Development of Spheroid-FPOP: An In-Cell Protein Footprinting Method for 3D Tumor Spheroids.

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.

THE MAKING OF A FOOTPRINT IN PROTEIN FOOTPRINTING: A REVIEW IN HONOR OF MICHAEL L. GROSS.

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. Footprinting-based 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. © 2020 The Authors. Mass Spectrometry Reviews published by John Wiley & Sons Ltd. Mass Spec Rev.

Ultra-Rapid Crystallization of L-alanine Using Monomode Microwaves, Indium Tin Oxide and Metal-Assisted and Microwave-Accelerated Evaporative Crystallization.

The use of indium tin oxide (ITO) and focused monomode microwave heating for the ultra-rapid crystallization of L-alanine (a model amino acid) is reported. Commercially available ITO dots (< 5 mm) attached to blank poly(methyl)methacrylate (PMMA, 5 cm in diameter with 21-well silicon isolators: referred to as the iCrystal plates) were found to withstand prolonged microwave heating during crystallization experiments. Crystallization of L-alanine was performed at room temperature (a control experiment), with the use of two microwave sources: a 2.45 GHz conventional microwave (900 W, power level 1, a control experiment) and 8 GHz (20 W) solid state, monomode microwave source with an applicator tip that focuses the microwave field to a 5-mm cavity. Initial appearance of L-alanine crystals and on iCrystal plates with ITO dots took 47 ± 2.9 min, 12 ± 7.6 min and 1.5 ± 0.5 min at room temperature, using a conventional microwave and focused monomode microwave heating, respectively. Complete evaporation of the solvent using the focused microwaves was achieved in 3.2 ± 0.5 min, which is ~52-fold and ~172-fold faster than that observed at room temperature and using conventional microwave heating, respectively. The size and number of L-alanine crystals was dependent on the type of the 21-well iCrystal plates and the microwave heating method: 33 crystals of 585 ± 137 µm in size at room temperature > 37 crystals of 542 ± 100 µm in size with conventional microwave heating > 331 crystals of 311 ± 190 µm in size with focused monomode microwave. FTIR, optical microscopy and powder X-ray diffraction analysis showed that the chemical composition and crystallinity of the L-alanine crystals did not change when exposed to microwave heating and ITO surfaces. In addition, theoretical simulations for the binding of L-alanine molecules to ITO and other metals showed the predicted nature of hydrogen bonds formed between L-alanine and these surfaces.

Decrystallization of Crystals Using Gold "Nano-Bullets" and the Metal-Assisted and Microwave-Accelerated Decrystallization Technique.

Gout is caused by the overproduction of uric acid and the inefficient metabolism of dietary purines in humans. Current treatments of gout, which include anti-inflammatory drugs, cyclooxygenase-2 inhibitors, and systemic glucocorticoids, have harmful side-effects. Our research laboratory has recently introduced an innovative approach for the decrystallization of biological and chemical crystals using the Metal-Assisted and Microwave-Accelerated Evaporative Decrystallization (MAMAD) technique. In the MAMAD technique, microwave energy is used to heat and activate gold nanoparticles that behave as "nano-bullets" to rapidly disrupt the crystal structure of biological crystals placed on planar surfaces. In this study, crystals of various sizes and compositions were studied as models for tophaceous gout at different stages (i.e., uric acid as small crystals (~10-100 µm) and l-alanine as medium (~300 µm) and large crystals (~4400 µm). Our results showed that the use of the MAMAD technique resulted in the reduction of the size and number of uric acid and l-alanine crystals up to >40% when exposed to intermittent microwave heating (up to 20 W power at 8 GHz) in the presence of 20 nm gold nanoparticles up to 120 s. This study demonstrates that the MAMAD technique can be potentially used as an alternative therapeutic method for the treatment of gout by effective decrystallization of large crystals, similar in size to those that often occur in gout.