Time-resolved assessment of single-cell protein secretion by sequencing

Secreted proteins play critical roles in cellular communication and functional orchestration. Methods enabling concurrent measurement of cellular protein secretion, phenotypes and transcriptomes are still unavailable. Here, we describe time-resolved assessment of protein secretion from single cells by sequencing (TRAPS-seq). Released proteins are trapped onto cell surface via affinity matrices, and the captured analytes together with phenotypic markers can be probed by oligonucleotide-barcoded antibodies and simultaneously sequenced with transcriptomes. We used TRAPS-seq to interrogate secretion dynamics of pleiotropic cytokines (IFN-γ, IL-2 and TNF-α) of early activated human T lymphocytes, unraveling limited correlation between cytokine secretion and its transcript abundance with regard to timing and strength. We found that early central memory T cells with CD45RA expression (TCMRA) are the most effective responders in multiple cytokine secretion, and polyfunctionality involves unique yet dynamic combinations of gene signatures over time. TRAPS-seq presents a useful tool for cellular indexing of secretions, phenotypes, and transcriptomes at single-cell resolution.

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NOMA: a high-throughput microchip for robust sequential measurements of secretions from the same single-cells

10.1101/2021.01.21.427049 ◽

2021 ◽

Author(s):

Meimei Liu ◽

Yahui Ji ◽

Fengjiao Zhu ◽

Xue Bai ◽

Linmei Li ◽

...

Keyword(s):

Single Cell ◽

Protein Secretion ◽

Single Cells ◽

Extended Period ◽

Extracellular Vesicle ◽

Single Cell Protein ◽

Cell Protein ◽

Cell Secretion ◽

Suspension Cells ◽

Sequential Measurements

AbstractDespite advances in single-cell secretion analysis technologies, lacking simple methods to reliably keep the live single-cells traceable for longitudinal detection poses a significant obstacle. Here we developed the high-density NOMA (narrow-opening microwell array) microchip that realized the retention of ≥97% of trapped single cells during repetitive detection procedures, regardless of adherent or suspension cells. We demonstrated its use to decode the correlation of protein abundance between secreted extracellular vesicles (EVs) and its donor cells at the same single-cell level, in which we found that these two were poorly correlated with each other. We further applied it in monitoring single-cell protein secretions sequentially from the same single cells. Notably, we observed the digital protein secretion patterns dominate the protein secretion. We also applied the microchip for longitudinally tracking of the single-cell integrative secretions over days, which revealed the presence of “super secretors” within the cell population that could be more persistent to secrete protein or extracellular vesicle for an extended period. The NOMA platform reported here is simple, robust, and easy to operate for realizing sequential measurements from the same single cells, representing a novel and informative tool to inspire new observations in biomedical research.

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Interfacing Droplet Microfluidics with Antibody Barcodes for Multiplexed Single-Cell Protein Secretion Profiling

Lab on a Chip ◽

10.1039/d1lc00567g ◽

2021 ◽

Author(s):

Tahereh Khajvand ◽

Peifeng Huang ◽

Linmei Li ◽

Mingxia Zhang ◽

Fengjiao Zhu ◽

...

Keyword(s):

Single Cell ◽

Protein Secretion ◽

Single Cells ◽

Single Cell Protein ◽

Droplet Microfluidics ◽

Cell Protein ◽

Cellular Functions ◽

Disease States

Multiplexed protein secretion analysis of single cells is important to understand the heterogeneity of cellular functions and processes in healthy and disease states. However, current single-cell platforms, such as microwell,...

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Single-Cell Protein Secretion Detection and Profiling

Annual Review of Analytical Chemistry ◽

10.1146/annurev-anchem-061318-115055 ◽

2019 ◽

Vol 12 (1) ◽

pp. 431-449 ◽

Cited By ~ 9

Author(s):

Zhuo Chen ◽

Jonathan J. Chen ◽

Rong Fan

Keyword(s):

