Applications of ATR-FTIR Spectroscopic Imaging to Proteins

Abstract

Protein aggregation and crystallisation play an important role in the development of biopharmaceuticals and for structural proteomics but both processes are still poorly understood. There is a demand for new methods to screen the extensive range of conditions that promote crystallisation and aggregation as well as provide insight into the behaviour of the proteins. Attenuated Total Reflection (ATR) – Fourier Transform Infrared (FTIR) spectroscopic imaging is a powerful analytical tool which can be applied to study proteins. This technique combines ATR-FTIR spectroscopy with an infrared array detector allowing for both spatial and chemical information to be obtained from the sample. There are a range of imaging fields of view and spatial resolution possible with ATR-FTIR spectroscopic imaging and this presents multiple opportunities for the study of proteins. The purpose of this research was to further develop the application of ATR-FTIR spectroscopic imaging within the field of protein studies. ATR-FTIR imaging has been applied to study the effects of different conditions for microbatch protein crystallisation in a high throughput manner, where many samples can be analysed at the same time on the surface of a Macro ATR crystal by building a wax grid with multiple wells for different samples. Additionally, Micro ATR-FTIR imaging was combined with hanging drop protein crystallisation for high spatial resolution imaging of the growth of protein crystals. The surface properties of Silicon ATR crystals were modified to create a gradient of hydrophobicity allowing the effect of different surface properties on protein adsorption and crystallisation to be studied in situ. The development of these approaches will advance the use of spectroscopic imaging within the field of biopharmaceuticals, where it is has the potential to help the optimisation of both biopharmaceutical drug discovery processes and structural proteomics studies.