Proteomics studies play an increasing role in the field of biology. The use of mass spectrometry (MS) in combination with a range of separation methods is the main principal methodology for proteomics. The two principal approaches to identifying and characterizing proteins using MS are the “bottom-up”, which analyzes peptides by proteolytic digestion, and “top-down”, which analyzes intact proteins.
Figure 1. Divergent workflows in top-down and bottom-up proteomics. (Toby T K, et al.; 2016)
What Is Top Down Proteomics?
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.
Table 1. Comparison between bottom-up and top-down proteomics
|Bottom-up Proteomics||Top-down Proteomics|
|Detection of PTM||Yes||Yes|
|Analyzed sample forms||Peptide fragments||Full-length proteins|
|Detection efficiency||Relatively low||Significantly high|
|Strength||Well-developed for analyzing small peptides||Superior method for protein level sequence and PTM identification|
Top-down proteomics analyzes intact proteins with high-throughput quality. Whereas, in bottom-up proteomics, proteins are digested into peptide fragments before MS analysis, and therefore, has higher resolution. 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 (post-translational modification) 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 Proteomics at Creative Proteomics
At Creative Proteomics, we can provide top-down based services, including protein sequencing, PTMs characterization, and protein structure characterization.
With the advances in protein separation and mass spectrometry, top-down mass spectrometry moved from analyzing single proteins to analyzing complex samples and identifying hundreds and even thousands of proteins. Creative Proteomics can provide both N- and C-terminal sequencing services under top-down strategy, which is useful even when the termini are blocked by modifications.
In top down strategy, the protein is ionized directly, which makes it possible for improved sequence coverage and detection of PTMs. At Creative Proteomics, top-down mass spectrometry with ECD & ETD has been successfully applied to proteomics research, especially for mapping modifications with higher coverage, identifying unpredicted PTMs, and determining the order of multiple modifications.
The protein structure characterization can help us understand the functions of proteins. In addition, characterization of protein structure plays an important role in biologic development and ongoing quality control. Currently, MS is commonly used to determine both the primary and higher order structures of proteins. By the top-down strategy, due to no digestion of proteins before analysis, the information that is lost in bottom-up strategy can be maintained and detected, such as the quaternary structure with disulfide bonds.
Advantages of Top-down Proteomics
At Creative Proteomics, we have professional technology platforms to offer top down-based services, including but not limited to:
Based on experienced staff and advanced instruments, Creative Proteomics provides a wide range of top-down proteomics in a time-saving and cost-efficient manner. Our ordering procedure is as follows. If you have any questions or specific requirements, please feel free to contact us.
1. Toby T K, et al.; Progress in top-down proteomics and the analysis of proteoforms. Annual review of analytical chemistry, 2016, 9: 499-519.