Spatiotemporally Resolved Proteomics and Dynamic Biomaterial Control through Bioorthogonal Photochemistry

Abstract

Biological processes are staggeringly dynamic and heterogeneous, exhibiting regular change across a variety of time and length scales. Though all cells within an organism share a common genome, differential expression of genes into proteins regulate developmental processes, tissue morphogenesis and function, disease susceptibility and response, and a wide variety of intra- and extra-cellular signaling events. This thesis introduces the first tools for spatiotemporally resolved proteomics, enabling visualization and quantification of proteins produced in vitro and in vivo within user-defined regions of time and 3D space (i.e., 4D). Light-activated bioorthogonal non-canonical amino acid tagging (laBONCAT) exploits the photouncaging of a reactive amino acid prior to translational incorporation, while photoactivated in vitro/vivo tagging (PAINT) utilizes light to spatially tag proteins that were previously metabolically labeled. In both cases, affinity purification and quantitative mass spectrometric analysis permits 4D mapping of global protein production within living samples. Relying on bioorthogonal photochemistries that can be performed on demand and with single-micron resolution, this work demonstrates the ability to label newly synthesized proteins in 2D/3D culture and in zebrafish models with subcellular resolution. This unique approach is likely to prove useful in determining new diagnostic markers for disease, as well as expanding our knowledge of fundamental biology. Recognizing the precise 4D control that photochemistry affords, this thesis further expands the toolbox of photochemical reactions available, creating biomaterials whose chemical and physical properties can be modulated in a wavelength-dependent manner. Here, a photoorthogonal reaction scheme is introduced that combines photocleavable linkers based on ortho-nitrobenzyl ester (oNB) and a boron-dipyrromethene (BODIPY) that respond to UV and visible light, respectively. oNB remains undisturbed under 505 nm irradiation, while BODIPY undergoes rapid photolysis. Although BODIPY also cleaves with 365 nm irradiation, its comparative slow degradation kinetics permit reaction orthogonality with oNB under physiological conditions. This pair permits on-demand and spatially defined regulation of protein presentation within and degradation of hydrogel biomaterials, providing exciting opportunities for controlled drug delivery and tissue engineering applications.