Quantification of extracellular proteins, protein complexes and mRNAs in single cells by proximity sequencing

Vistain, Luke; Phan, Hoang Van; Keisham, Bijentimala; Jordi, Claudio; Chen, Meng-Jie; Reddy, Sai T.; Tay, Savaş

doi:10.1038/s41592-022-01684-z

Cited by 21 publications

(23 citation statements)

References 59 publications

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“…We found that VEGFA transcripts and VEGF-A secretion have very low correlation in normoxic and hypoxic MSCs. While surprising, the uncoupling of transcript level and protein secretion is in line with similar studies using CITE-seq where certain surface protein and transcripts feature low correlation 14,15,45 .…”

Section: Discussionsupporting

confidence: 84%

Secretion encoded single-cell sequencing (SEC-seq) uncovers gene expression signatures associated with high VEGF-A secretion in mesenchymal stromal cells

Udani

Langerman

Koo

et al. 2023

Preprint

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Cells secrete numerous bioactive molecules essential for the function of healthy organisms. However, there are no scalable methods to link individual cell secretions to their transcriptional state. By developing and using secretion encoded single-cell sequencing (SEC-seq), which exploits hydrogel nanovials to capture individual cells and their secretions, we simultaneously measured the secretion of vascular endothelial growth factor A (VEGF-A) and the transcriptome for thousands of individual mesenchymal stromal cells (MSCs). We found that VEGF-A secretion is heterogeneous across the cell population and lowly correlated with the VEGFA transcript level. While there is a modest population-wide increase in VEGF-A secretion by hypoxic induction, highest VEGF-A secretion across normoxic and hypoxic culture conditions occurs in a subpopulation of MSCs characterized by a unique gene expression signature. Taken together, SEC-seq enables the identification of specific genes involved in the control of secretory states, which may be exploited for developing means to modulate cellular secretion for disease treatment.

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Section: Discussionsupporting

confidence: 84%

Secretion encoded single-cell sequencing (SEC-seq) uncovers gene expression signatures associated with high VEGF-A secretion in mesenchymal stromal cells

Udani

Langerman

Koo

et al. 2023

Preprint

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“…One of the advantages of using padlock probes is the power of signal amplification by RCA. Therefore, ARTseq-FISH has the potential to detect DNA (i.e., single nucleotide polymorphisms (SNPs)) 74 , miRNAs 75, 76 , viral RNA 77 , and proximity of proteins 37, 78 or even proximity of RNA and protein. In addition, due to the signal amplification of RCA, the results of ARTseq-FISH can also be read out by flow cytometry 79, 80 , which makes this and future techniques more broadly accessible.…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, the resolution of protein detection is often below the level of RNA detection proving a barrier to quantitative insights into protein location or spatial correlation of RNA and protein abundance 24,26,[28][29][30][31][32] . Conversely, DNA-barcoding dependent methods (for example, CODEX 33 , ImmunoSABER 34 and InSituPlex® 35 ) have shown their multiplexing capacity in protein detection, but have thus far not been integrated with RNA detection 36,37 .…”

Section: Introductionmentioning

confidence: 99%

ARTseq-FISH reveals position-dependent fate decisions driven by cell cycle changes

Sluijs¹,

Blay²,

Stepanov³

et al. 2022

Preprint

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Cell fate decisions are ubiquitous and play a critical role throughout development, yet how localization impacts cellular decision making remains unclear. To identify the drivers of position-dependent fate decisions at a molecular level, we developed a scalable antibody and mRNA targeting sequential fluorescence in situ hybridization (ARTseq-FISH) method capable of simultaneously profiling mRNAs, proteins and phosphoproteins in single cells at sub-micrometre spatial resolution. We studied 67 unique (phospho-)protein and mRNA targets in individual mouse embryonic stem cells (mESCs) cultured on circular micropatterns, yielding quantification of both abundance and localization of mRNAs and (phospho-)proteins during the first 48 hours of differentiation. ARTseq-FISH revealed a fate decision between continued self-renewal and differentiation that relies solely on the position of each mESC on the micropattern. Our results demonstrate that temporal changes in cell cycle orchestrate these position-dependent cell fate decisions.

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“…To investigate the roles of protein interactions in greater depth, we recently developed a method called proximity sequencing (Prox-seq) for simultaneous quantification of mRNA, surface proteins and protein complexes at the single-cell level [ 4 ] Prox-seq captures protein complex information in barcoded DNA oligonucleotides (oligos) using a proximity ligation assay [ 5 , 6 ] (PLA). Each protein in Prox-seq is targeted by two DNA-conjugated antibodies, called Prox-seq probes A and B ( Fig 1a ).…”

Section: Introductionmentioning

confidence: 99%

Computational prediction of protein interactions in single cells by proximity sequencing

Xia,

Phan,

Vistain

et al. 2024

PLoS Comput Biol

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Proximity sequencing (Prox-seq) simultaneously measures gene expression, protein expression and protein complexes on single cells. Using information from dual-antibody binding events, Prox-seq infers surface protein dimers at the single-cell level. Prox-seq provides multi-dimensional phenotyping of single cells in high throughput, and was recently used to track the formation of receptor complexes during cell signaling and discovered a novel interaction between CD9 and CD8 in naïve T cells. The distribution of protein abundance can affect identification of protein complexes in a complicated manner in dual-binding assays like Prox-seq. These effects are difficult to explore with experiments, yet important for accurate quantification of protein complexes. Here, we introduce a physical model of Prox-seq and computationally evaluate several different methods for reducing background noise when quantifying protein complexes. Furthermore, we developed an improved method for analysis of Prox-seq data, which resulted in more accurate and robust quantification of protein complexes. Finally, our Prox-seq model offers a simple way to investigate the behavior of Prox-seq data under various biological conditions and guide users toward selecting the best analysis method for their data.

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Quantification of extracellular proteins, protein complexes and mRNAs in single cells by proximity sequencing

Cited by 21 publications

References 59 publications

Secretion encoded single-cell sequencing (SEC-seq) uncovers gene expression signatures associated with high VEGF-A secretion in mesenchymal stromal cells

Secretion encoded single-cell sequencing (SEC-seq) uncovers gene expression signatures associated with high VEGF-A secretion in mesenchymal stromal cells

ARTseq-FISH reveals position-dependent fate decisions driven by cell cycle changes

Computational prediction of protein interactions in single cells by proximity sequencing

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