Quantification of protein glycation using vibrational spectroscopy.

Area of Science:

• Biophysical Chemistry
• Analytical Chemistry
• Biotechnology

Background:

• Protein glycation is a significant post-translational modification implicated in diseases such as diabetes and Alzheimer's.
• Glycation also occurs during the production of therapeutic proteins, like monoclonal antibodies (mAbs), necessitating quality control measures.
• Accurate quantification of glycated proteins is crucial for disease diagnosis and ensuring the efficacy and safety of protein-based therapeutics.

Purpose of the Study:

• To investigate the utility of vibrational spectroscopy, specifically FTIR-ATR and Raman spectroscopy, for detecting and quantifying protein glycation.
• To demonstrate the potential of these techniques for rapid, minimally invasive analysis of glycated proteins.
• To develop a robust quantification model for glycated proteins using Raman spectroscopy.

Main Methods:

• Glycated lysozyme and albumin samples were synthesized by incubation with glucose.
• Fourier-transform infrared-attenuated total reflectance (FTIR-ATR) and Raman spectroscopy were employed to analyze protein samples.
• Principal component analysis (PCA) was used to differentiate between glycated and non-glycated proteins.
• Partial least squares regression (PLSR) was applied for quantitative analysis of glycation levels in lysozyme using Raman spectra.

Main Results:

• Both FTIR-ATR and Raman spectroscopy successfully distinguished between glycated and non-glycated protein samples.
• PCA revealed distinct clustering of control and glycated samples, with specific spectral peaks identified as key contributors to this separation.
• Raman spectroscopy, coupled with PLSR, generated a highly accurate quantification model (R² = 0.99) for glycated lysozyme in solution.
• Analysis highlighted spectral changes in the sugar C-O/C-C/C-N region (1200-800 cm⁻¹) for glycated proteins.

Conclusions:

• FTIR-ATR and Raman spectroscopy are effective methods for identifying protein glycation.
• Raman spectroscopy shows significant potential as a high-throughput method for quantifying glycated proteins.
• These vibrational spectroscopy techniques offer valuable applications in both clinical diagnostics and pharmaceutical quality control for protein therapeutics.