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PROTEIN STRUCTURAL CHARACTERIZATION
High Resolution Analysis of Protein & Antibody Structure
Technology
Hydrogen/deuterium exchange mass spectrometry (HDX-MS) has now become an indispensable technique for the structural and functional characterization of proteins.
In a typical HDX workflow, a protein sample is immersed in D2O, at which time labile hydrogens (e.g., on the amides) begin to exchange with deuterium from the solvent. This exchange process is structure sensitive – the hydrogens on residues that are protected from solvent or involved in stable hydrogen-bonding exchange at a much slower rate. Specialized techniques in mass spectrometry can be used to reveal the detailed pattern of deuterium occupancy on individual residues. This information can be used to elucidate or confirm secondary structure, identify binding sites, reveal conformational changes and compare the structural similarity of antibodies or protein therapeutics.
Technological Advantages
MRM Proteomics specializes in “top-down” HDX-MS. Existing HDX approaches commonly use “bottom-up” methods that require digestion of the protein prior to analysis. Our top-down approach bypasses this digestion step, so that the intact protein can be ionized and fragmented in the gas phase. By analyzing intact proteins instead of peptides, it is possible to achieve complete sequence coverage, residue-level resolution (instead of peptide-level resolution), and to minimize signal loss associated with back-exchange of deuterium atoms.
We have further optimized this HDX approach using subzero temperatures to minimize back exchange and specialized instrumentation to maximize coverage and generate comprehensive residue-by-residue information about protein structure. Our top-down HDX technologies are now compatible with proteins up to 150 KDa, including intact antibodies that were much too large to be analyzed by previous methods. This approach is extremely valuable for fine-grained characterization of biosimilars or antibodies. Our top-down methods can also be used for PTM analysis (including rapid global glycosylation profiling).
FEATURES & BENEFITS:
Higher-order structural characterization of proteins including hydrogen bonding patterns
Detailed structural information with close-to-single-residue resolution and high sequence-coverage
Pinpoint conformational changes, bonding patterns, or binding sites in comparative studies
Generate isoform-specific information not available through bottom-up methods
Analyze intact antibodies up to 150 kDa using “top-down” or “middle-down” approaches
Additional structural and binding information can be generated through supplementary structural proteomics techniques (crosslinking, surface modification, etc.)
Pinpoint conformational changes, bonding patterns, or binding sites in comparative studies
Additional Tools & Techniques for Structural Proteomics
In addition to our leading-edge technologies for HDX, MRM Proteomics has also developed and implemented additional mass spectrometry-based strategies to determine the secondary, tertiary, and quaternary structure of proteins and protein aggregates. We offer services for crosslinking, surface modification, and limited proteolysis, which can be used to determine structural constraints for studying protein folding, protein complex assembly, and protein-protein interactions. Our specialized in-house tools include >50 custom reagents and custom software for automated analysis of datasets from crosslinking experiments.
Top/Middle-Down Analysis with Hydrogen-Deuterium Exchange & FT-ICR-MS (Intact or partially proteolyzed protein)
Features & Benefits
- Higher-order structural characterization – reveals hydrogen bonding patterns and solvent exposure
- Close-to-single-residue resolution
- High sequence-coverage
- Reduced back-exchange
- Isoform-specific analysis
Applications
- Analyze proteins & antibodies <150 kDa
- Compare biosimilars to originator drugs
- Pinpoint conformational changes, bonding patterns, or binding sites
Bottom-Up Analysis with Hydrogen-Deuterium Exchange & FT-ICR-MS
Features & Benefits
- Higher-order structural characterization
- Reveals hydrogen bonding patterns
- No size limitation on the proteins that can be analyzed
Applications
- Analyze proteins & antibodies <150 kDa
- Compare biosimilars to originator drugs
- Pinpoint conformational changes, bonding patterns, or binding sites
Post-Translational Modification Analysis with FT-ICR-MS
Features & Benefits
- Characterize post-translational modifications (PTMs)
- Generate isoform-specific information
Applications
- Global glycosylation profiling
- Determine disulphide bonding patterns
Intact Mass Measurement
Features & Benefits
- Ultrahigh resolution
Applications
- Determine mass of intact protein with extremely high accuracy
Protein/peptide sequencing
Features & Benefits
- Approaching single-residue resolution
Applications
- Determine or confirm primary structure
Crosslinking with Bottom-Up MS Analysis
Features & Benefits
- Characterize 3D protein structures (tertiary & quaternary) for difficult-to-crystalize proteins
- >40 custom crosslinking reagents for generating diverse constraint data
- In-house custom software for rapid and automated data analysis
Applications
- Analyze protein folding or misfolding
- Determine protein complex assemblys
- Study protein-protein interactions
- Generate constraint data for 3D structure modeling
Surface Modification with Bottom-Up MS Analysis
Features & Benefits
- Characterize 3D protein structures (tertiary & quaternary) for difficult-to-crystalize proteins
- >10 custom reagents for surface modification
Applications
- Analyze protein folding or misfolding
- Determine protein complex assemblys
- Generate additional constraint data for 3D structure modelings
Limited Proteolysis
Features & Benefits
- Characterize 3D protein structures (tertiary & quaternary)
Applications
- Analyze protein folding or misfolding
- Determine protein complex assemblys
- Generate additional constraint data for 3D structure modelings
Combination Approaches
Features & Benefits
- Multiple methods for lots of constraint data (crosslinking, surface medication, limited proteolysis, etc.)
