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A free online tool for protein cleavage sites prediction with a chosen enzyme.
Calculate monoisotopic and average mass of a custom peptide or peptides predicted from a protein.
The cleavage specificities of selected enzymes.
A comprehensive and freely accessible resource of protein sequence and functional information.
Mass spectrum simulation tool for peptide/protein sequences. The number of charge can be custom defined.
An online tool predicts m/z values for SMS ions generated from selected fragmentation methods such as CID, ECD, or ETD. The ion type and maximum number of charge can be pre-defined.
PDB provides the most complete collection of information about the 3D structures of proteins, protein complexes and other biomolecules.
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HDX-MS
Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) is one of our key technologies used for accurate and comprehensive antibody protein structural characterization. Our HDX-MS service platform is founded on the technologies developed by our senior scientists for over a decade, and we continually develop innovative methodologies and proprietary workflows for our services. Here at NovoAb we have three different HDX-MS strategies, namely "bottom-up HDX-MS", "top-down HDX-MS" and "middle-down HDX-MS" to meet your needs. One or a combination of two of the strategies can be chosen based on the objective of your specific project. Below is a schematic overview of our HDX-MS technologies.
"Bottom-Up HDX" "Middle-Down HDX" "Top-Down HDX"
1. Bottom-Up HDX-MS
The ‘bottom-up’ HDX-MS workflow includes isotope labeling of the protein, followed by acid quenching of the solution at pH 2.5 and 0 °C at specific labeling time points. An enzyme that is active at acidic pH (e.g. pepsin) is then added to induce limited proteolysis. The resulting peptides are separated by HPLC and analyzed by ESI-MS under cold acidic conditions. Subsequent off-line analysis of the deuteration status of these individual protein segments provides structural information at peptide-level resolution.
2. Top-Down HDX-MS
The advantage of the bottom-up approach is that there is effectively no limit on protein size. However, to reach a more complete sequence coverage and enhanced spatial resolution, the protein based ‘top-down’ HDX-MS approach is a powerful alternative option. These goals are achieved through direct gas-phase fragmentation (‘digestion’) of intact proteins using electron based dissociation mechanism. This top-down approach has been used to produce amino acid-level HDX information with complete sequence coverage. Because this approach bypasses in-solution digestion, the analytes in the sample are much simpler compared to the bottom-up approach. Taking this advantage, the HPLC step of top-down HDX-MS analysis can be performed under subzero temperature (e.g. -20 °C), which significantly reduces the level of back exchange down to a minimal level (2%). Using this method, amino acid level structural information can be obtained for the entire sequence of the antibody light chain. In combination with the bottom-up approach, the top-down HDX-MS technology can be used for a more complete and high-resolution structural characterization of antibodies and biosimilars, where their higher order structure (HOS) is compared against each other side-by-side.
3. Middle-Down HDX-MS
Despite the above mentioned advantages of the top-down HDX-MS technology, its success declines in the high-mass region. Therefore, for very large proteins or proteins that top-down doesn't work well, we employ the newest hybrid ‘middle-down HDX’ technology, which combines the positive aspects of both bottom-up and top-down approaches. In our innovative middle-down HDX-MS approach, large proteins such as monoclonal antibodies are digested in solution into a limited number of specific fragments under cold acidic conditions, followed by HPLC separation and online nonergodic fragmentation inside the mass spectrometer. This method can provide single residue level structural information with 100% coverage for 150 kDa mAb, while still keeping the back-exchange at a minimal level by using the advanced subzero temperature HPLC technology.
