Abstract
One of the areas in which bioorthogonal chemistry-chemistry performed inside a cell or organism-has become of pivotal importance is in the study of host-pathogen interactions. The incorporation of bioorthogonal groups into the cell wall or proteome of intracellular pathogens has allowed study within the endolysosomal system. However, for the approach to be successful, the incorporated bioorthogonal groups must be stable to chemical conditions found within these organelles, which are some of the harshest found in metazoans: the groups are exposed to oxidizing species, acidic conditions, and reactive thiols. Here we present an assay that allows the assessment of the stability of bioorthogonal groups within host cell phagosomes. Using a flow cytometry-based assay, we have quantified the relative label stability inside dendritic cell phagosomes of strained and unstrained alkynes. We show that groups that were shown to be stable in other systems were degraded by as much as 79% after maturation of the phagosome.