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

Area of Science:

• Biochemistry
• Structural Biology
• Spectroscopy

Background:

• Protein structural integrity is crucial for biotherapeutic efficacy and safety.
• Protein aggregation, characterized by altered structures like intermolecular beta-sheets, poses significant challenges.
• High-throughput methods for identifying protein structural changes, particularly aggregation, are needed.

Purpose of the Study:

• To develop a high-throughput method for identifying protein structural modifications, specifically aggregation.
• To utilize protein microarrays and Fourier transform infrared (FTIR) imaging for structural analysis.
• To apply Multivariate Curve Resolution alternating least squares (MCR-ALS) for spectral deconvolution and component identification.

Main Methods:

• Protein microarrays with high sample density (ca 2500 samples/cm²) were employed.
• Fourier transform infrared (FTIR) imaging was used to acquire spectral data from protein samples.
• Multivariate Curve Resolution alternating least squares (MCR-ALS) was applied to analyze spectral components.

Main Results:

• Six spectral components were sufficient to model native protein structures (alpha-helix, beta-sheet).
• A new spectral component, identified by MCR-ALS in denatured proteins, was assigned to intermolecular beta-sheets.
• This new component serves as a spectroscopic marker for protein aggregation.

Conclusions:

• MCR-ALS analysis of FTIR imaging data provides a novel method for detecting protein aggregation.
• The study successfully identified a spectroscopic signature for intermolecular beta-sheet formation, indicative of aggregation.
• This approach allows for the semiquantitative evaluation of protein aggregation, aiding in biotherapeutic development and safety assessment.