- Recent high profile publications for protein structure determination
Applications
- Determine tertiary and quaternary structure for difficult-to-crystalize proteins
- Map binding sites and orientations
APPLICATIONS
Many new drugs are “biologics,” meaning that they are proteins or antibodies produced in biological expressions systems, rather than the more traditional small molecule drug entities produced by organic chemists and chemical synthesis. Part of the approval process for any drug, whether it is a biological or small molecule entity is for the manufacturer to prove that it has fully characterized the structure, purity, etc. of the drug compound. Generic drugs (called biosimilars in the case of biologics) have additional requirements: a generic or biosimilar manufacturer must prove that it has synthesized a drug molecule that has the same structure as the original (originator) drug. This is very difficult for antibodies which have complex structures but are too small for direct imaging. Our clients have used the HDX-MS data generated by MRM Proteomics to confirm that the structure of a biosimilar protein drug matches the original biologic. These results have been used in FDA filings.
HDX and other structural proteomics approaches have been successfully combined to fully elucidate the structure of high-molecular-weight protein complexes for which there was no high quality x-ray crystallography data (e.g., RNA polymerase II-Mediator core initiation complex). There are almost limitless possibilities for the application of structural proteomics techniques for investigating protein structure, function, and interactions.
PUBLICATIONS
2014
Pan, J.; Zhang, S.; Parker, C. E.; Borchers, C. H.
Subzero temperature chromatography and top-down mass spectrometry for protein higher-order structure characterization: method validation and application to therapeutic antibodies.
2016
Pan, J.; Zhang, S.; Chou, A.; Borchers, C. H.
Higher-order structural interrogation of antibodies using middle-down hydrogen/deuterium exchange mass spectrometry.
2016
Pan, J.; Zhang, S.; Borchers, C. H.
Protein species-specific characterization of conformational change induced by multisite phosphorylation.
2015
Plaschka, C.; Larivière, L.; Wenzeck, L.; Seizl, M.; Hemann, M.; Tegunov, D.; Petrotchenko, E. V.; Borchers, C. H.; Baumeister, W.; Herzog, F.; Villa, E.; Cramer, P.
Architecture of the RNA polymerase II-Mediator core initiation complex.
2015
Solomonson, M.; Setiaputra, D.; Makepeace, K. A.; Lameignere, E.; Petrotchenko, E. V.; Conrady, D. G.; Bergeron, J. R.; Vuckovic, M.; DiMaio, F.; Borchers, C. H.; Yip, C. K.; Strynadka, N. C.
Structure of EspB from the ESX-1 type VII secretion system and insights into its export mechanism.
2016
Groitl, B.; Horowitz, S.; Makepeace, K. A.; Petrotchenko, E. V.; Borchers, C. H.; Reichmann, D.; Bardwell, J. C.; Jakob, U..
Protein unfolding as a switch from self-recognition to high-affinity client binding.
2014
Quan, S.; Wang, L.; Petrotchenko, E. V.; Makepeace, K. A.; Horowitz, S.; Yang, J.; Zhang, Y.; Borchers, C. H.; Bardwell, J. C.
Super Spy variants implicate flexibility in chaperone action
2014
Brodie, N. I.; Makepeace, K. A.; Petrotchenko, E. V.; Borchers, C.
Super Spy variants implicate flexibility in chaperone action
2016
Brodie, N. I.; Petrotchenko, E. V.; Borchers, C. H.
Isotopically-coded shortrange hetero-bifunctional photo-reactive crosslinkers for studying protein structure.