Protein Structural Denaturation Evaluated by MCR-ALS of Protein Microarray FTIR Spectra

• Published in: Analytical chemistry (Washington) Vol. 93; no. 40; pp. 13441 - 13449
• Main Authors: De Meutter, Joëlle, Goormaghtigh, Erik
• Format: Journal Article
• Language: English
• Published: Washington American Chemical Society 12.10.2021
• Subjects: Agglomeration; analytical chemistry; Chemistry; Denaturation; Feature recognition; Fourier transform infrared spectroscopy; Fourier transforms; Infrared analysis; Infrared imaging; Infrared spectroscopy; Protein arrays; Protein denaturation; Protein interaction; protein microarrays; Protein structure; Proteins; Secondary structure; Spectra
• ISSN: 0003-2700; 1520-6882; 1520-6882
• DOI: 10.1021/acs.analchem.1c01416

The loss of native structure is common in proteins. Among others, aggregation is one structural modification of particular importance as it is a major concern for the efficiency and safety of biotherapeutic proteins. Yet, recognizing the structural features associated with intermolecular bridging in a high-throughput manner remains a challenge. We combined here the use of protein microarrays spotted at a density of ca 2500 samples per cm2 and Fourier transform infrared (FTIR) imaging to analyze structural modifications in a set of 85 proteins characterized by widely different secondary structure contents, submitted or not to mild denaturing conditions. Multivariate curve resolution alternating least squares (MCR-ALS) was used to model a new spectral component appearing in the protein set subject to denaturing conditions. In the native protein set, 6 components were found to be sufficient to obtain good modeling of the spectra. Furthermore, their shape allowed them to be assigned to α-helix, β-sheet, and other structures. Their content in each protein was correlated with the known secondary structure, confirming these assignments. In the denatured proteins, a new component was necessary and modeled by MCR-ALS. This new component could be assigned to the intermolecular β-sheet, bridging protein molecules. MCR-ALS, therefore, unveiled a potential spectroscopic marker of protein aggregation and allowed a semiquantitative evaluation of its content. Insight into other structural rearrangements was also obtained.