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Overlapping Peptide Library to Map Qa-1 Epitopes in a Protein
Published on: December 20, 2017
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Development of an LC-MS/MS peptide mapping protocol for the NISTmAb
Trina Mouchahoir1,2, John E Schiel3,4
1Biomolecular Measurement Division, National Institute of Standards and Technology, 100 Bureau Drive, Gaithersburg, MD, 20899, USA. trina.mouchahoir@nist.gov.
Analytical and Bioanalytical Chemistry
|February 8, 2018
Summary
Optimizing tryptic digestion for peptide mapping of protein therapeutics minimizes artificial modifications. This enhanced method confirms the identity of monoclonal antibodies at the primary structure level.
Area of Science:
- Biopharmaceutical analysis
- Protein characterization
- Analytical chemistry
Background:
- Peptide mapping confirms protein therapeutic identity and monitors modifications.
- Sample preparation can induce artificial modifications, confounding results.
- Optimizing conditions to balance digestion efficiency and minimize artifacts is crucial.
Purpose of the Study:
- To optimize a tryptic digestion protocol for peptide mapping of the NISTmAb IgG1κ.
- To minimize artificial modifications during sample preparation.
- To balance digestion efficiency with artifact reduction.
Main Methods:
- Focused on optimizing buffer concentration, digestion time, and temperature.
- Evaluated different sources and types of trypsin (recombinant vs. pancreatic, bovine vs. porcine).
- Applied the optimized protocol to the NISTmAb for peptide mapping.
Main Results:
- Developed an optimized tryptic digestion protocol for NISTmAb.
- Successfully generated a peptide map of the NISTmAb.
- Confirmed the identity of the NISTmAb at the primary structure level.
Conclusions:
- The optimized protocol effectively balances digestion efficiency with minimal artificial modifications.
- Peptide mapping using this protocol is a reliable method for protein therapeutic identity confirmation.
- This approach is vital for accurate assessment of protein modifications.
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