Structural characterization of amyloid aggregates with spatially resolved infrared spectroscopy.

The self-assembly of proteins and peptides into ordered structures called amyloid fibrils is a hallmark of numerous diseases, impacting the brain, heart, and other organs. The structure of amyloid aggregates is central to their function and thus has been extensively studied. However, the structural heterogeneities between aggregates as they evolve throughout the aggregation pathway are still not well understood. Conventional biophysical spectroscopic methods are bulk techniques and only report on the average structural parameters. Understanding the structure of individual aggregate species in a heterogeneous ensemble necessitates spatial resolution on the length scale of the aggregates. Recent technological advances have led to augmentation of infrared (IR) spectroscopy with imaging modalities, wherein the photothermal response of the sample upon vibrational excitation is leveraged to provide spatial resolution beyond the diffraction limit. These combined approaches are ideally suited to map out the structural heterogeneity of amyloid ensembles. AFM-IR, which integrates IR spectroscopy with atomic force microscopy enables identification of the structural facets the oligomers and fibrils at individual aggregate level with nanoscale resolution. These capabilities can be extended to chemical mapping in diseased tissue specimens with submicron resolution using optical photothermal microscopy, which combines IR spectroscopy with optical imaging. This book chapter provides the basic premise of these novel techniques and provides the typical methodology for using these approaches for amyloid structure determination. Detailed procedures pertaining to sample preparation and data acquisition and analysis are discussed and the aggregation of the amyloid ß peptide is provided as a case study to provide the reader the experimental parameters necessary to use these techniques to complement their research efforts.

Colocalization of ß-Sheets and Carotenoids in Aß Plaques Revealed with Multimodal Spatially Resolved Vibrational Spectroscopy.

The aggregation of amyloid ß(Aß) peptides is at the heart of Alzheimer's disease development and progression. As a result, amyloid aggregates have been studied extensively in vitro, and detailed structural information on fibrillar amyloid aggregates is available. However, forwarding these structural models to amyloid plaques in the human brain is still a major challenge. The chemistry of amyloid plaques, particularly in terms of the protein secondary structure and associated chemical moieties, remains poorly understood. In this report, we use Raman microspectroscopy to identify the presence of carotenoids in amyloid plaques and demonstrate that the abundance of carotenoids is correlated with the overall protein secondary structure of plaques, specifically to the population of ß-sheets. While the association of carotenoids with plaques has been previously identified, their correlation with the ß structure has never been identified. To further validate these findings, we have used optical photothermal infrared (O-PTIR) spectroscopy, which is a spatially resolved technique that yields complementary infrared contrast to Raman. O-PTIR unequivocally demonstrates the presence of elevated ß-sheets in carotenoid-containing plaques and the lack of ß structure in noncarotenoid plaques. Our findings underscore the potential link between anti-inflammatory species as carotenoids to specific secondary structural motifs within Aß plaques and highlight the possible role of chemically distinct plaques in neuroinflammation, which can uncover new mechanistic insights and lead to new therapeutic strategies for AD.