Advances of Single-Cell Protein Analysis

Proteins play a significant role in the key activities of cells. Single-cell protein analysis provides crucial insights in studying cellular heterogeneities. However, the low abundance and enormous complexity of the proteome posit challenges in analyzing protein expressions at the single-cell level. This review summarizes recent advances of various approaches to single-cell protein analysis. We begin by discussing conventional characterization approaches, including fluorescence flow cytometry, mass cytometry, enzyme-linked immunospot assay, and capillary electrophoresis. We then detail the landmark advances of microfluidic approaches for analyzing single-cell protein expressions, including microfluidic fluorescent flow cytometry, droplet-based microfluidics, microwell-based assay (microengraving), microchamber-based assay (barcoding microchips), and single-cell Western blotting, among which the advantages and limitations are compared. Looking forward, we discuss future research opportunities and challenges for multiplexity, analyte, throughput, and sensitivity of the microfluidic approaches, which we believe will prompt the research of single-cell proteins such as the molecular mechanism of cell biology, as well as the clinical applications for tumor treatment and drug development.

[1]  Joshua M. Finkelstein,et al.  The protein world , 2016, Nature.

[2]  Z. Grossman,et al.  HIV preferentially infects HIV-specific CD4+ T cells , 2002, Nature.

[3]  Hui Zeng,et al.  Flow Cytometric and Cytokine ELISpot Approaches To Characterize the Cell-Mediated Immune Response in Ferrets following Influenza Virus Infection , 2016, Journal of Virology.

[4]  A. Oudenaarden,et al.  Every Cell Is Special: Genome-wide Studies Add a New Dimension to Single-Cell Biology , 2014, Cell.

[5]  R. Fan,et al.  Single Cell Functional Proteomics for Assessing Immune Response in Cancer Therapy: Technology, Methods, and Applications , 2013, Front. Oncol..

[6]  Junbo Wang,et al.  Development of microfluidic platform capable of high-throughput absolute quantification of single-cell multiple intracellular proteins from tumor cell lines and patient tumor samples. , 2020, Biosensors & bioelectronics.

[7]  Douglas S Kwon,et al.  Profiling human antibody responses by integrated single-cell analysis. , 2014, Vaccine.

[8]  O Ouchterlony,et al.  A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells. , 1983, Journal of immunological methods.

[9]  S. Quake,et al.  A microfabricated fluorescence-activated cell sorter , 1999, Nature Biotechnology.

[10]  Lung-Ming Fu,et al.  Review and perspectives on microfluidic flow cytometers , 2018, Sensors and Actuators B: Chemical.

[11]  G. Nolan,et al.  Mass Cytometry: Single Cells, Many Features , 2016, Cell.

[12]  Poul Jennum,et al.  Absence of autoreactive CD4+ T-cells targeting HLA-DQA1*01:02/DQB1*06:02 restricted hypocretin/orexin epitopes in narcolepsy type 1 when detected by EliSpot , 2017, Journal of Neuroimmunology.

[13]  Antoni Ribas,et al.  Single-cell analysis tools for drug discovery and development , 2015, Nature Reviews Drug Discovery.

[14]  Barbara Marte,et al.  Molecular cancer diagnostics , 2008, Nature.

[15]  J. Buhmann,et al.  Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry , 2014, Nature Methods.

[16]  Junbo Wang,et al.  A microfluidic flow cytometer enabling absolute quantification of single-cell intracellular proteins. , 2017, Lab on a chip.

[17]  William G Telford,et al.  Flow cytometry of fluorescent proteins. , 2012, Methods.

[18]  Jun Wang,et al.  Chemical analysis of single cells. , 2013, Analytical chemistry.

[19]  Peng Chen,et al.  Microfluidics towards single cell resolution protein analysis , 2019, TrAC Trends in Analytical Chemistry.

[20]  A. Abate,et al.  Ultrahigh-throughput screening in drop-based microfluidics for directed evolution , 2010, Proceedings of the National Academy of Sciences.

[21]  Peggy P Y Chan,et al.  Microparticle Delivery of Protein Markers for Single-Cell Western Blotting from Microwells. , 2018, Small.

[22]  Robert A. Hoffman,et al.  NIST/ISAC standardization study: Variability in assignment of intensity values to fluorescence standard beads and in cross calibration of standard beads to hard dyed beads , 2012, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[23]  Anne Rascle,et al.  An optimized IFN-γ ELISpot assay for the sensitive and standardized monitoring of CMV protein-reactive effector cells of cell-mediated immunity , 2017, BMC Immunology.

