SIFTER: Electrophoretic complex fractionation protocol

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.

scholarly journals Quantifying cytoskeletal heterogeneity via single-cell protein-complex fractionation

2020 ◽  
Author(s):  
Julea Vlassakis ◽  
Louise L. Hansen ◽  
Ryo Higuchi-Sanabria ◽  
Yun Zhou ◽  
C. Kimberly Tsui ◽  
...  
Keyword(s):  
Single Cell ◽  
Protein Complex ◽  
Protein Complexes ◽  
Single Cells ◽  
Single Cell Protein ◽  
Cellular Heterogeneity ◽  
Size Exclusion ◽  
Cell Protein ◽  
Intermediate Filament Protein ◽  
Filament Protein

AbstractMultimeric cytoskeletal protein complexes, including filamentous F-actin, orchestrate normal cellular function. However, protein-complex distributions in stressed, heterogeneous cell populations remain unknown. Cell staining and proximity-based methods have limited selectivity and/or sensitivity for robust endogenous multimeric protein-complex quantification from single cells. We introduce micro-arrayed differential detergent fractionation to simultaneously detect protein complexes in 100s of individual cells. Fractionation occurs by 60s size-exclusion electrophoresis with protein complex-stabilizing buffer that minimizes depolymerization. Validating with actin-destabilizing Latrunculin A (LatA), we quantify 2.7-fold lower median F-actin complex-levels in LatA-treated single cells. Further clustering analysis of U2OS cells treated with LatA detects a subpopulation (∼11%) exhibiting downregulated F-actin, but upregulated microtubule and intermediate filament protein complexes. Thus, some cells upregulate other cytoskeletal complexes to counteract the stress of LatA treatment. We also sought to understand the effect of non-chemical stress on cellular heterogeneity of F-actin, and find heat shock dysregulates F and monomeric G-actin correlation. The assay overcomes selectivity limitations of existing methods to biochemically quantify single-cell protein complexes perturbed with diverse stimuli.

scholarly journals Direct correlation between motile behavior and protein abundance in single cells

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.

scholarly journals A self-exciting point process to study multi-cellular spatial signaling patterns

2020 ◽  
Author(s):  
Archit Verma ◽  
Siddhartha G. Jena ◽  
Danielle R. Isakov ◽  
Kazuhiro Aoki ◽  
Jared E. Toettcher ◽  
...  
Keyword(s):  
Point Process ◽  
Point Processes ◽  
Single Cells ◽  
Cell Types ◽  
Single Cell Protein ◽  
Cellular Systems ◽  
Cell Protein ◽  
Drug Induced ◽  
Spatiotemporal Model ◽  
A Cell

Multi-cellular organisms rely on spatial signaling among cells to drive their organization, development, and response to stimuli. Several models have been proposed to capture the behavior of spatial signaling in multi-cellular systems, but existing approaches fail to capture both the autonomous behavior of single cells and the interactions of a cell with its neighbors simultaneously. We propose a spatiotemporal model of dynamic cell signaling based on Hawkes processes—self-exciting point processes—that model the signaling processes within a cell and spatial couplings between cells. With this cellular point process (CPP) model, we capture both the single-cell protein bursting rate and the magnitude and duration of signaling between cells relative to spatial locations. Furthermore, our model captures tissues composed of heterogeneous cell types with different bursting rates and signaling behaviors across multiple signaling proteins. We apply our model to epithelial cell systems that exhibit a range of autonomous and spatial signaling behaviors basally and under pharmacological exposure. Our model identifies known drug-induced signaling deficits, characterizes differences in signaling across a wound front, and generalizes to multi-channel observations.

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

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.

scholarly journals Single-cell mobility shift electrophoresis reports protein localization to the cell membrane

The Analyst ◽  
2019 ◽  
Vol 144 (3) ◽  
pp. 972-979 ◽  
Author(s):  
Elly Sinkala ◽  
Elisabet Rosàs-Canyelles ◽  
Amy E. Herr
Keyword(s):  
Single Cell ◽  
Gel Electrophoresis ◽  
Protein Localization ◽  
Single Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mobility Shift ◽  
Receptor Proteins ◽  
Cell Mobility ◽  
Regulatory Systems ◽  
Surface Receptor

Prepending surface receptor immunostaining with single-cell polyacrylamide gel electrophoresis provides a new tool with which to understand how localization of surface receptor proteins controls the complex regulatory systems in single cells.

scholarly journals Measuring expression heterogeneity of single-cell cytoskeletal protein complexes

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Julea Vlassakis ◽  
Louise L. Hansen ◽  
Ryo Higuchi-Sanabria ◽  
Yun Zhou ◽  
C. Kimberly Tsui ◽  
...  
Keyword(s):  
Single Cell ◽  
Protein Complex ◽  
Protein Complexes ◽  
Single Cells ◽  
Cellular Heterogeneity ◽  
Size Exclusion ◽  
Cell Protein ◽  
Cytoskeletal Protein ◽  
Filamentous Actin ◽  
Latrunculin A

AbstractMultimeric cytoskeletal protein complexes orchestrate normal cellular function. However, protein-complex distributions in stressed, heterogeneous cell populations remain unknown. Cell staining and proximity-based methods have limited selectivity and/or sensitivity for endogenous multimeric protein-complex quantification from single cells. We introduce micro-arrayed, differential detergent fractionation to simultaneously detect protein complexes in hundreds of individual cells. Fractionation occurs by 60 s size-exclusion electrophoresis with protein complex-stabilizing buffer that minimizes depolymerization. Proteins are measured with a ~5-hour immunoassay. Co-detection of cytoskeletal protein complexes in U2OS cells treated with filamentous actin (F-actin) destabilizing Latrunculin A detects a unique subpopulation (~2%) exhibiting downregulated F-actin, but upregulated microtubules. Thus, some cells may upregulate other cytoskeletal complexes to counteract the stress of Latrunculin A treatment. We also sought to understand the effect of non-chemical stress on cellular heterogeneity of F-actin. We find heat shock may dysregulate filamentous and globular actin correlation. In this work, our assay overcomes selectivity limitations to biochemically quantify single-cell protein complexes perturbed with diverse stimuli.

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

2021 ◽  
Vol 22 (1) ◽  
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.

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

2021 ◽  
Author(s):  
Tongjin Wu ◽  
Howard John Womersley ◽  
Jiehao Wang ◽  
Jonathan Adam Scolnick ◽  
Lih Feng Cheow
Keyword(s):  
Single Cell ◽  
Protein Secretion ◽  
Single Cells ◽  
Transcript Abundance ◽  
Cellular Protein ◽  
Single Cell Protein ◽  
Cytokine Secretion ◽  
Cell Protein ◽  
Time Resolved ◽  
Tnf Α

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.

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

Lab on a Chip ◽  
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,...

scholarly journals Single-cell proteomic and transcriptomic analysis of macrophage heterogeneity

2019 ◽  
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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