Analysis of protein microarrays by FTIR imaging

Abstract

BACKGROUND: Proteins are sensitive to environmental conditions. Whether they are produced for therapeutic purposes or for fundamental research, the integrity of their structure and post-traductional modifications are key issues. Measuring glycosylation or phosphorylation level as well as their secondary structure most often rely on complex and indirect experiments. Infrared spectroscopy presents a series of advantages related to its multivariate character. There is a lack of high-throughput methods able to analyse these parameters. OBJECTIVE: In this paper we attempted to combine protein microarrays and infrared imaging for high throughput analysis of proteins. METHODS A protein microarrayer was used to produce protein microarrays on BaF2 slides transparent in the mid-infrared. Spot density was about 25 spots / mm2. A 128x128 focal plane array infrared detector was used to record images of the protein microarrays. RESULTS We show that 100 µm diameter spot are easily analyzed. Spots obtained with low protein concentrations, resulting in an average of a single protein monolayer (ca 3 fg/µm2 for a 66 kDa protein) provided good quality spectra. CONCLUSIONS Infrared imaging is a label free, high throughput method, able to analyse protein microarrays and to take advantage from the wide information available in the infrared spectra.