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Spatial proteomics at cellular and subcellular resolution by coupling deep ultraviolet laser ablation with nanodroplet sample preparation

ABSTRACT: Spatial proteomics can provide an in-depth molecular-level interpretation of cellular functions in both physiological and pathological environments. Despite great potential, spatial proteomics is challenged by not only limited proteome coverage but also poor spatial resolution and low throughput. We have previously developed a spatial proteomics platform by integrating nanodroplet sample preparation with laser capture microdissection, and deployed it to robustly profile 500-2000 proteins from microdissected tissue samples as small as 50 um (~8 cells). Herein, we push the spatial resolution to subcellular scale (~7 um) by developing an image-guided deep UV ablation system. This novel spatial proteomics platform allowed us to globally profile organelles inside single cells.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Ljiljana Pasa-Tolic

PROVIDER: MSV000092014 | MassIVE | Tue May 23 09:44:00 BST 2023

REPOSITORIES: MassIVE

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Project description:Spatial organization of the transcriptome has emerged as a powerful means for regulating the post-transcriptional fate of RNA in eukaryotes; however, whether prokaryotes use RNA spatial organization as a mechanism for post-transcriptional regulation remains unclear. Here we used super-resolution microscopy to image the E. coli transcriptome and observed a genome-wide spatial organization of RNA: mRNAs encoding inner-membrane proteins are enriched at the membrane, whereas mRNAs encoding outer-membrane, cytoplasmic and periplasmic proteins are distributed throughout the cytoplasm. Membrane enrichment is caused by co-translational insertion of signal peptides recognized by the signal-recognition particle. Our time-resolved RNA-sequencing and live-cell super-resolution imaging experiments revealed a physiological consequence of this spatial organization and the underlying mechanism: membrane localization enhances degradation rates of inner-membrane-protein mRNAs by placing them in proximity to membrane-bound RNA degradation enzymes. Together, these results demonstrate that the bacterial transcriptome is spatially organized and that this organization shapes the posttranscriptional Spatial organization of the transcriptome has emerged as a powerful means for regulating the post-transcriptional fate of RNA in eukaryotes; however, whether prokaryotes use RNA spatial organization as a mechanism for post-transcriptional regulation remains unclear. Here we used super-resolution microscopy to image the E. coli transcriptome and observed a genome-wide spatial organization of RNA: mRNAs encoding inner-membrane proteins are enriched at the membrane, whereas mRNAs encoding outer-membrane, cytoplasmic and periplasmic proteins are distributed throughout the cytoplasm. Membrane enrichment is caused by co-translational insertion of signal peptides recognized by the signal-recognition particle. Our time-resolved RNA-sequencing and live-cell super-resolution imaging experiments revealed a physiological consequence of this spatial organization and the underlying mechanism: membrane localization enhances degradation rates of inner-membrane-protein mRNAs by placing them in proximity to membrane-bound RNA degradation enzymes. Together, these results demonstrate that the bacterial transcriptome is spatially organized and that this organization shapes the post-transcriptional dynamics of mRNAs.

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Spatial proteomics at cellular and subcellular resolution by coupling deep ultraviolet laser ablation with nanodroplet sample preparation

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