Combining SDS-PAGE to capillary zone electrophoresis-tandem mass spectrometry for high-resolution top-down proteomics analysis of intact histone proteoforms

IF 3.4 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Proteomics Pub Date : 2024-07-17 DOI:10.1002/pmic.202300650
Fei Fang, Guangyao Gao, Qianyi Wang, Qianjie Wang, Liangliang Sun
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Abstract

Mass spectrometry (MS)-based top-down proteomics (TDP) analysis of histone proteoforms provides critical information about combinatorial post-translational modifications (PTMs), which is vital for pursuing a better understanding of epigenetic regulation of gene expression. It requires high-resolution separations of histone proteoforms before MS and tandem MS (MS/MS) analysis. In this work, for the first time, we combined SDS-PAGE-based protein fractionation (passively eluting proteins from polyacrylamide gels as intact species for mass spectrometry, PEPPI-MS) with capillary zone electrophoresis (CZE)-MS/MS for high-resolution characterization of histone proteoforms. We systematically studied the histone proteoform extraction from SDS-PAGE gel and follow-up cleanup as well as CZE-MS/MS, to determine an optimal procedure. The optimal procedure showed reproducible and high-resolution separation and characterization of histone proteoforms. SDS-PAGE separated histone proteins (H1, H2, H3, and H4) based on their molecular weight and CZE provided additional separations of proteoforms of each histone protein based on their electrophoretic mobility, which was affected by PTMs, for example, acetylation and phosphorylation. Using the technique, we identified over 200 histone proteoforms from a commercial calf thymus histone sample with good reproducibility. The orthogonal and high-resolution separations of SDS-PAGE and CZE made our technique attractive for the delineation of histone proteoforms extracted from complex biological systems.

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将 SDS-PAGE 与毛细管区带电泳-串联质谱相结合,对完整的组蛋白蛋白形式进行高分辨率自上而下的蛋白质组学分析。
基于质谱(MS)的组蛋白蛋白组学(TDP)分析提供了有关组合翻译后修饰(PTMs)的重要信息,这对于更好地了解基因表达的表观遗传调控至关重要。这需要在质谱和串联质谱(MS/MS)分析之前对组蛋白蛋白形式进行高分辨率分离。在这项工作中,我们首次将基于 SDS-PAGE 的蛋白质分馏(从聚丙烯酰胺凝胶中被动洗脱蛋白质作为完整的质谱物种,PEPPI-MS)与毛细管区带电泳(CZE)-MS/MS 结合起来,对组蛋白蛋白形式进行了高分辨率表征。我们系统地研究了从 SDS-PAGE 凝胶中提取组蛋白蛋白形式、后续净化以及 CZE-MS/MS,以确定最佳程序。最佳程序显示组蛋白蛋白形式的分离和表征具有可重复性和高分辨率。SDS-PAGE 根据分子量分离组蛋白(H1、H2、H3 和 H4),而 CZE 则根据电泳迁移率(受 PTMs(如乙酰化和磷酸化)的影响)对每种组蛋白的蛋白形态进行额外分离。利用这项技术,我们从商业化的小牛胸腺组蛋白样本中鉴定出了 200 多种组蛋白蛋白形式,而且重现性良好。SDS-PAGE 和 CZE 的正交和高分辨率分离技术使我们的技术在描述从复杂生物系统中提取的组蛋白蛋白形式时具有吸引力。
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来源期刊
Proteomics
Proteomics 生物-生化研究方法
CiteScore
6.30
自引率
5.90%
发文量
193
审稿时长
3 months
期刊介绍: PROTEOMICS is the premier international source for information on all aspects of applications and technologies, including software, in proteomics and other "omics". The journal includes but is not limited to proteomics, genomics, transcriptomics, metabolomics and lipidomics, and systems biology approaches. Papers describing novel applications of proteomics and integration of multi-omics data and approaches are especially welcome.
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