In the past decade, scientific studies on protein profiling have grown exponentially. This is directly linked to the rapid development of protein profiling technologies with the field expanding dramatically. The main goal of protein profiling study is aimed at discovery of protein biomarkers, whose expression level is highly associated with disease occurrence and progress. These biomarkers are being extensively investigated to search for the therapy of diseases, in terms of prognosis and diagnosis of disease.
Currently, there are two major types of approaches to protein biomarker discovery: top-down and bottom-up proteomics. Top-down proteomics analyzes intact proteins and has high-throughput quality. Whereas, in bottom-up proteomics, proteins are digested into peptide fragments before MS analysis, therefore, has higher resolution.
Comparison between bottom-up and top-down proteomics:
Top-down proteomics involves separating intact proteins from complex biological samples using traditional separation techniques such as liquid chromatography or 2-D gel electrophoresis, followed by mass spectrometry to analyze intact proteins. Both electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI) can be used to generate ions and then fragment ions with collision introduced dissociation (CID), higher energy collision introduced dissociation (HCD), electron-capture dissociation (ECD) or electron-transfer dissociation (ETD) and analyze with tandem mass spectrometry, making it a promising alternative strategy for protein identification, profiling, sequencing and PTM characterization. Moreover, the top-down approach allows MS analysis of intact proteins that have not been cleaved, meaning the labile structural protein characteristics that are mostly destroyed in bottom-up MS are preserved. Hence, universal detection of all existing modifications can be achieved simultaneously in one spectrum and any existing correlations between those modifications can also be determined. In the top-down approach, elimination of protein digestion also results in significant time saving.
Top-down mass spectrometry, as an emerging approach for characterization of intact proteins, is established as a contrast to the bottom-up strategy, which is also called shotgun method, for analysis of peptides from enzymatic or chemical cleavage of intact proteins. Currently the bottom-up strategy is the most mature and most widely used approach for protein identification, characterization of post-translational modifications, and even relative and absolute quantification. But meanwhile, the application of the bottom-up strategy is limited by its inherent shortages.
Strength of top-down proteomics
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