NIH VideoCast - Extension of Hydroxyl Radical-Based Footprinting Coupled with Mass Spectrometry for In Cell and In Vivo Protein Analysis

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Extension of Hydroxyl Radical-Based Footprinting Coupled with Mass Spectrometry for In Cell and In Vivo Protein Analysis

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Air date:	Thursday, December 7, 2017, 9:30:00 AM Time displayed is Eastern Time, Washington DC Local
Views:	Total views: 152 (16 Live, 136 On-demand)
Category:	Proteomics
Runtime:	00:52:07
Closed captioning:	Check box to display CC outside of the video Note: You can drag the captioning window around and resize it
Description:	Proteomics Interest Group In recent years, protein footprinting coupled with mass spectrometry has been used to identify protein-protein interaction sites and regions of conformational change through modification of solvent accessible sites in proteins. Hydroxyl radical-based footprinting (HRBF) approaches utilize hydroxyl radicals to oxidatively modify the side chains of solvent accessible amino acids. There are several approaches to generate radicals for oxidation including synchrotron radiation and electrochemistry. One HRBF method, fast photochemical oxidation of proteins (FPOP), utilizes an excimer laser for photolysis of hydrogen peroxide to generate hydroxyl radicals. To date, HRBF methods have been used in vitro on relatively pure protein systems. We have further extended the FPOP method for in cell analysis of proteins. This will allow for study of proteins in their native cellular environment and be especially useful for the study of membrane proteins which can be difficult to purify for in vitro studies. We have designed and built a single cell flow system to enable uniform access of cells to the laser. Results demonstrate that in cell FPOP (IC-FPOP) can oxidatively modify over 1300 proteins in various cellular compartments. Further, the method successfully probes solvent accessibility similarly to in vitro FPOP. We have further extended the method for in vivo analysis using C. elegans, members of the nematode family. C. elegans are widely used as model systems for human diseases including cancer, Parkinson’s disease, and diabetes. Preliminary results indicate a number of proteins can be oxidatively modified in C. elegans byin vivo FPOP (IV-FPOP) leading to the possibility of studying protein structure in human diseases directly in animal model systems. However, further optimization of the method is required to increase the number of oxidatively modified proteins.

Author:	Lisa M. Jones, Ph. D., Assistant Professor, Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy

CIT Live ID:	26779
Permanent link:	https://videocast.nih.gov/watch=26779