Using peptide barcodes for simultaneous profiling of protein expression from mRNA

IF 1.8 3区化学 Q4 BIOCHEMICAL RESEARCH METHODS

Rapid Communications in Mass Spectrometry Pub Date : 2024-07-07 DOI:10.1002/rcm.9867

Shun Kumano, Kazuki Tanaka, Rena Akahori, Akiko Yanagiya, Akihiro Nojima

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引用次数: 0

Abstract

Rationale

mRNA technology has begun to play a significant role in the areas of therapeutic intervention and vaccine development. However, optimizing the mRNA sequence that influences protein expression levels is a resource-intensive and time-consuming process. This study introduces a new method to accelerate the selection of sequences of mRNA for optimal protein expression.

Methods

We designed the mRNA sequences in such a way that a unique peptide barcode, corresponding to each mRNA sequence, is attached to the expressed protein. These barcodes, cleaved off by a protease and simultaneously quantified by mass spectrometry, reflect the protein expression, enabling a parallel analysis. We validated this method using two mRNAs, each with different untranslated regions (UTRs) but encoding enhanced green fluorescence protein (eGFP), and investigated whether the peptide barcodes could analyze the differential eGFP expression levels.

Results

The fluorescence intensity of eGFP, a marker of its expression level, has shown noticeable changes between the two UTR sequences in mRNA-transfected cells when measured using flow cytometry. This suggests alterations in the expression level of eGFP due to the influence of different UTR sequences. Furthermore, the quantified amount of peptide barcodes that were released from eGFP showed consistent patterns with these changes.

Conclusions

The experimental findings suggest that peptide barcodes serve as a valuable tool for assessing protein expression levels. The process of mRNA sequence selection, aimed at maximizing protein expression, can be enhanced by the parallel analysis of peptide barcodes using mass spectrometry.

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使用肽条形码同步分析 mRNA 蛋白表达。

理由：mRNA 技术已开始在治疗干预和疫苗开发领域发挥重要作用。然而，优化影响蛋白质表达水平的 mRNA 序列是一个资源密集且耗时的过程。本研究介绍了一种新方法，可加快选择最佳蛋白质表达的 mRNA 序列：我们在设计 mRNA 序列时，将与每个 mRNA 序列相对应的独特肽条形码附在表达的蛋白质上。这些条形码经蛋白酶裂解后，同时用质谱仪定量，从而反映蛋白质的表达情况，实现平行分析。我们使用两种mRNA验证了这种方法，每种mRNA都有不同的非翻译区（UTR），但都编码增强型绿色荧光蛋白（eGFP），并研究了肽条形码是否能分析不同的eGFP表达水平：结果：在使用流式细胞仪测量经 mRNA 转染的细胞中 eGFP 的荧光强度（其表达水平的标记）时，两个 UTR 序列之间出现了明显的变化。这表明 eGFP 的表达水平受到不同 UTR 序列的影响而发生了变化。此外，从 eGFP 中释放的肽条形码的量化量也显示出与这些变化一致的模式：实验结果表明，肽条形码是评估蛋白质表达水平的重要工具。利用质谱对肽条形码进行平行分析，可以加强旨在最大限度提高蛋白质表达量的 mRNA 序列选择过程。

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

Rapid Communications in Mass Spectrometry 化学-分析化学

CiteScore

4.10

自引率

5.00%

发文量

219

审稿时长

2.6 months

期刊介绍： Rapid Communications in Mass Spectrometry is a journal whose aim is the rapid publication of original research results and ideas on all aspects of the science of gas-phase ions; it covers all the associated scientific disciplines. There is no formal limit on paper length ("rapid" is not synonymous with "brief"), but papers should be of a length that is commensurate with the importance and complexity of the results being reported. Contributions may be theoretical or practical in nature; they may deal with methods, techniques and applications, or with the interpretation of results; they may cover any area in science that depends directly on measurements made upon gaseous ions or that is associated with such measurements.