Journal
ELECTROPHORESIS
Volume 44, Issue 3-4, Pages 472-480Publisher
WILEY
DOI: 10.1002/elps.202200257
Keywords
IPG strips; isoelectric focusing; proteoforms; two-dimensional gel electrophoresis
Ask authors/readers for more resources
Integrative top-down proteomics aims to comprehensively analyze the breadth and depth of proteomes. This study disproves long-held beliefs about protein losses during front-end proteome resolution and confirms that standard facedown rehydration method has minimal impact on overall proteome resolution. This research demonstrates the importance of quantitative front-end 2DE/LC/MS/MS in analyzing proteomes by resolving their constituent proteoforms.
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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available