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A combined approach for single-cell mRNA and intracellular protein expression analysis.

ABSTRACT: Combined measurements of mRNA and protein expression in single cells enable in-depth analysis of cellular states. We present SPARC, an approach that combines single-cell RNA-sequencing with proximity extension essays to simultaneously measure global mRNA and 89 intracellular proteins in individual cells. We show that mRNA expression fails to accurately reflect protein abundance at the time of measurement, although the direction of changes is in agreement during neuronal differentiation. Moreover, protein levels of transcription factors better predict their downstream effects than do their corresponding transcripts. Finally, we highlight that protein expression variation is overall lower than mRNA variation, but relative protein variation does not reflect the mRNA level. Our results demonstrate that mRNA and protein measurements in single cells provide different and complementary information regarding cell states. SPARC presents a state-of-the-art co-profiling method that overcomes current limitations in throughput and protein localization, including removing the need for cell fixation.

SUBMITTER: Reimegard J

PROVIDER: S-EPMC8149646 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundHematopoietic stem and progenitor cell (HSPC) subsets in mice have previously been studied using cell surface markers, and more recently single-cell technologies. The recent revolution of single-cell analysis is substantially transforming our understanding of hematopoiesis, confirming the substantial heterogeneity of cells composing the hematopoietic system. While dynamic molecular changes at the DNA/RNA level underlying hematopoiesis have been extensively explored, a broad understanding of single-cell heterogeneity in hematopoietic signaling programs and landscapes, studied at protein level and reflecting post-transcriptional processing, is still lacking. Here, we accurately quantified the intracellular levels of 9 phosphorylated and 2 functional proteins at the single-cell level to systemically capture the activation dynamics of 8 signaling pathways, including EGFR, Jak/Stat, NF-κB, MAPK/ERK1/2, MAPK/p38, PI3K/Akt, Wnt, and mTOR pathways, during mouse hematopoiesis using mass cytometry.ResultsWith fine-grained analyses of 3.2 million of single hematopoietic stem and progenitor cells (HSPCs), and lineage cells in conjunction with multiparameter cellular phenotyping, we mapped trajectories of signaling programs during HSC differentiation and identified specific signaling biosignatures of cycling HSPC and multiple differentiation routes from stem cells to progenitor and lineage cells. We also investigated the recovery pattern of hematopoietic cell populations, as well as signaling regulation in these populations, during hematopoietic reconstruction. Overall, we found substantial heterogeneity of pathway activation within HSPC subsets, characterized by diverse patterns of signaling.ConclusionsThese comprehensive single-cell data provide a powerful insight into the intracellular signaling-regulated hematopoiesis and lay a solid foundation to dissect the nature of HSC fate decision. Future integration of transcriptomics and proteomics data, as well as functional validation, will be required to verify the heterogeneity in HSPC subsets during HSC differentiation and to identify robust markers to phenotype those HSPC subsets.

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A combined approach for single-cell mRNA and intracellular protein expression analysis.

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