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Macrophages play a vital role in the innate immune system, identifying and destroying unwanted cells. However, it has been difficult to attain a comprehensive understanding of macrophage protein abundance due to technical limitations. In addition, it remains unclear how changes in proteome composition are linked to phagocytic activity. In this study we developed methods to derive human macrophages and prepare them for mass spectrometry analysis in order to more-deeply understand the proteomic consequences of macrophage stimulation. Interferon gamma (IF-g), an immune stimulating cytokine, was used to induce macrophage activation, increasing phagocytosis of cancer cells by 2-fold. These conditions were used to perform comparative shotgun proteomics between resting macrophages and stimulated macrophages with increased phagocytic activity. Our analysis revealed that macrophages bias their protein production toward biological processes associated with phagocytosis and antigen processing in response to stimulation. We confirmed our findings by antibody-based western blotting experiments, validating both previously reported and novel proteins of interest. In addition to whole protein changes, we evaluated active protein synthesis by treating cells with the methionine surrogate probe homopropargylglycine (HPG). We saw increased rates of HPG incorporation during phagocytosis-inducing stimulation, suggesting protein synthesis rates are altered by stimulation. Together our findings provide the most comprehensive proteomic insight to date into primary human macrophages. We anticipate that this data can be used as a launchpoint to generate new hypotheses about innate immune function.

The genome of a living cell is continuously damaged by various exogenous and endogenous factors yielding multiple types of DNA damage including base damage and damage to the sugar-phosphate backbone of DNA. Double Strand Breaks (DSBs) are the most severe form of DNA damage and if left unchecked, may precipitate genomic rearrangements, cell death or contribute to malignancy. In clinical contexts, radiation is often used to induce DSBs as a form of genotoxic therapy. Despite the importance of DSBs and their repair, as yet there is no facile assay to detect DSBs in situ or to quantify their location or proximity to other cellular constituents. Such an assay would help to disentangle DDR signaling pathways and identify new molecular players involved in DSB repair. These efforts, in turn, may facilitate drug screening and accelerate the discovery of novel, more effective genotoxic agents. We have developed such an assay, presented here, and term it TdT-dUTP DSB End Labeling (TUDEL).TUDEL makes use of Terminal Deoxynucleotidyl Transferase (TdT), a template-independent DNA polymerase. TdT is commonly used in TUNEL assays to yield a binary output of DNA damage. We have adapted this approach, using TdT and EdUTP to label individual DNA double strand breaks in irradiated cells and detecting the incorporated EdU with fluorescent probes via Click chemistry. This tool complements and is compatible with existing, indirect methods to track DSBs such as immunofluorescent detection of γH2AX. TUDEL is also sufficiently specific, sensitive, quantitative, and robust to replace the neutral Comet assay for routine measurement of DSB formation and repair. Here we present a protocol for TUDEL., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)