Multidimensional infrared diffusion-ordered spectroscopy in depletion mode distinguishes protein amyloids and monomers

Conventional and two-dimensional infrared (2D-IR) spectroscopy are well suited to study amyloid aggregates, because the amide I mode is a sensitive probe of the aggregate structure. However, these methods are not so useful to study mixtures of aggregates and monomers, which generally have overlapping amide I spectra. Here, we show that IR-Diffusion-Ordered Spectroscopy can disentangle the contributions of protein monomers and aggregates (amyloids) in FTIR and 2D-IR spectra by separating the spectral contributions based on molecular size. We rely on the fact that the diffusion coefficient of a molecule is determined by its size through the Stokes-Einstein relation, and achieve sensitivity to the diffusion coefficient by creating a concentration gradient inside an IR sample cell and tracking its equilibration in an IR-frequency-resolved manner. The amyloid diffusion is too slow to be experimentally observable, so instead of tracking the arrival of molecular species diffusing into the initially empty region of the sample cell, we track the depletion of the more rapidly diffusing species as they leave the sample-filled region. This way, we can still obtain the spectrum of very slowly diffusing species, although we cannot determine their diffusion coefficient. We first demonstrate this depletion method on a mixture of two small organic molecules and then show how it can be used to separate the spectrum of a mixture of bovine-serum-albumin amyloids and monomers into its component spectra, both in the FTIR and 2D-IR case.

Automated summary

Conventional and 2D-IR spectroscopy are useful for studying amyloid aggregates, but not for mixtures of aggregates and monomers with overlapping spectra. IR-Diffusion-Ordered Spectroscopy can separate the spectral contributions of monomers and aggregates based on molecular size. By tracking the depletion of rapidly diffusing species, it is possible to obtain the spectrum of slowly diffusing species in a mixture of amyloids and monomers.