[24]  John R. Haliburton,et al.  Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding , 2017, Scientific Reports.

[25]  Tze Ping Loh,et al.  Barcoded DNA nanostructures for the multiplexed profiling of subcellular protein distribution , 2019, Nature Biomedical Engineering.

[26]  Amy E. Reece,et al.  Microfluidic techniques for high throughput single cell analysis. , 2016, Current opinion in biotechnology.

[27]  Mustafa Khammash,et al.  Digital Quantification of Proteins and mRNA in Single Mammalian Cells. , 2016, Molecular cell.

[28]  Junbo Wang,et al.  Development of Droplet Microfluidics Enabling High-Throughput Single-Cell Analysis , 2016, Molecules.

[29]  Gary D Bader,et al.  A draft map of the human proteome , 2014, Nature.

[30]  P. Chattopadhyay,et al.  Cytometry: today's technology and tomorrow's horizons. , 2012, Methods.

[31]  J Christopher Love,et al.  Immuno-hybridization chain reaction for enhancing detection of individual cytokine-secreting human peripheral mononuclear cells. , 2011, Analytical chemistry.

[32]  J Christopher Love,et al.  Multidimensional analysis of the frequencies and rates of cytokine secretion from single cells by quantitative microengraving. , 2010, Lab on a chip.

[33]  D. Weitz,et al.  Single-cell analysis and sorting using droplet-based microfluidics , 2013, Nature Protocols.

[34]  L. Hood,et al.  Integrated barcode chips for rapid, multiplexed analysis of proteins in microliter quantities of blood , 2008, Nature Biotechnology.

[35]  Amy E Herr,et al.  Profiling protein expression in circulating tumour cells using microfluidic western blotting , 2017, Nature Communications.

[36]  M. Roederer,et al.  11-color, 13-parameter flow cytometry: Identification of human naive T cells by phenotype, function, and T-cell receptor diversity , 2001, Nature Medicine.

[37]  Douglas A. Lauffenburger,et al.  Polyfunctional responses by human T cells result from sequential release of cytokines , 2011, Proceedings of the National Academy of Sciences.

[38]  Amy E Herr,et al.  Microfluidic Western blotting , 2012, Proceedings of the National Academy of Sciences.

[39]  Nataly Kravchenko-Balasha,et al.  Intercellular signaling through secreted proteins induces free-energy gradient-directed cell movement , 2016, Proceedings of the National Academy of Sciences.

[40]  Mats Nilsson,et al.  Profiling surface proteins on individual exosomes using a proximity barcoding assay , 2019, Nature Communications.

[41]  Daniel Bratton,et al.  Development of quantitative cell-based enzyme assays in microdroplets. , 2008, Analytical chemistry.

[42]  Zoltan Megyesi,et al.  Reagent Tracker Dyes Permit Quality Control for Verifying Plating Accuracy in ELISPOT Tests , 2018, Cells.

[43]  Navin Varadarajan,et al.  Rapid, efficient functional characterization and recovery of HIV-specific human CD8+ T cells using microengraving , 2012, Proceedings of the National Academy of Sciences.

[44]  Wei-Hua Huang,et al.  Recent advances in single-cell analysis using capillary electrophoresis and microfluidic devices. , 2008, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[45]  A. deMello,et al.  Quantitative detection of protein expression in single cells using droplet microfluidics. , 2007, Chemical communications.

[46]  Priscillia Gourvil,et al.  Improvement of phytoplankton culture isolation using single cell sorting by flow cytometry , 2017, Journal of phycology.

[47]  Amy E Herr,et al.  Single-Cell Western Blotting. , 2015, Methods in molecular biology.

[48]  Rong Fan,et al.  Single-cell proteomic chip for profiling intracellular signaling pathways in single tumor cells , 2011, Proceedings of the National Academy of Sciences.

[49]  Jonathan M Irish,et al.  Single Cell Profiling of Potentiated Phospho-Protein Networks in Cancer Cells , 2004, Cell.

[50]  Thomas D. Perroud,et al.  Microfluidically-unified cell culture, sample preparation, imaging and flow cytometry for measurement of cell signaling pathways with single cell resolution. , 2012, Lab on a chip.

[51]  W. Bonner,et al.  Cell Sorting: Automated Separation of Mammalian Cells as a Function of Intracellular Fluorescence , 1969, Science.

[52]  Paul J. Choi,et al.  Quantifying E. coli Proteome and Transcriptome with Single-Molecule Sensitivity in Single Cells , 2010, Science.

