Hemoprotein discovery through proteomic techniques

Abstract

Heme is an essential cofactor and signaling molecule. Due to hydrophobicity and toxicity, heme must be tightly regulated to prevent cellular dysfunction. Despite this fact, the manner by which heme is regulated is not well known. Understanding the molecules and mechanisms required to maintain heme homeostasis would be greatly beneficial in understanding heme regulation in health and disease. Historically, hemin-agarose resin has been utilized to identify new hemoproteins. However, this approach is susceptible to the non-specific binding of a large number of proteins in the proteome, making the identification of new hemoproteins difficult. In order to improve upon this technique, we have utilized biorthogonally tagged heme analogs to enrich heme-binding proteins. These heme analogs, which contain an azide, can be fed to cells and distributed using the cell’s own trafficking machinery. Then, through phosphine-azide copper-free click chemistry, biotin can be conjugated to the azide-tagged heme analogs for subsequent enrichment on streptavidin-resin. We demonstrate that these azide-linked heme analogs can enrich model hemoproteins in vitro and can be imported by cells. We also were able to optimize the method of hemoprotein enrichment in Saccharomyces cerevisiae. This technique could be extended to many cell types, in numerous contexts to gain a more thorough understanding of the heme-binding proteome in health and disease.