Using polarized Total Synchronous Fluorescence Spectroscopy (pTSFS) with PARAFAC analysis for characterizing intrinsic protein emission

Using polarized Excitation Emission Matrix (pEEM) spectroscopy to measure the intrinsic emission of proteins offers a potentially useful methodology for a wide variety of potential applications. However, the presence of Rayleigh light scatter causes significant problems when attempting to use Parallel Factor (PARAFAC) and for anisotropy calculations. The use of polarized Total Synchronous Fluorescence Spectroscopy (pTSFS) can minimize Rayleigh scatter and avoid the use of complex data correction methods. Here, we investigated for the first time the use of pTSFS and PARAFAC to analyze the intrinsic emission of an Immunoglobulin (IgG) type protein in its native state. To enable PARAFAC analysis however, TSFS data (which is not trilinear) must first be transformed into an EEM like layout (t-EEM) and this generated a region with no experimentally acquired information (<8%). Here we critically evaluated several data handling methods and determined that interpolation was the best solution for dealing with the spectral regions with no experimentally acquired data at the blue edge of the emission. There were only subtle structural changes measured over the temperature range (15–35 °C) analyzed and PARAFAC only resolved two emitting components. A Trp emission component (average signal from all Trp present) which represented >92% of the explained variance, and a much weaker, mostly Tyr related emission with ~3% of the explained variance. The recovery of this Tyr component was only possible because pTSFS measurements were less contaminated by Rayleigh scattering. Changes in Tyr-to-Trp energy transfer rates caused by thermal motion were detected as an increase in Tyr contribution, which could not be resolved with the equivalent pEEM measurements due to light scatter contamination. The increased selectivity, sensitivity, and reproducibility of pTSFS measurements shows that this is a better option than pEEM for fluorescence emission based monitoring of protein structural change or lot-to-lot variance of IgG type proteins.