[53]  Bo Xu,et al.  Basics and recent advances of two dimensional- polyacrylamide gel electrophoresis , 2013, Clinical Proteomics.

[54]  Bernd Bodenmiller,et al.  Unraveling cell populations in tumors by single-cell mass cytometry. , 2015, Current opinion in biotechnology.

[55]  Sean C. Bendall,et al.  Single-Cell Mass Cytometry of Differential Immune and Drug Responses Across a Human Hematopoietic Continuum , 2011, Science.

[56]  Martin Jaeger,et al.  Integration of multi-omics data and deep phenotyping enables prediction of cytokine responses , 2018, Nature Immunology.

[57]  S. Hanash,et al.  Disease proteomics , 2003, Nature.

[58]  M. Roederer,et al.  The history and future of the fluorescence activated cell sorter and flow cytometry: a view from Stanford. , 2002, Clinical chemistry.

[59]  Amy E. Herr,et al.  Single-Cell Western Blotting after Whole-Cell Imaging to Assess Cancer Chemotherapeutic Response , 2014, Analytical chemistry.

[60]  A. Singh,et al.  Single Cell Protein Analysis , 2015, Methods in Molecular Biology.

[61]  G Gerken,et al.  Quantification of CD8+ T lymphocytes responsive to human immunodeficiency virus (HIV) peptide antigens in HIV-infected patients and seronegative persons at high risk for recent HIV exposure. , 1998, The Journal of infectious diseases.

[62]  Renjie Liao,et al.  Highly Multiplexed Single-Cell Protein Analysis. , 2018, Chemistry.

[63]  J. Sweedler,et al.  Capillary electrophoresis with electrospray ionization mass spectrometric detection for single-cell metabolomics. , 2009, Analytical chemistry.

[64]  Savaş Tay,et al.  Ultra-sensitive digital quantification of proteins and mRNA in single cells , 2019, Nature Communications.

[65]  James R Heath,et al.  Quantitating cell-cell interaction functions with applications to glioblastoma multiforme cancer cells. , 2012, Nano letters.

[66]  G. Whitesides.  The origins and the future of microfluidics , 2006, Nature.

[67]  Bernhard H. Weigl,et al.  Wash-Free, Digital Immunoassay in Polydisperse Droplets. , 2020, Analytical chemistry.

[68]  Matthew R Clutter,et al.  High-content single-cell drug screening with phosphospecific flow cytometry. , 2008, Nature chemical biology.

[69]  Thomas Schmidt,et al.  Research Techniques Made Simple: Monitoring of T-Cell Subsets using the ELISPOT Assay. , 2016, The Journal of investigative dermatology.

[70]  Neil Savage,et al.  Proteomics: High-protein research , 2015, Nature.

[71]  M. Monsur Ali,et al.  Droplet microfluidics for single-molecule and single-cell analysis in cancer research, diagnosis and therapy , 2014 .

[72]  David R Walt,et al.  Protein Counting in Single Cancer Cells. , 2016, Analytical chemistry.

[73]  Amy E Herr,et al.  Single cell–resolution western blotting , 2016, Nature Protocols.

[74]  R. Kennedy,et al.  Capillary electrophoresis-based immunoassay to determine insulin content and insulin secretion from single islets of Langerhans. , 1995, Analytical chemistry.

[75]  Brad T Cookson,et al.  Repeatability of chemical cytometry: 2‐DE analysis of single RAW 264.7 macrophage cells , 2007, Electrophoresis.

[76]  David S Lawrence,et al.  Measurement of protein tyrosine phosphatase activity in single cells by capillary electrophoresis. , 2013, Analytical chemistry.

[77]  Tobias Preckel,et al.  Cytometric analysis of protein expression and apoptosis in human primary cells with a novel microfluidic chip‐based system , 2003, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[78]  Christian G Hartinger,et al.  Capillary electrophoresis in metallodrug development. , 2015, Drug discovery today. Technologies.

[79]  Amy E Herr,et al.  Separation-encoded microparticles for single-cell western blotting. , 2019, Lab on a chip.

[80]  Shana O. Kelley,et al.  Single-cell analysis targeting the proteome , 2020, Nature Reviews Chemistry.

[81]  Dino Di Carlo,et al.  Functional profiling of circulating tumor cells with an integrated vortex capture and single-cell protease activity assay , 2018, Proceedings of the National Academy of Sciences.

[82]  Elizabeth Sinclair,et al.  Comparison of the ELISPOT and cytokine flow cytometry assays for the enumeration of antigen-specific T cells. , 2003, Journal of immunological methods.

