Top-Down Proteomics Service

Proteomics is considered a comprehensive technology for addressing biological problems and involves a range of technical methods, including mass spectrometry (MS). Two main approaches for protein identification and characterization by MS can be classified as "bottom-up," analyzing peptides through protein hydrolysis, and "top-down,"  a global analysis of intact proteins by fragmenting them.

 



Toby T K, et al. Annu Rev Anal Chem (Palo Alto Calif). 2016.

Figure 1

 

In bottom-up proteomics, proteins need to be digested into peptides before MS analysis, whereas in top-down proteomics, intact proteins are separated from complex biological samples through separation (e.g., liquid chromatography (LC) or 2-dimensional gel electrophoresis (2DGel)) and then analyzed by MS. By soft ionization methods such as electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI), ions and molecules are generated after fragmentation through multiple dissociation methods, such as higher-energy collision dissociation (HCD), electron-capture dissociation (ECD), and electron-transfer dissociation (ETD), followed by mass spectrometric analysis. Bottom-up proteomics analysis is a highly promising strategy for protein identification, analysis, sequencing, and post-translational modification (PTM). The top-down approach allows MS analysis of intact proteins without enzymatic digestion, meaning that it retains features of proteins with unstable structures that are mostly destroyed in the bottom-up strategy. Therefore, data on modification sites and modification patterns can be obtained simultaneously in a single spectral analysis to yield relational data between these modification sites and patterns. Additionally, compared with the bottom-up approach, the digestion-free protein reduces time consumption in experimental processes.

 



Table 1. Top-Down Proteomics 2

 

MtoZ Biolabs uses Thermo Fisher's Orbitrap Fusion Lumos mass spectrometer platform combined with nanoLC-MS/MS nanoscale chromatography to provide top-down proteomics-based services, including protein sequencing, PTM characterization, and protein structural characterization.

 

1. Top-Down Sequencing

Top-down MS can perform single protein analysis as well as protein complex characterization. Under the top-down strategy, MtoZ Biolabs supports both N- and C-terminal sequencing services, especially when protein sample terminals are modified and capped.

 

2. Top-Down PTM Characterization

Top-down MS analysis has been successfully applied to proteomics research with ECD and ETD, particularly in sequence coverage mapping, identification of unpredicted PTMs, and determination of multiple modifications.

 

3. Protein Structural Characterization

Protein structural characterization helps better understand protein function. Moreover, it plays an important role in biological development and quality control. Currently, MS is often used to determine the primary and advanced structures of proteins. For example, since no digestion process is applied to protein samples, the top-down approach can maintain and detect quaternary structure of proteins with disulfide bonds.

 

Service Advantages

1. Relatively Simple Sample Preparation

2. No Need for Protein Digestion

3. Direct Detection of Molecular Weight of Biological Proteins

4. Extensive Information to Retention, Such as PTMs

5. Reduction in Time Consumption