Proteomics has been considered a comprehensive technique to decipher biological questions. The methodology regards to a range of separation methods including mass spectrometry(MS) The two principal approaches for protein identification and characterization via MS can be categorized as “bottom-up”, which analyzing peptides by proteolytic digestion, and “top-down”, namely global analysis of intact proteins by fragmentation.
Figure 1. Divergent workflows in top-down and bottom-up proteomics. (Toby T K, et al.; 2016)
What Is Top Down Proteomics?
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 need to be digested into peptide fragments before MS analysis, Top-down proteomics involves separating intact proteins from complex biological samples using conventional separation techniques such as liquid chromatography or 2-D gel electrophoresis, followed by MS analysis. Both electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI) considered as soft ionization method to generate ion molecules following with fragmentation via diverse dissociation methods, likely higher energy collision introduced dissociation (HCD), electron-capture dissociation (ECD) and electron-transfer dissociation (ETD), through tandem mass spectrometry, As a promising alternative strategy for protein identification, profiling, sequencing and PTM (post-translational modification) characterization, 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, an universal characterization of modification sites and patterns can be achieved simultaneously in one spectrum with their supposed related correlations between these determined modifications. Meanwhile, protein digestion process elimination reduces time consumption during the experiment in contrast to bottom-up approach.
Top Down Proteomics at Creative Proteomics
Based on Creative Proteomics platform, we are capable of providing top-down proteomics services, including protein sequencing, PTMs characterization, and protein structure characterization.
Top-down MS maintains both single proteins analysis and protein complex characterization. Creative Proteomics supports both N- and C-terminal sequencing services under top-down strategy, particularly when the termini of your protein samples are blocked by modifications.
At Creative Proteomics, top-down mass spectrometry with ECD & ETD has been successfully applied to proteomics research, especially for sequence coverage mapping, identifying unpredicted PTMs identification, multiple modifications determination.
The protein structure characterization assist to a better understanding of protein fucntion. In addition, it 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. For instances, the quaternary structure with disulfide bonds can be maintained and detected via top-down approach due to no digestion process has been applied under the native state of the protein sample.
Advantages of Top-down Proteomics
- Relatively simple sample preparation
- No protein digestion needed
- Direct detection of the molecular mass of biological proteins
- Mass information is retained in a great extent, such as PTMs
- Less time-consuming
- Liquid Chromatography (LC)
- High Performance Liquid Chromatography (HPLC)
- Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS)
Based on experienced staff and advanced instruments, Creative Proteomics provides a wide range of top-down proteomics with reasonable price in a time-saving manner. Our ordering procedure describles 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.