[83]  Wilhelm T S Huck,et al.  Probing cellular heterogeneity in cytokine-secreting immune cells using droplet-based microfluidics. , 2013, Lab on a chip.

[84]  Wei Wei,et al.  Single cell proteomics in biomedicine: High‐dimensional data acquisition, visualization, and analysis , 2017, Proteomics.

[85]  Giuseppe Basso,et al.  Expression of CD58 in normal, regenerating and leukemic bone marrow B cells: implications for the detection of minimal residual disease in acute lymphocytic leukemia. , 2003, Haematologica.

[86]  Karen Sachs,et al.  Multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators , 2012, Nature Biotechnology.

[87]  Jonathan J. Chen,et al.  Single-Cell Protein Secretion Detection and Profiling. , 2019, Annual review of analytical chemistry.

[88]  Kevin Maher,et al.  Quantitative flow cytometry in the clinical laboratory , 2005 .

[89]  J Christopher Love,et al.  Longitudinal multiparameter single-cell analysis of macaques immunized with pneumococcal protein-conjugated or unconjugated polysaccharide vaccines , 2017 .

[90]  Cedrik M. Britten,et al.  Response definition criteria for ELISPOT assays revisited , 2010, Cancer Immunology, Immunotherapy.

[91]  A. Lusis,et al.  Considerations for the design of omics studies , 2017 .

[92]  Aysun Adan,et al.  Flow cytometry: basic principles and applications , 2017, Critical reviews in biotechnology.

[93]  Reinhard Dechant,et al.  Ultra High-Throughput Multiparametric Imaging Flow Cytometry: Towards Diffraction-Limited Sub-Cellular Detection , 2019, bioRxiv.

[94]  D. Price,et al.  Quantum dot semiconductor nanocrystals for immunophenotyping by polychromatic flow cytometry , 2006, Nature Medicine.

[95]  J L Haynes,et al.  Principles of flow cytometry. , 1988, Cytometry. Supplement : the journal of the Society for Analytical Cytology.

[96]  G E Marti,et al.  Quantitative flow cytometry: history, practice, theory, consensus, inter-laboratory variation and present status. , 2002, Cytotherapy.

[97]  Eli R. Zunder,et al.  A continuous molecular roadmap to iPSC reprogramming through progression analysis of single-cell mass cytometry. , 2015, Cell stem cell.

[98]  Rong Fan,et al.  A Clinical Microchip for Evaluation of Single Immune Cells Reveals High Functional Heterogeneity in Phenotypically Similar T Cells Nih Public Access Author Manuscript Design Rationale and Detection Limit of the Scbc Online Methods Microchip Fabrication On-chip Secretion Profiling Supplementary Mater , 2022 .

[99]  D. Pe’er,et al.  Highly multiplexed profiling of single-cell effector functions reveals deep functional heterogeneity in response to pathogenic ligands , 2015, Proceedings of the National Academy of Sciences.

[100]  P. Chattopadhyay,et al.  Seventeen-colour flow cytometry: unravelling the immune system , 2004, Nature Reviews Immunology.

[101]  Ruedi Aebersold,et al.  Identification of proteins in single-cell capillary electrophoresis fingerprints based on comigration with standard proteins. , 2003, Analytical chemistry.

[102]  Daniel J. Ehrlich,et al.  A Parallel Microfluidic Flow Cytometer for High Content Screening , 2011, Nature Methods.

[103]  S. Gan,et al.  Enzyme immunoassay and enzyme-linked immunosorbent assay. , 2013, The Journal of investigative dermatology.

[104]  Wei Wei,et al.  Microchip-based single-cell functional proteomics for biomedical applications. , 2017, Lab on a chip.

[105]  J. C. Love,et al.  A microengraving method for rapid selection of single cells producing antigen-specific antibodies , 2006, Nature Biotechnology.

[106]  S. Fields,et al.  Protein analysis on a proteomic scale , 2003, Nature.

[107]  H. Swerdlow,et al.  Large-scale simultaneous measurement of epitopes and transcriptomes in single cells , 2017, Nature Methods.

[108]  T. Nutman,et al.  ELISPOT Assay to Detect Cytokine‐Secreting Murine and Human Cells , 1994, Current protocols in immunology.

[109]  Sean C. Bendall,et al.  Multiplexed ion beam imaging of human breast tumors , 2014, Nature Medicine.

[110]  Rong Fan,et al.  Annual Review of Biomedical Engineering Single-Cell Omics Analyses Enabled by Microchip Technologies , 2019 .