Multiplexed single cell proteomics of hypoxia in HEK293 expressing PIP-FUCCI

This study aimed to characterize temporal proteome dynamics of hypoxia using single-cell mass spectrometry-based proteomics. HEK293 cells expressing PIP-FUCCI cell cycle markers were grown in suspension culture. Cells were exposed to hypoxia and temporally sampled for single-cell sorting into 384-well plates. Samples were processed and multiplexed using TMT labeling according to the SCoPE2 workflow and analyzed using the RETICLE acquisition method. In parallel, single-cell RNA-seq data were also collected. These datasets were used together to disentangle the effects of hypoxia from cell cycle progression and to construct joint transcriptional and translational trajectories along the hypoxia response.