Abstract
De novo sequencing of a full-length monoclonal antibody raised against OX40 ligand is described. Using a combination of overlapping complementary proteolytic and chemical digestions, with analysis by mass spectrometry and Edman degradation, both the heavy and light chains were fully sequenced. Particular attention was paid to those modifications that could be susceptible to degradation in the complementarity determining region and Fc region. An overview of the protocol is described, and suggestions for improvements to aid in such sequencing projects in the future are discussed.
MeSH terms
- Amino Acid Sequence
- Antibodies, Monoclonal / chemistry*
- Antibodies, Monoclonal / pharmacology
- Complementarity Determining Regions / chemistry
- Dose-Response Relationship, Drug
- Humans
- Immunoglobulin Variable Region / chemistry
- Mass Spectrometry
- Membrane Glycoproteins / antagonists & inhibitors
- Membrane Glycoproteins / immunology*
- Models, Biological
- Molecular Sequence Data
- OX40 Ligand
- Organophosphorus Compounds
- Polyvinyls / chemistry
- Polyvinyls / metabolism
- Proteomics / methods*
- Sequence Alignment
- Sequence Analysis, Protein / methods*
- Tumor Necrosis Factor Inhibitors
- Tumor Necrosis Factors / immunology*
Substances
- 2-(4-isothiocyanatophenoxy)-1,3,2-dioxaphosphinene 2-oxide
- Antibodies, Monoclonal
- Complementarity Determining Regions
- Immunoglobulin Variable Region
- Membrane Glycoproteins
- OX40 Ligand
- Organophosphorus Compounds
- Polyvinyls
- TNFSF4 protein, human
- Tumor Necrosis Factor Inhibitors
- Tumor Necrosis Factors
- polyvinylidene fluoride