Abstract
Single-cell proteomics refers to the analysis and characterization of proteins expressed in individual cells. The purpose of studying single-cell proteomes is to better understand the activity and function of cells at the single-cell level. Since proteins are the main molecules responsible for cellular functions, analysis of single-cell proteomes gives a most accurate understanding of how cells operate and communicate and how protein expression alters in individual cells due to environmental/pathological stimuli. Although single-cell transcriptomics is an important approach for analysis of gene expression at single-cell level, it does not take into account posttranscriptional regulation, and gene expression levels may not always correlate well with protein expression levels. Single-cell proteomics is particularly important in development biology, stem cell biology, neurochemistry, and cancer biology fields. Comparing different stem/progenitor cell proteomes can reveal proteins that are important for stem cell self-renewal and differentiation. Single neuron proteome analysis will facilitate our understanding of how neurons transmit signals among their heterogeneous cell populations. Lastly, single-cell proteomics may help elucidate how cancer is developed from a single mutated cell and how cancer cells invade and metastasize. Single-cell proteome analysis represents a great challenge for analytical chemistry community. In the past two decades, we have witnessed new technology developments in this field, such as capillary electrophoresis, microfluidics, mass spectrometry, and flow cytometry. This chapter will provide an overview of the recent advancement of technology and methodology for single-cell proteomics and related applications. We will also discuss current challenges and future direction for single-cell proteomics.
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References
Abiko M, Furuta K, Yamauchi Y, Fujita C, Taoka M, Isobe T, Okamoto T (2013) Identification of proteins enriched in rice egg or sperm cells by single-cell proteomics. PLoS One 8:e69578
Arkhipov SN, Berezovski M, Jitkova J, Krylov SN (2005) Chemical cytometry for monitoring metabolism of a Ras-mimicking substrate in single cells. Cytometry A 63A:41–47
Begley CG, Ellis LM (2012) Drug development: raise standards for preclinical cancer research. Nature 483:531–533
Bendall SC et al (2011) Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Science 332:687–696
Boardman AK, McQuaide SC, Zhu C, Whitmore CD, Lidstrom ME, Dovichi NJ (2008) Interface of an array of five capillaries with an array of one-nanoliter wells for high-resolution electrophoretic analysis as an approach to high-throughput chemical cytometry. Anal Chem 80:7631–7634
Boardman A, Chang T, Folch A, Dovichi NJ (2010) Indium-tin oxide coated microfabricated device for the injection of a single cell into a fused silica capillary for chemical cytometry. Anal Chem 82:9959–9961
Brayboy LM, Wessel GM (2016) The double-edged sword of the mammalian oocyte – advantages, drawbacks and approaches for basic and clinical analysis at the single cell level. Mol Hum Reprod 22:200–207
Budnik B, Levy E, Harmange G, Slavov N (2018) Mass-spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation. Genome Biology 19:161
Chen D et al (2016) Single cell chemical proteomics with membrane-permeable activity-based probe for identification of functional proteins in lysosome of tumors. Anal Chem 88:2466–2471
Clutter MR, Heffner GC, Krutzik PO, Sachen KL, Nolan GP (2010) Tyramide signal amplification for analysis of kinase activity by intracellular flow cytometry. Cytometry A 77:1020–1031
