDS-PAGE分离蛋白水解片段，然后使用LC-MS/MS进行鉴定。因此，GeLC-MS/MS是鉴定片段的极好方法。然而，凝胶提取和纳米流LC-MS/MS的较低重现性可能会在样品之间的蛋白质定量比较分析中产生不准确的结果。本研究开发了一种新型的GeLC-MS/MS方法，结合稳定同位素二甲基标记。在该方法中，将轻标记和重标记样品的混合物加载到SDS-PAGE凝胶上，并通过一次性注射定量分析一个提取条带中具有不同同位素的蛋白质。即使在低肽丰度的情况下，该方法也能够准确测定两个样品之间肽的丰度比，并且不受凝胶提取或LC-MS方法的再现性的影响。因此，我们的新GeLC-MS/MS方法结合稳定同位素二甲基标记，提供了高准确性和全面的肽比较，能够检测由疾病或生理过程引起的蛋白水解事件。(c)2021作者爱思唯尔公司出版这是一个在CC BY-NC-ND许可证下的开放获取文章（http：//creativecommons.org/licenses/by-nc-nd/4.0/）。
Shotgun proteomics is a powerful method for comprehensively identifying and quantifying tryptic peptides, but it is difficult to analyze proteolytic events. One-dimensional gel and liquid chromatography-tandem mass spectrometry (GeLC-MS/MS) enables the separation of proteolytic fragments using SDS-PAGE followed by identification using LC-MS/MS. GeLC-MS/MS is thus an excellent method for identifying fragmentation. However, the lower reproducibility of gel extraction and nano flow LC-MS/MS can produce inaccurate results in comparative analyses of protein quantification among samples. In this study, a novel GeLC-MS/MS method coupled with stable isotope dimethyl labeling was developed. In the method, a mixture of light-and heavy-labeled samples is loaded onto an SDS-PAGE gel, and proteins with different isotopes in one extracted band are quantitatively analyzed by one-shot injection. This procedure enables accurate determination of the abundance ratio of peptides between two samples, even in cases of low peptide abundance, and it is not affected by the reproducibility of the gel extraction or LC-MS procedures. Therefore, our new GeLC-MS/MS method coupled with stable isotope dimethyl labeling provides high accuracy and comprehensive peptide comparisons, enabling the detection of proteolysis events caused by disease or physiological processes.(c) 2021 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).