Peptides presented by the class-I major histocompatibility complex (MHC-I) are important targets for immunotherapy. The identification of these peptide targets greatly facilitates the generation of T-cell-based therapeutics. Herein, we report the capability of proteolysis targeting chimera (PROTAC) compounds to induce the presentation of specific MHC class-I peptides derived from endogenous cellular proteins. Using LC-MS/MS, we identified several BET-derived MHC-I peptides induced by treatment with three BET-directed PROTAC compounds. To understand our ability to tune this process, we measured the relative rate of presentation of these peptides under varying treatment conditions using label-free mass spectrometry quantification. We found that the rate of peptide presentation reflected the rate of protein degradation, indicating a direct relationship between PROTAC treatment and peptide presentation. We additionally analyzed the effect of PROTAC treatment on the entire immunopeptidome and found many new peptides that were displayed in a PROTAC-specific fashion: we determined that these identifications map to the BET pathway, as well as, potential off-target or unique-to-PROTAC pathways. This work represents the first evidence of the use of PROTAC compounds to induce the presentation of MHC-I peptides from endogenous cellular proteins, highlighting the capability of PROTAC compounds for the discovery and generation of new targets for immunotherapy.
A triarylphosphine reagent that reacts with organic azides to install amide-linked diazirines is reported. This traceless Staudinger reagent reacts with complex organic azides to yield amide-linked diazirines, thus expanding the scope of the utility of both azide and diazirine chemistry.
The use of small-molecule fluorophores to label proteins with minimal perturbation in response to an external stimulus is a powerful tool to probe chemical and biochemical environments. Herein we describe first the use of a coumarin modified triazabutadiene that can deliver aryl diazonium ions to fluorescently label proteins via tyrosine selective modification. The labeling can be triggered by low pH induced liberation of the diazonium species, making the fluorophore specifically useful in labeling biochemical surroundings such as those found within the late endosome. Additionally, we show that a variety of coumarin triazabutadienes may also be prone to releasing their diazonium cargo via irradiation with UV light.
Chemical crosslinking is a versatile tool for the examination of biochemical interactions, in particular host-pathogen interactions. Herein we report a critical first step toward the goal of probing these interactions with the synthesis and use of a new heterobifunctional crosslinker containing a triazabutadiene scaffold. The triazabutadiene is stable to protein conjugation and liberates a reactive aryl diazonium species upon irradiation with 350 nm light. We highlight the use of this technology by modifiying the surface of several proteins, including dengue virus.
Dengue virus (DENV) is a mosquito-transmitted flavivirus that infects approximately 100 million people annually. Multi-day protocols for purification of DENV reduce the infective titer due to viral sensitivity to both temperature and pH. Herein we describe a 5-h protocol for the purification of all DENV serotypes, utilizing traditional gradient-free ultracentrifugation followed by selective virion precipitation. This protocol allows for the separation of DENV from contaminating proteins – including intact C6/36 densovirus, for the production of infective virus at high concentration for protein-level analysis.
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