title: A Comprehensive Guide for Performing Sample Preparation and Top-Down Protein Analysis.
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.
authors.affiliation: Proteomics Core Facility, University of Technology Sydney PO Box 123, Broadway 2007, Australia. matthew.padula@uts.edu.au.
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Padula
authors.affiliation: Proteomics Core Facility, University of Technology Sydney PO Box 123, Broadway 2007, Australia. iain.j.berry@student.uts.edu.au.|Infection, Immunity and Innovation Institute, University of Technology Sydney. PO Box 123, Broadway 2007, Australia. iain.j.berry@student.uts.edu.au.
authors.forename: Iain J
authors.initials: IJ
authors.lastname: Berry
authors.affiliation: Proteomics Core Facility, University of Technology Sydney PO Box 123, Broadway 2007, Australia. matthew.orourke@sydney.edu.au.|Mass Spectrometry Core Facility, Charles Perkins Centre, University of Sydney. The Hub D17, Sydney 2006, Australia. matthew.orourke@sydney.edu.au.
authors.forename: Matthew B
authors.initials: MB
authors.lastname: O Rourke
authors.affiliation: Proteomics Core Facility, University of Technology Sydney PO Box 123, Broadway 2007, Australia. benjamin.raymond@uts.edu.au.|Infection, Immunity and Innovation Institute, University of Technology Sydney. PO Box 123, Broadway 2007, Australia. benjamin.raymond@uts.edu.au.
authors.forename: Benjamin B A
authors.initials: BB
authors.lastname: Raymond
authors.affiliation: Proteomics Core Facility, University of Technology Sydney PO Box 123, Broadway 2007, Australia. jerran.santos@uts.edu.au.|Advanced Tissue Regeneration & Drug Delivery Group, University of Technology Sydney. PO Box 123, Broadway 2007, Australia. jerran.santos@uts.edu.au.
authors.forename: Jerran
authors.initials: J
authors.lastname: Santos
authors.affiliation: Proteomics Core Facility, University of Technology Sydney PO Box 123, Broadway 2007, Australia. steven.djordjevic@uts.edu.au.|Infection, Immunity and Innovation Institute, University of Technology Sydney. PO Box 123, Broadway 2007, Australia. steven.djordjevic@uts.edu.au.
authors.forename: Steven P
authors.initials: SP
authors.lastname: Djordjevic
keywords: Bottom-up; Chromatography; Electrophoresis; Isoelectric focusing; Mass spectrometry; Proteoform; Proteomics; Top-Down