Integrated Top-Down Proteomics for Comprehensive Proteome Analysis at the Proteoform Level.
- 1School of Life Sciences, Faculty of Science, University of Technology Sydney, Ultimo, NSW 2007, Australia.
- 2Institute for Globally Distributed Open Research and Education (IGDORE), St. Catharines, ON L2N 4X2, Canada.
Abstract
Background
Proteoforms are the critical functional species that drive biological processes. However, conventional proteogenomic ("shotgun") analyses discard relevant proteoform-level information through bulk protein digestion. This study presents a proof-of-principle integrative Top-Down Proteomics workflow designed to identify and characterise proteoforms across the breadth of a proteome.Methods
This workflow combined two-dimensional electrophoresis, a custom grid-cutting lattice for reproducible gel sectioning, in-gel digestion, liquid chromatography-tandem mass spectrometry, and open-search algorithms. Gel-grid locations, isoelectric points, and molecular weights were integrated with mass-spectrometry results to further characterise identified proteoforms.Results
The approach enabled identification of a broad range of proteoforms and their inherent post-translational modifications, including phosphorylation, acetylation, methylation, and glycosylation. Notably, substantial numbers of proteoforms were identified in visually "blank regions" of replicate two-dimensional gels. This eliminated bias associated with assessing only visually detectable protein spots and substantially expanded proteome-level proteoform detection and characterisation.Conclusions
This integrative workflow provides a reproducible and comprehensive strategy for addressing the often-overlooked complexity of proteomes at the proteoform level. By extending analysis beyond visually detectable gel spots, it enables deeper characterisation of proteoform diversity and associated post-translational modifications.
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BibTeX
@article{farrell2026integrated,
title = {{Integrated Top-Down Proteomics for Comprehensive Proteome Analysis at the Proteoform Level.}},
author = {Luke A Farrell and Breyer Woodland and Aiden Brennan and Jens R Coorssen and Matthew B O'Rourke and Matthew P Padula},
journal = {Proteomes},
year = {2026},
month = oct,
doi = {10.3390/proteomes14040053},
url = {https://europepmc.org/article/MED/42924751},
note = {CC BY},
}
RIS
TY - JOUR
TI - Integrated Top-Down Proteomics for Comprehensive Proteome Analysis at the Proteoform Level.
AU - Luke A Farrell
AU - Breyer Woodland
AU - Aiden Brennan
AU - Jens R Coorssen
AU - Matthew B O'Rourke
AU - Matthew P Padula
PY - 2026
DA - 2026/10/09
JO - Proteomes
DO - 10.3390/proteomes14040053
UR - https://europepmc.org/article/MED/42924751
AB - Background Proteoforms are the critical functional species that drive biological processes. However, conventional proteogenomic ("shotgun") analyses discard relevant proteoform-level information through bulk protein digestion. This study presents a proof-of-principle integrative Top-Down Proteomics workflow designed to identify and characterise proteoforms across the breadth of a proteome.Methods This workflow combined two-dimensional electrophoresis, a custom grid-cutting lattice for reproducible gel sectioning, in-gel digestion, liquid chromatography-tandem mass spectrometry, and open-search algorithms. Gel-grid locations, isoelectric points, and molecular weights were integrated with mass-spectrometry results to further characterise identified proteoforms.Results The approach enabled identification of a broad range of proteoforms and their inherent post-translational modifications, including phosphorylation, acetylation, methylation, and glycosylation. Notably, substantial numbers of proteoforms were identified in visually "blank regions" of replicate two-dimensional gels. This eliminated bias associated with assessing only visually detectable protein spots and substantially expanded proteome-level proteoform detection and characterisation.Conclusions This integrative workflow provides a reproducible and comprehensive strategy for addressing the often-overlooked complexity of proteomes at the proteoform level. By extending analysis beyond visually detectable gel spots, it enables deeper characterisation of proteoform diversity and associated post-translational modifications.
SN - 2227-7382
ER -