Quantitation using 2D-PAGE is then able to be carried out prior to identification through the densitometric analysis of the gel. At first glance this may seem odd but it revolves around performing proteome analysis by fractionating intact proteoforms until single proteoforms are isolated, a workflow routinely performed in 2D-PAGE fractionation but also able to be achieved through multiple, orthogonal methods of chromatography.

Here, we define Top-Down as the quantitation of an intact proteoform before its identification. Shotgun methods require that a peptide be identified and assigned to a gene product, the amino acid sequence that is bioinformatically determined from a gene, before quantitation is performed using a limited number of peptides to infer the abundance of a gene product rather than a proteoform. The definition of Top-Down proteomics that we will adopt here places more weight on the quantitation of intact proteoforms and less on the need to identify the proteoform as an intact molecule, allowing the isolated proteoforms identification through its peptides. However, this use of the term ‘Top-Down’ ignores a large number of techniques that also allow the analysis of a proteome from intact proteoforms. Top-Down was coined by those seeking to identify intact proteins using a mass spectrometer, performing no enzymatic digestion events prior to introducing the proteoform molecules into the mass spectrometer. The term Top-Down came about to distinguish protein identification using intact protein isoforms, or proteoforms, from the very widely used term ‘Bottom-Up’, that is used to describe the analysis of a proteome by first enzymatically digesting all of the proteins into peptides and subjecting them to a ‘shotgun’ analysis (a term first used in DNA sequencing ). This is mainly due to the lack of a precise definition of what the ‘Top-Down’ analysis of a protein actually means. The mere mention of the phrase ‘Top-Down Proteomics’ is likely to incite some strong and varied opinions from proteomics researchers. 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. 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.

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. 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’. 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. 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).

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