Multiplexed single-cell proteomics using SCoPE2

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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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 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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Single cell protein analysis for systems biology

Essays in Biochemistry ◽

10.1042/ebc20180014 ◽

2018 ◽

Vol 62 (4) ◽

pp. 595-605 ◽

Cited By ~ 18

Author(s):

Ezra Levy ◽

Nikolai Slavov

Keyword(s):

Systems Biology ◽

Single Cell ◽

Cell Fate ◽

Fluorescent Proteins ◽

Single Cells ◽

Protein Analysis ◽

Single Cell Protein ◽

Cell Protein ◽

Protein Levels ◽

Trade Offs

The cellular abundance of proteins can vary even between isogenic single cells. This variability between single-cell protein levels can have regulatory roles, such as controlling cell fate during apoptosis induction or the proliferation/quiescence decision. Here, we review examples connecting protein levels and their dynamics in single cells to cellular functions. Such findings were made possible by the introduction of antibodies, and subsequently fluorescent proteins, for tracking protein levels in single cells. However, in heterogeneous cell populations, such as tumors or differentiating stem cells, cellular decisions are controlled by hundreds, even thousands of proteins acting in concert. Characterizing such complex systems demands measurements of thousands of proteins across thousands of single cells. This demand has inspired the development of new methods for single-cell protein analysis, and we discuss their trade-offs, with an emphasis on their specificity and coverage. We finish by highlighting the potential of emerging mass-spec methods to enable systems-level measurement of single-cell proteomes with unprecedented coverage and specificity. Combining such methods with methods for quantitating the transcriptomes and metabolomes of single cells will provide essential data for advancing quantitative systems biology.

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Production of Single Cell Protein Using Mixed Culture of Toddy with Banana Fruit Juice: A Cost Effective Method

Proceedings of International Forestry and Environment Symposium ◽

10.31357/fesympo.v22i0.3449 ◽

2018 ◽

Vol 22 (0) ◽

Author(s):

S. Rajendran ◽

R. Kapilan ◽

S. Vasantharuba

Keyword(s):

Single Cell ◽

Mixed Culture ◽

Fruit Juice ◽

Cost Effective ◽

Single Cell Protein ◽

Cell Protein ◽

Banana Fruit ◽

Cost Effective Method

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Single-cell protein profiling in microchambers with barcoded beads

Microsystems & Nanoengineering ◽

10.1038/s41378-019-0099-5 ◽

2019 ◽

Vol 5 (1) ◽

Cited By ~ 6

Author(s):

Lucas Armbrecht ◽

Rafael Sebastian Müller ◽

Jonas Nikoloff ◽

Petra Stephanie Dittrich

Keyword(s):

Single Cell ◽

Cell Lines ◽

Single Cells ◽

Single Cell Protein ◽

Protein Quantification ◽

Protein Profiling ◽

Cell Protein ◽

Breast Cancer Cell Lines ◽

Mammalian Cell Lines ◽

Galectin 3

Abstract Single-cell profiling provides insights into cellular behaviour that macroscale cell cultures and bulk measurements cannot reveal. In the context of personalized cancer treatment, the profiling of individual tumour cells may lead to higher success rates for therapies by rapidly selecting the most efficacious drugs. Currently, genomic analysis at the single-cell level is available through highly sensitive sequencing approaches. However, the identification and quantification of intracellular or secreted proteins or metabolites remains challenging. Here, we introduce a microfluidic method that facilitates capture, automated data acquisition and the multiplexed quantification of proteins from individual cells. The microfluidic platform comprises 1026 chambers with a volume of 152 pL each, in which single cells and barcoded beads are co-immobilized. We demonstrated multiplexed single-cell protein quantification with three different mammalian cell lines, including two model breast cancer cell lines. We established on-chip immunoassays for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), galectin-3 (Gal-3) and galectin-3 binding protein (Gal-3bp) with detection limits as low as 7.0 × 104, 2.3 × 105 and 1.8 × 103 molecules per cell, respectively. The three investigated cell types had high cytosolic levels of GAPDH and could be clearly differentiated by their expression levels of Gal-3 and Gal-3bp, which are important factors that contribute to cancer metastasis. Because it employed commercially available barcoded beads for this study, our platform could be easily used for the single-cell protein profiling of several hundred different targets. Moreover, this versatile method is applicable to the analysis of bacteria, yeast and mammalian cells and nanometre-sized lipid vesicles.

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Single cell protein analysis for systems biology

10.7287/peerj.preprints.26965 ◽

2018 ◽

Cited By ~ 1

Author(s):

Ezra Levy ◽

Nikolai Slavov

Keyword(s):

Systems Biology ◽

Single Cell ◽

Cell Fate ◽

Fluorescent Proteins ◽

Single Cells ◽

Protein Analysis ◽

Single Cell Protein ◽

Cell Protein ◽

Protein Levels ◽

Trade Offs

The cellular abundance of proteins can vary even between isogenic single cells. This variability between single-cell protein levels can have functional roles, such as controlling cell fate during apoptosis induction or the proliferation/quiescence decision. Here, we review such examples of connecting protein levels and their dynamics in single cells to cellular functions. Such findings were made possible by the introduction of antibodies, and subsequently fluorescent proteins, for tracking protein levels in single cells. However, in heterogeneous cell populations, such as tumors or differentiating stem cells, cellular decisions are controlled by hundreds, even thousands of proteins acting in concert. Characterizing such complex systems demands measurements of thousands of proteins across thousands of single cells. This demand has inspired the development of new methods for single cell protein analysis, and we discuss their trade-offs, with emphasis on their specificity and coverage. We finish by highlighting the potential of emerging mass-spec methods to enable systems-level measurement of single-cell proteomes with unprecedented coverage and specificity. Combining such methods with methods for quantifying the trasncriptomes and metabolomes of single cells will provide essential data for advancing quantitative systems biology.

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Single cell protein analysis for systems biology

10.7287/peerj.preprints.26965v1 ◽

2018 ◽

Cited By ~ 2

Author(s):

Ezra Levy ◽

Nikolai Slavov

Keyword(s):

Systems Biology ◽

Single Cell ◽

Cell Fate ◽

Fluorescent Proteins ◽

Single Cells ◽

Protein Analysis ◽

Single Cell Protein ◽

Cell Protein ◽

Protein Levels ◽

Trade Offs

The cellular abundance of proteins can vary even between isogenic single cells. This variability between single-cell protein levels can have functional roles, such as controlling cell fate during apoptosis induction or the proliferation/quiescence decision. Here, we review such examples of connecting protein levels and their dynamics in single cells to cellular functions. Such findings were made possible by the introduction of antibodies, and subsequently fluorescent proteins, for tracking protein levels in single cells. However, in heterogeneous cell populations, such as tumors or differentiating stem cells, cellular decisions are controlled by hundreds, even thousands of proteins acting in concert. Characterizing such complex systems demands measurements of thousands of proteins across thousands of single cells. This demand has inspired the development of new methods for single cell protein analysis, and we discuss their trade-offs, with emphasis on their specificity and coverage. We finish by highlighting the potential of emerging mass-spec methods to enable systems-level measurement of single-cell proteomes with unprecedented coverage and specificity. Combining such methods with methods for quantifying the trasncriptomes and metabolomes of single cells will provide essential data for advancing quantitative systems biology.

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