通过结合 SDS-PAGE、全凝胶切片、定量 LC-MS/MS 和数千种蛋白质的凝胶分布重建，进行蛋白质组相关蛋白质组学分析的 1DE-MS 分析,Journal of Proteome Research

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通过结合 SDS-PAGE、全凝胶切片、定量 LC-MS/MS 和数千种蛋白质的凝胶分布重建，进行蛋白质组相关蛋白质组学分析的 1DE-MS 分析
Journal of Proteome Research ( IF 3.8 ) Pub Date : 2022-08-26 , DOI: 10.1021/acs.jproteome.2c00180
Changming Chen ₁ , Meiling Wen ₁ , Ya Jin ₁

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SDS-PAGE 通常用于蛋白质组学分析，但通常用于样品预分离，尽管该技术按分子质量 ( M _w s) 分离蛋白质，并且该信息将有助于蛋白质组水平分析。在这里，我们报告了一种结合 SDS-PAGE、全凝胶切片和定量 LC-MS/MS 的方法，用于建立蛋白质组中数千种蛋白质的凝胶分布。先前获得的脑缺血再灌注损伤大鼠大脑皮层数据集¹进行了分析，并重建了 5906 种蛋白质的凝胶分布。这些分布被称为 1DE-MS 图，显示大约 30% 的蛋白质在凝胶中检测到不止一种蛋白质形式。根据分布（窄、分散或宽）和丰度峰表观Mw s_与计算的_Mws之间的相对偏差，这些图谱分为六种类型。只有 56% 的蛋白质表现出窄分布并_匹配Mws，而其他人则有相当复杂的个人资料。对示例概况的生物信息学分析表明，解析的蛋白质形式涉及可变剪接、蛋白水解加工、糖基化和泛素化、片段化，可能还包括跨膜结构。基于配置文件的差异分析表明，许多疾病引起的变化都依赖于蛋白质。这项工作提供了 SDS-PAGE 凝胶中蛋白质分布的蛋白质组尺度视图，该方法可用于获取蛋白质组学相关信息以进行深入的蛋白质组学研究。

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1DE-MS Profiling for Proteoform-Correlated Proteomic Analysis, by Combining SDS-PAGE, Whole-Gel Slicing, Quantitative LC–MS/MS, and Reconstruction of Gel Distributions of Several Thousands of Proteins

SDS-PAGE has often been used in proteomic analysis, but generally for sample prefractionation although the technique separates proteins by molecular masses (M_ws) and the information would contribute to proteoform-level analysis. Here, we report a method that combines SDS-PAGE, whole-gel slicing, and quantitative LC–MS/MS for establishing gel distributions of several thousand proteins in a proteome. A previously obtained data set on rat cerebral cortex with cerebral ischemia-reperfusion injury¹ was analyzed, and the gel distributions of 5906 proteins were reconstructed. These distributions, referred to as 1DE-MS profiles, revealed that about 30% of the proteins had more than one proteoform detected in the gels. The profiles were categorized into six types by distribution (narrow, dispersed, or broad) and relative deviations between the abundance-peak apparent M_ws and calculated M_ws. Only 56% of the proteins showed narrow distributions and matched M_ws, while the others had rather complex profiles. Bioinformatic analysis on example profiles showed the resolved proteoforms involved alternative splicing, proteolytic processing, glycosylation and ubiquitination, fragmentation, and probably transmembrane structures. Profile-based differential analysis revealed that many of the disease-caused changes were proteoform dependent. This work provided a proteome-scale view of protein distributions in SDS-PAGE gels, and the method would be useful to obtain proteoform-correlated information for in-depth proteomics.

更新日期：2022-08-26

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