The spike protein of the SARS-CoV-2 virus is a very commonly researched target in COVID-19 vaccine and therapeutic studies because it is an integral part of host cell entry through interactions between the S1 subunit of the spike protein with the ACE2 protein on the target cell surface. Viral proteins important in host cell entry are typically highly glycosylated. Looking at the sequence of the SARS-CoV-2 virus, researchers predict that the spike protein is highly glycosylated. In a recent study, researchers conducted a glycosylation analysis of SARS-CoV-2 proteins using mass spec analysis to determine the N-glycosylation profile of the subunits that make up the spike protein.
Glycans assist in protein folding and help the virus avoid immune recognition by the host. Glycosylation can also have an impact on the antigenicity of the virus, as well as potential effects on vaccine safety and efficacy. Mass spectrometry is widely used for viral characterization studies of influenza viruses. Specifically, mass spec has been used to study influenza protein glycosylation, antigen quantification, and determination of vaccine potency.
The use of mass spectrometry for the characterization of individual or complex protein samples continues to be one of the fastest growing fields in the life science market.
Bottom-up proteomics is the traditional approach to address these questions. Optimization of each the individual steps (e.g. sample prep, digestion and instrument performance) is critical to the overall success of the entire experiment.
To address issues that may arise in your experimental design, Promega has developed unique tools and complementary webinars to help you along the way.
Here you can find a summary of individual webinars for the following topics:
Owing to efficient proteolysis and particular advantages of trypsin-generated peptides for mass spectrometry analysis, trypsin is the most widely used proteomic protease. Recently, however, Lys-C has been increasingly used as either a trypsin alternate or as supplement. Its increasing favor is largely due to its ability to perform proteolytic digestion under protein denaturing conditions, an attribute that can greatly extend the observable proteome.
Lys-C is found in number of bacterial hosts with Lysobacterenzymogenes being used as a most popular source of commercially available Lys-C. We have now developed a recombinant form of Lys-C from Pseudomonas aeruginosa. We have compared performance of Pseudomonas and Lysobacter Lys-C.
Surprisingly, we found difference between Pseudomonas and Lysobacter Lys-C proteases on peptide level. The peptides generated by the proteases had much smaller overlap (25%) than typically observed between runs for the same sample indicating different bias toward lysine cleavage sites for Pseudomonas and Lysobacter Lys-C.
The proteases might have different proteolytic mechanisms. In fact, difference in proteolytic mechanisms is not unexpected considering the limited homology between these two proteases
Therefore we recommend combined digestion with Pseudomonas and Lysobacter Lys-C to maximize peptide and protein identification.
For a detailed technical review of these two protease visit: http://www.promega.com/resources/scientific_posters/posters/a-novel-recombinant-lysc-protease-for-proteomic-sample-preparation-scientific-poster/
Efficient proteolysis is a major requirement for protein mass spectrometry analysis. Incomplete digestion has multiple ramifications including decreased number of identified proteins, compromised analytical reproducibility and protein quantitation, etc. Trypsin is one of the most robust proteases and is characterized by efficient proteolysis. Typical trypsin reactions do not digest proteins to completion, missing 15–30% of cleavage sites. Incomplete digestion affects protein identification, reproducibility of mass spectrometry analysis and accuracy of protein quantitation. Supplementing Trypsin with Lys-C compensates for the majority of missed cleavages. Continue reading “Enhanced Protein Mass Spectrometry Analysis with Trypsin/Lys-C Mix”
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