Analysis of protein microarrays by FTIR imaging

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 128 × 128 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.