Single Cell ◽

Protein Secretion ◽

Single Cells ◽

Cell Communication ◽

Basic Research ◽

Immune Monitoring ◽

Single Cell Protein ◽

Droplet Microfluidics ◽

Cell Protein ◽

Cell Cell

Secreted proteins play important roles in mediating various biological processes such as cell–cell communication, differentiation, migration, and homeostasis at the population or tissue level. Here, we review bioanalytical technologies and devices for detecting protein secretions from single cells. We begin by discussing conventional approaches followed by detailing the latest advances in microengineered systems for detecting single-cell protein secretions with an emphasis on multiplex measurement. These platforms include droplet microfluidics, micro-/nanowell-based assays, and microchamber-based assays, among which the advantages and limitations are compared. Microscale systems also enable the tracking of protein secretion dynamics in single cells, further empowering the study of the cell–cell communication network. Looking forward, we discuss the remaining challenges and future opportunities that will transform basic research of cellular secretion functions at the systems level and the clinical applications for immune monitoring and cancer treatment.

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Direct correlation between motile behavior and protein abundance in single cells

10.1101/067918 ◽

2016 ◽

Author(s):

Yann S Dufour ◽

Sébastien Gillet ◽

Nicholas W Frankel ◽

Douglas B Weibel ◽

Thierry Emonet

Keyword(s):

Protein Expression ◽

Single Cell ◽

Phenotypic Diversity ◽

Single Cells ◽

Selective Advantage ◽

Single Cell Protein ◽

Cell Protein ◽

Chemotaxis Proteins

AbstractUnderstanding how stochastic molecular fluctuations affect cell behavior requires the quantification of both behavior and protein numbers in the same cells. Here, we combine automated microscopy with in situ hydrogel polymerization to measure single-cell protein expression after tracking swimming behavior. We characterized the distribution of non-genetic phenotypic diversity in Escherichia coli motility, which affects single-cell exploration. By expressing fluorescently tagged chemotaxis proteins (CheR and CheB) at different levels, we quantitatively mapped motile phenotype (tumble bias) to protein numbers using thousands of single-cell measurements. Our results disagreed with established models until we incorporated the role of CheB in receptor deamidation and the slow fluctuations in receptor methylation. Beyond refining models, our central finding is that changes in numbers of CheR and CheB affect the population mean tumble bias and its variance independently. Therefore, it is possible to adjust the degree of phenotypic diversity of a population by adjusting the global level of expression of CheR and CheB while keeping their ratio constant, which, as shown in previous studies, confers functional robustness to the system. Since genetic control of protein expression is heritable, our results suggest that non-genetic diversity in motile behavior is selectable, supporting earlier hypotheses that such diversity confers a selective advantage.

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SIFTER: Electrophoretic complex fractionation protocol

10.21203/rs.3.pex-1582/v1 ◽

2021 ◽

Author(s):

Julea Vlassakis ◽

Louise L Hansen ◽

Amy E Herr

Keyword(s):

Single Cell ◽

Protein Interaction ◽

Gel Electrophoresis ◽

Protein Complexes ◽

Single Cells ◽

Polyacrylamide Gel Electrophoresis ◽

Simultaneous Detection ◽

Single Cell Protein ◽

Cell Protein ◽

A Cell

Abstract We introduce micro-arrayed, differential detergent fractionation for the simultaneous detection of protein complexes in 100s of individual cells with SIFTER (Single-cell protein Interaction Fractionation Through Electrophoresis and immunoassay Readout). Size-based fractionation of protein complexes is accomplished with five assay steps. First, a cell suspension generated by trypsinization is introduced onto a microwell array, and single cells are settled into the microwells by gravity. Cells are lysed in F-actin stabilization buffer that is delivered by a hydrogel lid. Second, the protein complexes are fractionated from the smaller monomers by polyacrylamide gel electrophoresis. Monomers are electrophoresed into the gel and are immobilized using a UV-induced covalent reaction to benzophenone. Third, a protein-complex depolymerization buffer is introduced by another hydrogel lid. Fourth, the recently depolymerized complexes are electrophoresed into a region of the gel separate from the immobilized monomers, where the complex fraction are in turn immobilized. Fifth, in-gel immunoprobing detects the immobilized populations of monomer and depolymerized complexes. These general steps are built on previously published protocols for bulk actin studies, single-cell western blotting, and bidirectional separations1-4.

