Title:Exploring the Potential of Molecular Spectroscopy for the Detection of
Post-translational Modifications of a Stressed Biopharmaceutical Protein
Volume: 22
Issue: 11
Author(s): Marco P. Corujo, Vivian Lindo, Nikola Chmel and Alison Rodger*
Affiliation:
- Department of Chemistry, University of Warwick, Coventry, UK
- Department of Molecular Sciences, Macquarie
University, North Ryde, 2109, New South Wales, Australia
Keywords:
Post translational modifications, biopharmaceuticals, protein secondary structure, spectroscopy, raman, circular dichroism, mass spectrometry, infrared absorbance.
Abstract:
Background: Proteins are biomolecules that consist of sequences of amino acids (primary
structure) which can further interact and cause the backbone to fold into more complex arrangements
(secondary and tertiary structures). Any chemical alterations of the molecules after the
translation of the messenger RNA code into a protein primary sequence are known as posttranslational
modifications (PTMs). PTMs may affect the protein’s functionality; thus it is necessary
to identify them. PTMs of particular interest to the pharmaceutical industry include deamidation,
oxidation, deglycosylation and isomerization, which may occur due to environmental stressors.
However, they have proved challenging to identify quickly. Electronic and vibrational spectroscopies
have proved valuable tools for studying higher-order structure and stability of proteins.
Methods: In this work, circular dichroism (CD), infrared absorbance (IR) and Raman spectroscopies
were applied to characterize antibody (mAb NIP 228) PTMs as a result of different stressors.
Mass spectrometry was used to confirm the identity of modifications including the targeted ones.
Results: Room temperature CD showed that the secondary structure was the same after all treatments,
and temperature-controlled CD showed how protein stability was affected by modifications.
Both Raman and IR analysis detected small differences between the reference and deglycosylated
proteins, and clearly indicated the presence of other PTMs.
Conclusion: This work required some novel computational approaches to pre-process Raman and
IR spectra and a review of the band assignments for proteins existing in the literature.