Cohen D, Dickerson JA, Whitmore CD, Turner EH, Palcic MM, Hindsgaul O, Dovichi NJ (2008) Chemical cytometry: fluorescence-based single-cell analysis. Annu Rev Anal Chem 1:165–190
Copley MR, Eaves CJ (2013) Developmental changes in hematopoietic stem cell properties. Exp Mol Med 45:e55
Darmanis S et al (2016) Simultaneous multiplexed measurement of RNA and proteins in single cells. Cell Rep 14:380–389
De Rosa SC, Herzenberg LA, Herzenberg LA, Roederer M (2001) 11-color, 13-parameter flow cytometry: identification of human naive T cells by phenotype, function, and T-cell receptor diversity. Nat Med 7:245–248
Di Carlo D, Aghdam N, Lee LP (2006) Single-cell enzyme concentrations, kinetics, and inhibition analysis using high-density hydrodynamic cell isolation arrays. Anal Chem 78:4925–4930
Finck R et al (2013) Normalization of mass cytometry data with bead standards. Cytometry A 83:483–494
Garcia-Berrocoso T et al (2018) Single cell immuno-laser microdissection coupled to label-free proteomics to reveal the proteotypes of human brain cells after ischemia. Mol Cell Proteomics 17:175–189
Hu S, Zhang ZR, Cook LM, Carpenter EJ, Dovichi NJ (2000) Separation of proteins by sodium dodecyl sulfate capillary electrophoresis in hydroxypropyl cellulose sieving matrix with laser-induced fluorescence detection. J Chromatogr A 894:291–296
Hu S, Jiang J, Cook LM, Richards DP, Horlick L, Wong B, Dovichi NJ (2002a) Capillary sodium dodecyl sulfate-DALT electrophoresis with laser-induced fluorescence detection for size-based analysis of proteins in human colon cancer cells. Electrophoresis 23:3136–3142
Hu S, Ye YL, Surh G, Clark JI, Dovichi NJ (2002b) Analysis of proteins by capillary SDS-DALT electrophoresis with laser-induced fluorescence detection. LC-GC Eur 15:166
Hu K, Zarrine-Afsar A, Ahmadzadeh H, Krylov SN (2004a) Single-cell analysis by chemical cytometry combined with fluorescence microscopy. Instrum Sci Technol 32:31–41
Hu S, Michels DA, Fazal MA, Ratisoontorn C, Cunningham ML, Dovichi NJ (2004b) Capillary sieving electrophoresis/micellar electrokinetic capillary chromatography for two-dimensional protein fingerprinting of single mammalian cells. Anal Chem 76:4044–4049
Huebner A, Srisa-Art M, Holt D, Abell C, Hollfelder F, Demello AJ, Edel JB (2007) Quantitative detection of protein expression in single cells using droplet microfluidics. Chem Commun 12:1218–1220
Hughes AJ, Spelke DP, Xu ZC, Kang CC, Schaffer DV, Herr AE (2014a) Single-cell western blotting. Nat Methods 11:749–U794
Hughes CS, Foehr S, Garfield DA, Furlong EE, Steinmetz LM, Krijgsveld J (2014b) Ultrasensitive proteome analysis using paramagnetic bead technology. Mol Syst Biol 10:757
Irish JM, Hovland R, Krutzik PO, Perez OD, Bruserud O, Gjertsen BT, Nolan GP (2004) Single cell profiling of potentiated phospho-protein networks in cancer cells. Cell 118:217–228
Karlsson AC et al (2003) Comparison of the ELISPOT and cytokine flow cytometry assays for the enumeration of antigen-specific T cells. J Immunol Methods 283:141–153
Krieg C et al (2018) High-dimensional single-cell analysis predicts response to anti-PD-1 immunotherapy. Nat Med 24:144–153
Krutzik PO, Nolan GP (2006) Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling. Nat Methods 3:361–368
Krylov SN, Starke DA, Arriaga EA, Zhang Z, Chan NW, Palcic MM, Dovichi NJ (2000) Instrumentation for chemical cytometry. Anal Chem 72(4):872–877
Labas V, Teixeira-Gomes AP, Bouguereau L, Gargaros A, Spina L, Marestaing A, Uzbekova S (2018) Intact cell MALDI-TOF mass spectrometry on single bovine oocyte and follicular cells combined with top-down proteomics: a novel approach to characterise markers of oocyte maturation. J Proteomics 175:56–74
Li SY et al (2015) An integrated platform for isolation, processing, and mass spectrometry-based proteomic profiling of rare cells in whole blood. Mol Cell Proteomics 14:1672–1683