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Single-cell proteomic and transcriptomic analysis of macrophage heterogeneity using SCoPE2

Genome Biology ◽

10.1186/s13059-021-02267-5 ◽

2021 ◽

Vol 22 (1) ◽

Cited By ~ 4

Author(s):

Harrison Specht ◽

Edward Emmott ◽

Aleksandra A. Petelski ◽

R. Gray Huffman ◽

David H. Perlman ◽

...

Keyword(s):

Single Cell ◽

Single Cell Analysis ◽

Single Cells ◽

Single Cell Protein ◽

Cellular Heterogeneity ◽

Cell Protein ◽

Alternatively Activated Macrophages ◽

Monocytes And Macrophages ◽

Molecular Phenotypes ◽

Post Transcriptional Regulation

Abstract Background Macrophages are innate immune cells with diverse functional and molecular phenotypes. This diversity is largely unexplored at the level of single-cell proteomes because of the limitations of quantitative single-cell protein analysis. Results To overcome this limitation, we develop SCoPE2, which substantially increases quantitative accuracy and throughput while lowering cost and hands-on time by introducing automated and miniaturized sample preparation. These advances enable us to analyze the emergence of cellular heterogeneity as homogeneous monocytes differentiate into macrophage-like cells in the absence of polarizing cytokines. SCoPE2 quantifies over 3042 proteins in 1490 single monocytes and macrophages in 10 days of instrument time, and the quantified proteins allow us to discern single cells by cell type. Furthermore, the data uncover a continuous gradient of proteome states for the macrophages, suggesting that macrophage heterogeneity may emerge in the absence of polarizing cytokines. Parallel measurements of transcripts by 10× Genomics suggest that our measurements sample 20-fold more protein copies than RNA copies per gene, and thus, SCoPE2 supports quantification with improved count statistics. This allowed exploring regulatory interactions, such as interactions between the tumor suppressor p53, its transcript, and the transcripts of genes regulated by p53. Conclusions Even in a homogeneous environment, macrophage proteomes are heterogeneous. This heterogeneity correlates to the inflammatory axis of classically and alternatively activated macrophages. Our methodology lays the foundation for automated and quantitative single-cell analysis of proteins by mass spectrometry and demonstrates the potential for inferring transcriptional and post-transcriptional regulation from variability across single cells.

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Advanced Biosensing towards Real-Time Imaging of Protein Secretion from Single Cells

10.5772/intechopen.94248 ◽

2020 ◽

Author(s):

Lang Zhou ◽

Pengyu Chen ◽

Aleksandr Simonian

Keyword(s):

Single Cell ◽

Real Time ◽

Protein Secretion ◽

Intercellular Communication ◽

Single Cells ◽

Bone Marrow Failure ◽

Cellular Protein ◽

Label Free ◽

Immune Effector ◽

Real Time Imaging

Protein secretion of cells plays a vital role in intercellular communication. The abnormality and dysfunction of cellular protein secretion are associated with various physiological disorders, such as malignant proliferation of cells, aberrant immune function, and bone marrow failure. The heterogeneity of protein secretion exists not only between varying populations of cells, but also in the same phenotype of cells. Therefore, characterization of protein secretion from single cell contributes not only to the understanding of intercellular communication in immune effector, carcinogenesis and metastasis, but also to the development and improvement of diagnosis and therapy of relative diseases. In spite of abundant highly sensitive methods that have been developed for the detection of secreted proteins, majority of them fall short in providing sufficient spatial and temporal resolution for comprehensive profiling of protein secretion from single cells. The real-time imaging techniques allow rapid acquisition and manipulation of analyte information on a 2D plane, providing high spatiotemporal resolution. Here, we summarize recent advances in real-time imaging of secretory proteins from single cell, including label-free and labelling techniques, shedding light on the development of simple yet powerful methodology for real-time imaging of single-cell protein secretion.

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Single-cell proteomic and transcriptomic analysis of macrophage heterogeneity

10.1101/665307 ◽

2019 ◽

Cited By ~ 20

Author(s):

Harrison Specht ◽

Edward Emmott ◽

Aleksandra A. Petelski ◽

R. Gray Huffman ◽

David H. Perlman ◽

...

