Combining FTIR spectroscopy and microarray technology

Belgian researchers have discovered how to use Fourier transform infrared (FTIR) spectroscopy to increase the amount of information that can be extracted from a protein microarray. Writing in the journal Biomedical Spectroscopy and Imaging, they show how high-quality spectra can be obtained from spots of protein no larger than the diameter of a human hair.

The common use of protein microarrays requires the binding of proteins to other compounds, such as therapeutic drugs. A fluorescent molecule is attached to the protein so that if binding with the drug occurs, there will be a light signal from the bound pair. However, these fluorescent proteins can be difficult and expensive to produce, and the information is limited to determining whether the drug binds or not.

Infrared (IR) spectroscopy can probe the molecular structure of a substance. Different wavelengths of infrared light are absorbed by different chemical bonds in a molecule and, by scanning a range of wavelengths, the kinds of bonds can be measured. These measurements comprise a fingerprint of the molecules in the sample. Furthermore, IR spectra account not only for the chemical nature of cell molecules, but also for their shape. They are particularly sensitive to protein secondary structure.

Researchers at the Center for Structural Biology and Bioinformatics, based at the Université Libre de Bruxelles, created microarrays using a commercial tool in which about 100 pL of protein were deposited from solution. A 128x128 focal-plane array was used to collect a full infrared spectrum from each of the resulting protein spots in the microarray, leading to 16,384 complete spectra. These spectra were preprocessed to remove random noise and carry out background correction.

Lysozyme, albumin and haemoglobin solutions were prepared at concentrations from 10 to 0.1 mg/mL and deposited in multiple spots. Single spots provided structural and concentration information from a typical protein, albumin.

According to lead investigator Dr Erik Goormaghtigh, the combination of FTIR spectroscopy and microarray technology has three main advantages:

1. Label-free detection. Labelled proteins may be not commercially available or very expensive. It also enhances quality because the labelling procedure often destroys part of the protein structure.
2. Direct and absolute quantification of proteins. Infrared detection can quantify the amount of bound protein in a sample.
3. Full imprint of proteins. Infrared imaging provides a complete vibrational spectrum of the binding molecule, which includes information on chemical reactions and protein secondary structure.

“The results of this study show that high-quality spectra can be obtained from minute amounts of proteins, ie, below a single monolayer of proteins,” said Dr Goormaghtigh. “This is important as it opens the way to use infrared imaging, instead of fluorescence, for instance, for detection of binding. This work also shows that it is now possible to produce high-throughput protein analysis by combination of microarrays technology and infrared spectroscopy imaging, allowing hundreds of proteins to be quantitatively analysed in a few minutes.”

Image caption: Example of single-pixel spectra extracted from albumin spots obtained from 1, 0.8, 0.5 and 0.25 mg/mL solutions, as indicated by the arrows. The concentration resulting, on the average, in a one-protein monolayer is indicated by the dotted line.