Li QL, Chen PL, Fan YY, Wang X, Xu KH, Li L, Tang B (2016) Multicolor fluorescence detection-based microfluidic device for single-cell metabolomics: simultaneous quantitation of multiple small molecules in primary liver cells. Anal Chem 88:8610–8616
Lillard SJ, Yeung ES (1996) Analysis of single erythrocytes by injection-based capillary isoelectric focusing with laser-induced native fluorescence detection. J Chromatogr B Biomed Appl 687: 363–369
Lillard SJ, Yeung ES, Lautamo RM, Mao DT (1995) Separation of hemoglobin variants in single human erythrocytes by capillary electrophoresis with laser-induced native fluorescence detection. J Chromatogr A 718(2):397–404
Lombard-Banek C, Moody SA, Nemes P (2016a) Single-cell mass spectrometry for discovery proteomics: quantifying translational cell heterogeneity in the 16-cell frog (Xenopus) embryo. Angew Chem Int Ed Engl 55:2454–2458
Lombard-Banek C, Reddy S, Moody SA, Nemes P (2016b) Label-free quantification of proteins in single embryonic cells with neural fate in the cleavage-stage frog (Xenopus laevis) embryo using capillary electrophoresis electrospray ionization high-resolution mass spectrometry (CE-ESI-HRMS). Mol Cell Proteomics 15:2756–2768
Love JC, Ronan JL, Grotenbreg GM, van der Veen AG, Ploegh HL (2006) A microengraving method for rapid selection of single cells producing antigen-specific antibodies. Nat Biotechnol 24:703–707
Lucy CA, MacDonald AM, Gulcev MD (2008) Non-covalent capillary coatings for protein separations in capillary electrophoresis. J Chromatogr A 1184:81–105
Ma C et al (2011) A clinical microchip for evaluation of single immune cells reveals high functional heterogeneity in phenotypically similar T cells. Nat Med 17:738–743
Mainz ER, Wang QZ, Lawrence DS, Allbritton NL (2016) An integrated chemical cytometry method: shining a light on Akt activity in single cells. Angew Chem Int Ed Engl 55: 13095–13098
Michels DA, Hu S, Dambrowitz KA, Eggertson MJ, Lauterbach K, Dovichi NJ (2004) Capillary sieving electrophoresis-micellar electrokinetic chromatography fully automated two-dimensional capillary electrophoresis analysis of Deinococcus radiodurans protein homogenate. Electrophoresis 25:3098–3105
Ornatsky OI et al (2008) Study of cell antigens and intracellular DNA by identification of element-containing labels and metallointercalators using inductively coupled plasma mass spectrometry. Anal Chem 80:2539–2547
Perez OD, Nolan GP (2002) Simultaneous measurement of multiple active kinase states using polychromatic flow cytometry. Nat Biotechnol 20:155–162
Ramos-Payan M, Ocana-Gonzalez JA, Fernandez-Torres RM, Llobera A, Bello-Lopez MA (2018) Recent trends in capillary electrophoresis for complex samples analysis: a review. Electrophoresis 39:111–125
Sachs K, Perez O, Pe’er D, Lauffenburger DA, Nolan GP (2005) Causal protein-signaling networks derived from multiparameter single-cell data. Science 308:523–529
Shin YS et al (2010) Chemistries for patterning robust DNA microbarcodes enable multiplex assays of cytoplasm proteins from single cancer cells. Chemphyschem 11:3063–3069
Sinkala E et al (2017) Profiling protein expression in circulating tumour cells using microfluidic western blotting. Nat Commun 8:14622
Sobhani K, Fink SL, Cookson BT, Dovichi NJ (2007) Repeatability of chemical cytometry: 2-DE analysis of single RAW 264.7 macrophage cells. Electrophoresis 28:2308–2313
Srivastava N, Brennan JS, Renzi RF, Wu M, Branda SS, Singh AK, Herr AE (2009) Fully integrated microfluidic platform enabling automated phosphoprofiling of macrophage response. Anal Chem 81:3261–3269
