Abstract
Methodologies for the global analysis of proteins in a sample, or proteome analysis, have been available since 1975 when Patrick O′Farrell published the first paper describing two-dimensional gel electrophoresis (2D-PAGE). This technique allowed the resolution of single protein isoforms, or proteoforms, into single ‘spots’ in a polyacrylamide gel, allowing the quantitation of changes in a proteoform′s abundance to ascertain changes in an organism′s phenotype when conditions change. In pursuit of the comprehensive profiling of the proteome, significant advances in technology have made the identification and quantitation of intact proteoforms from complex mixtures of proteins more routine, allowing analysis of the proteome from the ‘Top-Down’. However, the number of proteoforms detected by Top-Down methodologies such as 2D-PAGE or mass spectrometry has not significantly increased since O’Farrell’s paper when compared to Bottom-Up, peptide-centric techniques. This article explores and explains the numerous methodologies and technologies available to analyse the proteome from the Top-Down with a strong emphasis on the necessity to analyse intact proteoforms as a better indicator of changes in biology and phenotype. We arrive at the conclusion that the complete and comprehensive profiling of an organism′s proteome is still, at present, beyond our reach but the continuing evolution of protein fractionation techniques and mass spectrometry brings comprehensive Top-Down proteome profiling closer.
Highlights
The mere mention of the phrase ‘Top-Down Proteomics’ is likely to incite some strong and varied opinions from proteomics researchers
It is our firm belief that both approaches can be considered Top-Down proteomics and this review aims to define the current employed methodologies for the system-wide analysis and quantitation of intact proteoforms rather than analysis and quantitation through inferring the presence of a protein through its peptides
Despite significant advances in the sensitivity and speed attained by modern mass spectrometers, advances in the study of proteoforms and protein complexes remains in its infancy
Summary
O0 Rourke 1,3 , Benjamin B.A. Raymond 1,2 , Jerran Santos 1,4 and Steven P. Immunity and Innovation Institute, University of Technology Sydney, PO Box 123, Broadway, Ultimo NSW 2007, Australia. Mass Spectrometry Core Facility, Charles Perkins Centre, University of Sydney, The Hub D17, Sydney, Camperdown NSW 2006, Australia. Advanced Tissue Regeneration & Drug Delivery Group, University of Technology Sydney, PO Box 123, Broadway, Ultimo NSW 2007, Australia
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
