The goal of integrative top–down proteomics (i.e., two-dimensional gel electrophoresis [2DE] coupled with liquid chromatography and tandem mass spectrometry [LC/MS/MS]) is a routine analytical approach that fully addresses the breadth and depth of proteomes. To accomplish this, there should be no addition, removal, or modification to any constituent proteoforms. To address two-decade old claims of protein losses during front–end proteome resolution using 2DE, here we tested an alternate rehydration method for immobilized pH gradient strips prior to isoelectric focusing (IEF; i.e., faceup compared to facedown) and quantitatively assessed losses during the front–end of 2DE (rehydration and IEF). Using a well-established high-resolution, quantitative 2DE protocol, there were no detectable proteoform losses using the alternate faceup rehydration method. Although there is a <0.25% total loss of proteoforms during standard facedown rehydration, it is insignificant in terms of having any effect on overall proteome resolution (i.e., total spot count and total spot signal). This report is another milestone in integrative top–down proteomics, disproving long-held dogma in the field and confirming that quantitative front–end 2DE/LC/MS/MS is currently the only method to broadly and deeply analyze proteomes by resolving their constituent proteoforms.

中文翻译：

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定量评估通过自上而下的综合蛋白质组学证实了深度蛋白质组分析

综合自上而下蛋白质组学（即二维凝胶电泳 [2DE] 与液相色谱和串联质谱 [LC/MS/MS] 相结合）的目标是一种常规分析方法，可充分解决蛋白质组的广度和深度问题。为实现这一点，不应添加、删除或修改任何组成蛋白。为了解决使用 2DE 进行前端蛋白质组解析期间蛋白质损失的说法已有 20 年之久，我们在这里测试了等电聚焦（IEF；即面朝上与面朝下相比）之前固定化 pH 梯度条的替代补液方法，并定量评估了过程中的损失2DE 的前端（再水化和 IEF）。使用完善的高分辨率、定量 2DE 协议，使用替代面朝上补液方法没有可检测到的蛋白质损失。尽管在标准面朝下补液过程中蛋白质组的总损失小于 0.25%，但它对整体蛋白质组分辨率（即总斑点数和总斑点信号）的影响微不足道。这份报告是自上而下的综合蛋白质组学的又一里程碑，它推翻了该领域长期以来的教条，并证实定量前端 2DE/LC/MS/MS 是目前唯一通过解析其组成蛋白质组来广泛深入分析蛋白质组的方法.