Project Summary Understanding protein folding is critical for understanding proper protein function. It is well known that various diseases are associated with misfolding or folding deficiencies. Protein folding has been studied in vitro with residue resolution. However, it is well established that folding in the cell can be significantly different from folding studies in an isolated environment, notably due to the interactions of chaperones. Studying protein folding in a cellular context is limited with few approaches being able to tease out high resolution structural detail from a complex cellular environment. To overcome these limitations, we aim to develop a new method to study protein folding. This method, pulse chase in-cell fast photochemical oxidation of proteins (pcIC-FPOP), couples pulse- chase technology with mass spectrometry-based in cell footprinting which will allow for quick analysis of short- lived protein folding intermediates. This method aims to fill gaps in knowledge of protein folding and its role in various diseases. We designed a new platform for in cell hydroxyl radical protein footprinting. The system called Platform Incubator with an XY movable stage (PIXY) includes a sterile incubation system, XY movable stage with controllers, peristaltic pumps, a 248 nm KrF excimer laser, and optic mirrors. The system has been tested and optimized as the new platform for cell growth and IC-FPOP studies, referred to as PIXY IC- FPOP. Transient transfections were performed to assess and compare cell culture quality under standard incubator and stage top incubator conditions. Furthermore, a luciferase assay was performed to quantitate transfection efficiency. PIXY IC-FPOP experiments were also carried out to access comparability with the standard IC-FPOP flow system technique and results. PIXY will be configured into an automated system through LABVIEW software. Once optimized, PIXY will be further validated as a protein folding tool by observing the folding dynamics of well- studied abundant serpin protein, alpha-1 antitrypsin as well as its chaperone interaction. The sensitivity of the PIXY platform to decipher between wild type protein folding dynamics and subsequent mutants will allow for the expansion of this method to other protein groups. pcIC-FPOP will be used to study transthyretin (TTR) misfolding and aggregation known to be associated with the amyloid disease, hereditary transthyretin amyloidosis (hATTR).