Jessica Bane1, Olivier Mozziconacci1, Li Yi2, Y John Wang2, Alavattam Sreedhara3, Christian Schöneich4. 1. Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Avenue, Lawrence, Kansas, 66047, USA. 2. Late Stage Pharmaceutical Development, Genentech, Inc., 1 DNA Way, South San Francisco, California, 94080, USA. 3. Late Stage Pharmaceutical Development, Genentech, Inc., 1 DNA Way, South San Francisco, California, 94080, USA. sreedhaa@gene.com. 4. Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Avenue, Lawrence, Kansas, 66047, USA. schoneic@ku.edu.
Abstract
PURPOSE: Triply oxidized histidine in an IgG1 monoclonal antibody was noticed when exposed to ICH light conditions. In order to understand the role of light source, irradiation wavelengths and primary sequence, specifically those of a nearby tryptophan, we synthesized and exposed several peptides to ICH light conditions and analyzed the products using LC-MS analysis. METHODS: Protein and peptide samples were photo-irradiated under ICH conditions as well as with monochromatic light at λ = 254 nm and analyzed using either LTQ Orbitrap or a LTQ-FT ion cyclotron resonance mass spectrometer respectively. RESULTS: A triply oxidized His residue was detected along with a second doubly oxidized His residue in an IgG1. Both of these oxidized His residues are located near Trp residues. In order to investigate the role of Trp photosensitization in His oxidation we synthesized model peptides and Ala mutants. Peptides exposed to ICH light stress conditions revealed a small percent of triply oxidized His in the Trp-containing peptide sequences but not in their corresponding Ala mutants. CONCLUSIONS: The differences in product formation under different photo-irradiation conditions underline the importance of light source, irradiation wavelengths and primary sequence in the photosensitivity of proteins.
PURPOSE: Triply oxidized histidine in an IgG1 monoclonal antibody was noticed when exposed to ICH light conditions. In order to understand the role of light source, irradiation wavelengths and primary sequence, specifically those of a nearby tryptophan, we synthesized and exposed several peptides to ICH light conditions and analyzed the products using LC-MS analysis. METHODS: Protein and peptide samples were photo-irradiated under ICH conditions as well as with monochromatic light at λ = 254 nm and analyzed using either LTQ Orbitrap or a LTQ-FT ion cyclotron resonance mass spectrometer respectively. RESULTS: A triply oxidized His residue was detected along with a second doubly oxidized His residue in an IgG1. Both of these oxidized His residues are located near Trp residues. In order to investigate the role of Trp photosensitization in His oxidation we synthesized model peptides and Ala mutants. Peptides exposed to ICH light stress conditions revealed a small percent of triply oxidized His in the Trp-containing peptide sequences but not in their corresponding Ala mutants. CONCLUSIONS: The differences in product formation under different photo-irradiation conditions underline the importance of light source, irradiation wavelengths and primary sequence in the photosensitivity of proteins.
Entities:
Keywords:
His; Trp; mass spectrometry; monoclonal antibody; photo-oxidation; singlet oxygen
Authors: Vanessa V Agon; William A Bubb; Adam Wright; Clare L Hawkins; Michael J Davies Journal: Free Radic Biol Med Date: 2005-12-12 Impact factor: 7.376
Authors: Jessica Haywood; Olivier Mozziconacci; Kevin M Allegre; Bruce A Kerwin; Christian Schöneich Journal: Mol Pharm Date: 2013-02-06 Impact factor: 4.939
Authors: Daniel Steinmann; Olivier Mozziconacci; Rupesh Bommana; John F Stobaugh; Y John Wang; Christian Schöneich Journal: Pharm Res Date: 2017-09-18 Impact factor: 4.200