Su Y, Shi Q, Wei W (2017a) Single cell proteomics in biomedicine: high-dimensional data acquisition, visualization, and analysis. Proteomics 17(3–4). https://doi.org/10.1002/pmic.201600267
Su YP et al (2017b) Single-cell analysis resolves the cell state transition and signaling dynamics associated with melanoma drug-induced resistance. Proc Natl Acad Sci USA 114:13679–13684
Sun L et al (2014) Over 10,000 peptide identifications from the HeLa proteome by using single-shot capillary zone electrophoresis combined with tandem mass spectrometry. Angew Chem Int Ed Engl 53:13931–13933
Sun L, Dubiak KM, Peuchen EH, Zhang Z, Zhu G, Huber PW, Dovichi NJ (2016) Single cell proteomics using frog (Xenopus laevis) blastomeres isolated from early stage embryos, which form a geometric progression in protein content. Anal Chem 88:6653–6657
Sun B, Kovatch JR, Badiong A, Merbouh N (2017) Optimization and modeling of quadrupole orbitrap parameters for sensitive analysis toward single-cell proteomics. J Proteome Res 16: 3711–3721
Thorsen T, Maerkl SJ, Quake SR (2002) Microfluidic large-scale integration. Science 298:580–584
Torres AJ, Contento RL, Gordo S, Wucherpfennig KW, Love JC (2013) Functional single-cell analysis of T-cell activation by supported lipid bilayer-tethered ligands on arrays of nanowells. Lab Chip 13:90–99
Tricot S et al (2015) Evaluating the efficiency of isotope transmission for improved panel design and a comparison of the detection sensitivities of mass cytometer instruments. Cytometry A 87:357–368
Venable A, Mitalipova M, Lyons I, Jones K, Shin S, Pierce M, Stice S (2005) Lectin binding profiles of SSEA-4 enriched, pluripotent human embryonic stem cell surfaces. BMC Dev Biol 5:15
Vickerman BM, Anttila MM, Petersen BV, Allbritton NL, Lawrence DS (2018) Design and application of sensors for chemical cytometry. ACS Chem Biol 13(7):1741–1751
Virant-Klun I, Leicht S, Hughes C, Krijgsveld J (2016) Identification of maturation-specific proteins by single-cell proteomics of human oocytes. Mol Cell Proteomics 15:2616–2627
Wei W et al (2016) Single-cell phosphoproteomics resolves adaptive signaling dynamics and informs targeted combination therapy in glioblastoma. Cancer Cell 29:563–573
Wu M, Singh AK (2012) Single-cell protein analysis. Curr Opin Biotechnol 23:83–88
Wu M et al (2012) Microfluidically-unified cell culture, sample preparation, imaging and flow cytometry for measurement of cell signaling pathways with single cell resolution. Lab Chip 12:2823–2831
Xu F et al (2014) Single-cell chemical proteomics with an activity-based probe: identification of low-copy membrane proteins on primary neurons. Angew Chem Int Ed Engl 53:6730–6733
Yang MA, Nelson R, Ros A (2016) Toward analysis of proteins in single cells: a quantitative approach employing isobaric tags with MALDI mass spectrometry realized with a microfluidic platform. Anal Chem 88:6672–6679
Yeung ES (2011) Genome-wide correlation between mRNA and protein in a single cell. Angew Chem Int Ed Engl 50:583–585
Zhong X, Zhang Z, Jiang S, Li L (2014) Recent advances in coupling capillary electrophoresis-based separation techniques to ESI and MALDI-MS. Electrophoresis 35:1214–1225
Zhu Y, Li H, Bhatti S, Zhou S, Yang Y, Fish T, Thannhauser TW (2016) Development of a laser capture microscope-based single-cell-type proteomics tool for studying proteomes of individual cell layers of plant roots. Hortic Res 3:16026
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Xu, X., Hu, S. (2022). Single-Cell Proteomics. In: Santra, T.S., Tseng, FG. (eds) Handbook of Single-Cell Technologies. Springer, Singapore. https://doi.org/10.1007/978-981-10-8953-4_1
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DOI: https://doi.org/10.1007/978-981-10-8953-4_1
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