Highly multiplexed detection of proteins secreted by single cells is always challenging. Herein, a multiplexed in situ tagging technique based on single‐stranded DNA encoded microbead arrays and multicolor successive imaging for assaying single‐cell secreted proteins with high throughput and high sensitivity is presented. This technology is demonstrated to be capable of increasing the multiplexity exponentially. Upon integration with polydimethylsiloxane microwells, this platform is applied to detect ten immune effector proteins from differentiated single macrophages stimulated with lipopolysaccharide. Significant heterogeneity is observed when the derived human primary macrophages are analyzed. This versatile technology is expected to open new opportunities in systems biology, immune regulation studies, signaling analysis, and molecular diagnostics.
Transient, site-specific, or so-called quinary, interactions are omnipresent in live cells and modulate protein stability and activity. Quinary intreactions are readily detected by in-cell NMR spectroscopy as severe broadening of the NMR signals. Intact ribosome particles were shown to be necessary for the interactions that give rise to the NMR protein signal broadening observed in cell lysates and sufficient to mimic quinary interactions present in the crowded cytosol. Recovery of target protein NMR spectra that were broadened in lysates, in vitro and in the presence of purified ribosomes was achieved by RNase A digestion only after the structure of the ribosome was destabilized by removing magnesium ions from the system. Identifying intact ribosomal particles as the major protein-binding component of quinary interactions and consequent spectral peak broadening will facilitate quantitative characterization of macromolecular crowding effects in live cells and streamline models of metabolic activity.
Analytical tools to study cell physiology are critical for optimizing drug-host interactions. Real time pulse chase NMR spectroscopy, RTPC-NMR, was introduced to monitor the kinetics of metabolite production in HEK 293T cells treated with COVID-19 vaccine-like lipid nanoparticles, LNPs, with and without mRNA. Kinetic flux parameters were resolved for the incorporation of isotopic label into metabolites and clearance of labeled metabolites from the cells. Changes in the characteristic times for alanine production implicated mitochondrial dysfunction as a consequence of treating the cells with lipid nanoparticles, LNPs. Mitochondrial dysfunction was largely abated by inclusion of mRNA in the LNPs, the presence of which increased the size and uniformity of the LNPs. The methodology is applicable to all cultured cells.
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