FTIR spectroscopy obtains molecular structure, bonding information from protein microarrays

June 22, 2016

An IR spectroscopy method greatly increases the amount of information that can be extracted from a protein microarray.

An example of single pixel spectra extracted from albumin spots obtained from 1.0, 0.8, 0.5, and 0.25 mg/mL solutions, as indicated by the arrows. The resulting concentration, on the average, in a 1 protein monolayer is indicated by the dotted line.

By using Fourier transform infrared (FTIR) spectroscopy, researchers at the Center for Structural Biology and Bioinformatics at the Université Libre de Bruxelles (Brussels, Belgium) could greatly increase the amount of information that can be extracted from a protein microarray. In a new study, they showed 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.

FTIR spectroscopy can probe the molecular structure of a substance. Different wavelengths of IR 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 very sensitive to protein secondary structure.

The researchers created microarrays using a commercial tool in which about 100 pL of protein were deposited from solution. The resulting spots of protein were about 100 µm in diameter. A 128 × 128 focal-plane array was used to collect a full IR spectrum from each of the spots in the array, resulting in 16,384 complete spectra. These spectra were preprocessed to remove random noise and do background correction. To test the sensitivity of the method, lysozyme, albumin, and hemoglobin 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 albumin.

Combining FTIR and microarray technology allows for label-free detection (which is much lower in cost), direct and absolute quantification of proteins, and full imprint of proteins, according to Erik Goormaghtigh, Ph.D., who led the work. He adds that the combo approach also allows hundreds of proteins to be quantitatively analyzed in a few minutes.

Full details of the work appear in the journal Biomedical Spectroscopy and Imaging; for more information, please visit http://dx.doi.org/10.3233/bsi-160137.