Carbonara Katrina, Padula Matthew P, Coorssen Jens R
Department of Health Sciences, Faculty of Applied Health Sciences, Brock University, St. Catharines, Ontario, Canada.
School of Life Sciences and Proteomics, Lipidomics and Metabolomics Core Facility, Faculty of Science, University of Technology Sydney, Sydney, New South Wales, Australia.
Electrophoresis. 2023 Feb;44(3-4):472-480. doi: 10.1002/elps.202200257. Epub 2022 Dec 7.
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.
整合式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]联用）的目标是一种常规分析方法，可全面涵盖蛋白质组的广度和深度。要实现这一目标，不应对任何组成蛋白质异构体进行添加、去除或修饰。为回应二十年来关于在使用2DE进行前端蛋白质组分离过程中蛋白质损失的说法，我们在此测试了等电聚焦（IEF）之前用于固定pH梯度条带的另一种水化方法（即面朝上与面朝下相比），并定量评估了2DE前端（水化和IEF）过程中的损失。使用成熟的高分辨率定量2DE方案，采用另一种面朝上水化方法未检测到蛋白质异构体损失。尽管在标准面朝下水化过程中蛋白质异构体的总损失<0.25%，但就对整体蛋白质组分辨率（即总斑点数和总斑点信号）产生任何影响而言，这并不显著。本报告是整合式自上而下蛋白质组学的又一个里程碑，推翻了该领域长期以来的教条，并证实定量前端2DE/LC/MS/MS目前是通过解析其组成蛋白质异构体来广泛而深入分析蛋白质组的唯一方法。