Keyword(s):

Single Cell ◽

Single Cell Analysis ◽

Single Cells ◽

Single Cell Protein ◽

Cellular Heterogeneity ◽

Cell Protein ◽

Alternatively Activated Macrophages ◽

Monocytes And Macrophages ◽

Molecular Phenotypes ◽

Post Transcriptional Regulation

AbstractMacrophages are innate immune cells with diverse functional and molecular phenotypes. This diversity is largely unexplored at the level of single-cell proteomes because of limitations of quantitative single-cell protein analysis. To overcome this limitation, we developed SCoPE2, which substantially increases quantitative accuracy and throughput while lowering cost and hands-on time by introducing automated and miniaturized sample preparation. These advances enable us to analyze the emergence of cellular heterogeneity as homogeneous monocytes differentiate into macrophage-like cells in the absence of polarizing cytokines. SCoPE2 quantified over 3,042 proteins in 1,490 single monocytes and macrophages in ten days of instrument time, and the quantified proteins allow us to discern single cells by cell type. Furthermore, the data uncover a continuous gradient of proteome states for the macrophages, suggesting that macrophage heterogeneity may emerge in the absence of polarizing cytokines. This gradient correlates to the inflammatory axis of classically and alternatively activated macrophages. Parallel measurements of transcripts by 10x Genomics suggest that our measurements sample 20-fold more protein copies than RNA copies per gene, and thus SCoPE2 supports quantification with improved count statistics. The joint distributions of proteins and transcripts allowed exploring regulatory interactions, such as between the tumor suppressor p53, its transcript, and the transcripts of genes regulated by p53. Our methodology lays the foundation for quantitative single-cell analysis of proteins by mass-spectrometry and demonstrates the potential for inferring transcriptional and post-transcriptional regulation from variability across single cells.Abstract Figure

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Combined aptamer and transcriptome sequencing of single cells

10.1101/228338 ◽

2017 ◽

Cited By ~ 2

Author(s):

Cyrille L. Delley ◽

Leqian Liu ◽

Maen F. Sarhan ◽

Adam R. Abate

Keyword(s):

Single Cell ◽

Single Cells ◽

Expression Patterns ◽

Cell Types ◽

Single Cell Protein ◽

Protein Characterization ◽

Cell Protein ◽

Surface Binding ◽

Distinct Cell

AbstractThe transcriptome and proteome encode distinct information that is important for characterizing heterogeneous biological systems. We demonstrate a method to simultaneously characterize the transcriptomes and proteomes of single cells at high throughput using aptamer probes and droplet-based single cell sequencing. With our method, we differentiate distinct cell types based on aptamer surface binding and gene expression patterns. Aptamers provide advantages over antibodies for single cell protein characterization, including rapid, in vitro, and high-purity generation via SELEX, and the ability to amplify and detect them with PCR and sequencing.

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Combined mRNA and protein single cell analysis in a dynamic cellular system using SPARC

10.1101/749473 ◽

2019 ◽

Cited By ~ 1

Author(s):

Johan Reimegård ◽

Marcus Danielsson ◽

Marcel Tarbier ◽

Jens Schuster ◽

Sathishkumar Baskaran ◽

...

Keyword(s):

Protein Expression ◽

Mrna Expression ◽

Single Cell ◽

Single Cell Analysis ◽

Single Cells ◽

Embryonic Stem ◽

Single Cell Protein ◽

Cell Protein ◽

Depth Analysis ◽

Mrna And Protein Expression

ABSTRACTCombined measurements of mRNA and protein expression in single cells enables in-depth analysis of cellular states. We present single-cell protein and RNA co-profiling (SPARC), an approach to simultaneously measure global mRNA and large sets of intracellular protein in individual cells. Using SPARC, we show that mRNA expression fails to accurately reflect protein abundance at the time of measurement in human embryonic stem cells, although the direction of changes of mRNA and protein expression are in agreement during cellular differentiation. Moreover, protein levels of transcription factors better predict their downstream effects than do the corresponding transcripts. We further show that changes of the balance between protein and mRNA expression levels can be applied to infer expression kinetic trajectories, revealing future states of individual cells. Finally, we highlight that mRNA expression may be more varied among cells than levels of the corresponding proteins. Overall, our results demonstrate that mRNA and protein measurements in single cells provide different and complementary information regarding cell states. Accordingly, SPARC can offer valuable insights in gene expression programs of single cells.

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