Method Optimization of a New Automated Platform for Proteome-Wide Structural Biology

Abstract

Proteins adopt different higher-order structures (HOS) to enable their unique biological functions. Understanding the complexities of protein HOS and dynamics requires integrated approaches, including mass spectrometry (MS), which has evolved into an indispensable tool for proteomics research. One approach readily integrated with MS is protein footprinting. In-cell fast photochemical oxidation of proteins (IC-FPOP) is a protein footprinting method that utilizes hydroxyl radicals to oxidatively modify the side chains of solvent accessible amino acids. Liquid chromatography coupled to mass spectrometry is used to both identify modified amino acids and quantify the levels of labeling. Owing to solvent accessibility changing upon binding or changes in conformations, IC-FPOP can be used to identify protein-ligand and protein-protein interaction sites and regions of conformational change. The method can modify thousands of proteins in a single experiment leading to structural information across the proteome. IC-FPOP modifies proteins on the microsecond timescale making the method suitable to study fast biological processes. However, the single cell flow system developed for initial IC-FPOP experiments had temporal limitations motivating the design for a higher throughput platform. My research describes the development of a new platform for IC-FPOP entitled Platform Incubator with XY Movement (PIXY). PIXY permits IC-FPOP to occur in a sterile system using a temperature-controlled stage top incubator, peristaltic pumps for chemical transport, mirrors for laser beam guidance, and a mobile stage for XY movement. Automated communication amongst the entire PIXY system was made possible using LabVIEW software which allows the analysis of one sample in only 20 seconds. Well over 2000 proteins in HEK cells can be oxidatively modified by IC-FPOP in PIXY. This allows for a greater amount of structural information to be obtained. The capabilities of this high throughput platform permit other cell based experimental applications including fluorescent imaging and time-dependent solution transfer. PIXY’s ability to accommodate automated time points and subsequent changes over time make it a powerful tool for probing protein biochemistry in the native cellular environment.