Within the past decade protein footprinting in conjunction with mass spectrometry has become a powerful and versatile means to unravel the higher order structure of proteins. Footprintingbased approaches has demonstrated the capacity to inform on interaction sites and dynamic regions that participate in conformational changes. These findings when set in a biological perspective inform on protein folding/unfolding, protein-protein interactions, and protein-ligand interactions. In this review, we will look at the contribution of Dr. Michael L. Gross to protein footprinting approaches such as hydrogen deuterium exchange mass spectrometry and hydroxyl radical protein footprinting. This review details the development of novel footprinting methods as well as their applications to study higher order protein structure.
Many cancer drugs fail at treating solid epithelial tumors
with
hypoxia and insufficient drug penetration thought to be contributing
factors to the observed chemoresistance. Owing to this, it is imperative
to evaluate potential cancer drugs in conditions as close to in vivo as possible, which is not always done. To address
this, we developed a mass spectrometry-based protein footprinting
method for exploring the impact of hypoxia on protein in 3D colorectal
cancer cells. Our group has previously extended the protein footprinting
method fast photochemical oxidation of proteins (FPOP) for live cell
analysis (IC-FPOP); however, this is the first application of IC-FPOP
in a 3D cancer model. In this study, we perform IC-FPOP on intact
spheroids (Spheroid-FPOP) using a modified version of the static platform
incubator with an XY movable stage (PIXY) FPOP platform. We detected
modification in each of three spheroid layers, even the hypoxic core.
Pathway analysis revealed protein modifications in over 10 distinct
protein pathways, including some involved in protein ubiquitination;
a process modulated in cancer pathologies. These results demonstrate
the feasibility of Spheroid-FPOP to be utilized as a tool to interrogate
protein interactions within a native tumor microenvironment.
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