Hydroxyl radical protein footprinting (HRPF) coupled to mass spectrometry (MS) is a vital technique in determining the higher order structure of proteins. This method uses hydroxyl radicals to oxidatively modify solvent-accessible amino acids and has recently been extended to in-cell analysis. Specifically, HRPF via in-cell fast photochemical oxidation of proteins (IC-FPOP) is a relatively new approach to labelling proteins, with the ability to modify 19 of the 20 amino acids. FPOP uses an excimer laser to split hydrogen peroxide (H2O2) into hydroxyl radicals, which are then able to react with, and thus label solvent-accessible surface residues of a protein on the microsecond time scale. In Lisa Jones’s lab, in-cell protein footprinting has been coupled with mass spectrometry to discern protein structure in-cell. Specifically, IC-FPOP has been implemented in a Platform Incubator with a movable XY stage (PIXY) and used to successfully modify proteins in several commonly used cell lines. However, there remains a gap in the IC-FPOP methodology pertaining to the effective radical dose delivered to the cellular environments. While IC-FPOP is an effective avenue of HRPF, a challenge that remains is maintaining reproducibility as it pertains to the concentration of radical that is delivered to the system, or the effective radical dose, of hydroxyl radicals reacting with the protein. Effective radical dose is affected by the amount of radical delivered as well as the presence of other substances, such as radical scavengers. While dosimetry experiments for HRPF via FPOP have been done before in other labs, there is currently no protocol for dosimetry for IC-FPOP. CellROX Deep Red is a fluorescent probe that fluoresces only in the presence of reactive oxidative species (ROS). I propose probing the efficacy of the fluorophore CellROX Deep Red as a radical dosimeter for IC-FPOP. I will first troubleshoot and optimize the fluorescence imaging of HEK293 cells with CellROX Deep Red directly following their oxidation. I will then conduct imaging of the cells having been exposed to increasing values of hydrogen peroxide concentrations in order to demonstrate a linear response of the dosimeter CellROX Deep Red.

The Severe Acute Respiratory Virus 2 (SARS-CoV-2) virus is responsible for the Coronavirus Disease 2019 (COVID-19) pandemic that continues to plague present-day society as new variants with increased virulence emerge. Viral infection may only occur once the host cell’s angiotensin-converting enzyme 2 (ACE2) receptor binds to an exposed viral spike glycoprotein’s Receptor Binding Domain (RBD) in its “up” state. Over the years as new variants emerged, an increase in infectivity has been observed. We hypothesize that mutations observed in later variants contribute to RBD instability, resulting in a greater likelihood of an RBD up spike. To elucidate the relationship between RBD dynamics and increased virulence amongst SARS-CoV-2 Wuhan 2019/Wild-type (WT), Delta, and Omicron variants, the MS-based protein footprinting method fast photochemical oxidation of proteins (FPOP) was performed using GenNext® Technologies’ Flash Oxidation (FOX®) system. As glycans are preferentially labeled, this instrument simultaneously allows for sufficient glycoprotein labeling via UV-based radical dosimetry and ensures user safety by generating hydroxyl radicals via lamp-based photolysis of hydrogen peroxide. Preliminary results show the FOX® system may be utilized to perform in vitro FPOP studies on the SARS-CoV-2 spike glycoprotein, however, further optimization is needed for more accurate comparison across conditions to discuss virulence in relation to spike structure.