A Rapid FT-IR-Based Method for Monitoring Detergent Removal from Biological Samples

摘要

In life science research, detergents are primarily used in sample preparation to liberate cellular components through membrane disruption and to solubilize lipid-associated proteins. However, detergents can interfere with applications such as enzyme-linked immunosorbent assays, isoelectric focusing, nuclear magnetic resonance spectroscopy, and mass spectrometry, and therefore, often need to be removed before analysis. Here we report a novel infrared-based method that permits fast and impartial analysis of detergent removal from biological samples. Because this method is spectrally based and label-free, determination of protein concentration can be performed simultaneously. An additional benefit of this technique is the small volume (2 μL) required for analysis, a significant fact given the often precious nature of biological samples. FT-IR spectroscopy is a rapid, universal, and highly effective method for detection and analysis of biomolecules suspended in solution that is based on the unique spectral signatures of analyzed species. More specifically, the work presented here demonstrated the application of FT-IR for simultaneous determination of protein and detergent content in biological samples. Most notably, reported quantitation requires single-time instrument calibration with the detergent of interest permitting standardization across both sample types and processing runs. Protein quantitation is performed using preloaded, standard NIST BSA calibration. In addition, the reported method can be used in parallel with the majority of known detergent removal techniques. Overall, the results suggest that optimization of a sample preparation workflow, including detergent removal, are confounded not only by the nature of the starting material (cell or tissue lysate), but also by the detection requirements and sensitivities of the downstream analytical tools.