基于酰胺化的化学标记技术在磷酸化多肽分析中的应用

邹伦妃; 张启伟; 肖琛; 熊淼; 郑琦

doi:10.7538/zpxb.2021.0202

基于酰胺化的化学标记技术在磷酸化多肽分析中的应用

江汉大学光电化学材料与器件教育部重点实验室,湖北武汉430054

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出版历程
- 刊出日期: 2022-07-19

Chemical Labeling Method Based on Amidation for the Analysis of Phosphopeptides

Key Laboratory of Optoelectronic Chemical Materials and Devices of Ministry of Education, Jianghan University, Wuhan 430054, China

摘要

摘要: 蛋白质的翻译后修饰复杂多样，通过激酶与磷酸酶调控的磷酸化是最广泛、最重要的翻译后修饰之一。定量分析磷酸化蛋白质的动态变化，有利于阐明磷酸化修饰调控生命活动的机理，了解其在实现生物功能过程中的作用。以质谱为核心的分析方法是当前蛋白质翻译后修饰研究中最常用的定量技术，主要包括无标记定量、内标绝对定量、化学标记等。本文将一种用于蛋白质糖基化分析的标记方法应用于磷酸化多肽的研究。使用二甲胺标记蛋白质的所有酶解肽段，被酰胺化的羧基可提高TiO₂对磷酸化多肽的富集特异性以及液相色谱-质谱（LC-MS）检测的灵敏度。稳定同位素标记实验表明，该方法适用于高通量磷酸化多肽的相对定量分析。基于二甲胺衍生的化学标记法在磷酸化多肽的质谱分析中具有优势，有望成为生物组织中磷酸化蛋白质研究的有力工具。
- 磷酸化 /
- 酰胺化 /
- 稳定同位素标记 /
- 液相色谱-质谱（LC-MS）
Abstract: Post-translational modification of proteins is complex and diverse, with the phosphorylation regulated by kinases and phosphatases being one of the most widespread and important modifications. Dynamic change of quantitative analysis of phosphorylated proteins is helpful to clarify the mechanism of phosphorylation regulating life activities and understand its role in achieving biological functions. Mass spectrometry is the most commonly used strategy for the quantitative analysis of post-translational modification, involving label-free quantification, internal standard absolute quantification, and chemical labeling, etc. In this paper, a chemical labeling approach which has been used for protein glycosylation profiling was applied to the relative quantitative analysis of phosphopeptides. Using the method, carboxyl groups were located on the side chain of aspartic acid and glutamic acid residues, and the C-terminus of peptide were allowed to react with dimethylamine (d0-DMA)/deuterated dimethylamine (d6-DMA). As a result, the carboxyl groups were neutralized, and all enzymatic peptides were labeled with light/heavy tags. After the samples with different tags were mixed, the phosphopeptides were enriched using TiO₂ magnetic particles and then analyzed qualitatively and relatively quantitatively by liquid chromatography-mass spectrometry (LC-MS). Amidation could shield the negatively charged carboxyl groups of non-phosphorylated and phosphorylated peptides, so that the peptides with phosphate groups mainly exhibited negative charge property, thus improving the enrichment specificity of TiO₂ for phosphopeptides as well as its detection sensitivity in LC-MS analysis. Using 0.2 mg casein as a standard, 8 phosphopeptides were detected in the derivatized samples. A high abundance of b and y ions indicated that the amidation by dimethylamine was highly specific and usually occurs only at the carboxyl group without a significant effect on the phosphate group of peptides. A series of molar ratios (5∶1, 2∶1, 1∶1, 1∶2, 1∶5) of samples were derivatized using d0-DMA and d6-DMA, respectively. The results showed that the error was less than 20% for each ratio, indicating that the method possessed good relative quantitative range and accuracy. However, too many derivative sites in the peptides could result in incomplete derivatization, and the C-terminal lysine residues might self-condense to form lactam during the derivatization process. By selecting some individualized proteases during protein digestion, such as GluC, the number of derivative sites might be in an appropriate range and lysine residue could be avoided at the terminal of peptides, which should be useful for improving identification efficiency and quantitative accuracy of phosphopeptides. In summary, the chemical labeling method based on dimethylamine derivatization presents advantages for high-throughput relative quantitative analysis of phosphopeptides and it is expected to be a powerful tool for protein phosphorylation profiling in biological tissues.
- phosphorylation /
- amidation /
- stable isotope labeling /
- liquid chromatography-mass spectrometry (LC-MS)

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