Quantification of protein glycation using vibrational spectroscopy

Abstract

Glycation is a protein modification prevalent in the progression of diseases such as Diabetes and Alzheimer's, as well as a byproduct of therapeutic protein expression, notably for monoclonal antibodies (mAbs). Quantification of glycated protein is thus advantageous in both assessing the advancement of disease diagnosis and for quality control of protein therapeutics. Vibrational spectroscopy has been highlighted as a technique that can easily be modified for rapid analysis of the glycation state of proteins, and requires minimal sample preparation. Glycated samples of lysozyme and albumin were synthesised by incubation with 0.5 M glucose for 30 days. Here we show that both FTIR-ATR and Raman spectroscopy are able to distinguish between glycated and non-glycated proteins. Principal component analysis (PCA) was used to show separation between control and glycated samples. Loadings plots found specific peaks that accounted for the variation – notably a peak at 1027 cm⁻¹ for FTIR-ATR. In Raman spectroscopy, PCA emphasised peaks at 1040 cm⁻¹ and 1121 cm⁻¹. Therefore, both FTIR-ATR and Raman spectroscopy found changes in peak intensities and wavenumbers within the sugar C–O/C–C/C–N region (1200–800 cm⁻¹). For quantification of the level of glycation of lysozyme, partial least squares regression (PLSR), with statistical validation, was employed to analyse Raman spectra from solution samples containing 0–100% glycated lysozyme, generating a robust model with R² of 0.99. We therefore show the scope and potential of Raman spectroscopy as a high throughput quantification method for glycated proteins in solution that could be applied in disease diagnostics, as well as therapeutic protein quality control.