Abstract
Cellular heterogeneity is pervasive and of paramount importance in biology. Single-cell analysis techniques are indispensable for understanding the heterogeneity and functions of cells. Low-copy-number proteins (fewer than 1,000 molecules per cell) perform multiple crucial functions such as gene expression, cellular metabolism and cell signaling. The expression level of low-copy-number proteins of individual cells provides key information for the in-depth understanding of biological processes and diseases. However, the quantitative analysis of low-copy-number proteins in a single cell still remains challenging. To overcome this, we developed an approach called single-cell plasmonic immunosandwich assay (scPISA) for the quantitative measurement of low-copy-number proteins in single living cells. scPISA combines in vivo microextraction for specific enrichment of target proteins from cells and a state-of-the-art technique called plasmon-enhanced Raman scattering for ultrasensitive detection of low-copy-number proteins. Plasmon-enhanced Raman scattering detection relies on the plasmonic coupling effect (hot-spot) between silver-based plasmonic nanotags and a gold-based extraction microprobe, which dramatically enhances the signal intensity of the surface-enhanced Raman scattering of the nanotags and thereby enables sensitivity at the single-molecule level. scPISA is a straightforward and minimally invasive technique, taking only ~6–15 min (from in vivo extraction to Raman spectrum readout). It is generally applicable to all freely floating intracellular proteins provided that appropriate antibodies or alternatives (for example, molecularly imprinted polymers or aptamers) are available. The entire protocol takes ~4–7 d to complete, including material fabrication, single-cell manipulation, protein labeling, signal acquisition and data analysis.
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Data availability
The detailed raw data for Fig. 6 and their interpretation for the low-copy-number protein detection in single living cells by scPISA are available in the original publication24. Source data are provided with this paper.
References
Irish, J. M., Kotecha, N. & Nolan, G. P. Innovation-mapping normal and cancer cell signalling networks: towards single-cell proteomics. Nat. Rev. Cancer 6, 146–155 (2006).
Graf, T. & Stadtfeld, M. Heterogeneity of embryonic and adult stem cells. Cell Stem Cell 3, 480–483 (2008).
Easwaran, H., Tsai, H. C. & Baylin, S. B. Cancer epigenetics: tumor heterogeneity, plasticity of stem-like states, and drug resistance. Mol. Cell 54, 716–727 (2014).
Bonavia, R., Inda, M. D., Cavenee, W. K. & Furnari, F. B. Heterogeneity maintenance in glioblastoma: a social network. Cancer Res 71, 4055–4060 (2011).
Dagogo-Jack, I. & Shaw, A. T. Tumour heterogeneity and resistance to cancer therapies. Nat. Rev. Clin. Oncol. 15, 81–94 (2018).
Jacob, F. et al. A patient-derived glioblastoma organoid model and biobank recapitulates inter- and intra-tumoral heterogeneity. Cell 180, 188–204 (2020).
Almendro, V., Marusyk, A. & Polyak, K. Cellular heterogeneity and molecular evolution in cancer. Annu. Rev. Pathol. Mech. Dis. 8, 277–302 (2013).
Toriello, N. M. et al. Integrated microfluidic bioprocessor for single-cell gene expression analysis. Proc. Natl Acad. Sci. USA 105, 20173–20178 (2008).
Labib, M. & Kelley, S. O. Single-cell analysis targeting the proteome. Nat. Rev. Chem. 4, 143–158 (2020).
Wang, D. J. & Bodovitz, S. Single cell analysis: the new frontier in ‘omics’. Trends Biotechnol. 28, 281–290 (2010).
Pollen, A. A. et al. Low-coverage single-cell mRNA sequencing reveals cellular heterogeneity and activated signaling pathways in developing cerebral cortex. Nat. Biotechnol. 32, 1053–1058 (2014).
Zheng, X. T. & Li, C. M. Single cell analysis at the nanoscale. Chem. Soc. Rev. 41, 2061–2071 (2012).
Ahmed, Z. et al. Grb2 monomer-dimer equilibrium determines normal versus oncogenic function. Nat. Commun. 6, 7354 (2015).
Timsah, Z. et al. Competition between Grb2 and Plc gamma 1 for FGFR2 regulates basal phospholipase activity and invasion. Nat. Struct. Mol. Biol. 21, 180–188 (2014).
Timsah, Z. et al. Grb2 depletion under non-stimulated conditions inhibits PTEN, promotes Akt-induced tumor formation and contributes to poor prognosis in ovarian cancer. Oncogene 35, 2186–2196 (2016).
Revankar, C. M., Cimino, D. F., Sklar, L. A., Arterburn, J. B. & Prossnitz, E. R. A transmembrane intracellular estrogen receptor mediates rapid cell signaling. Science 307, 1625–1630 (2005).
Ghaemmaghami, S. et al. Global analysis of protein expression in yeast. Nature 425, 737–741 (2003).
Restrepo-Perez, L., Joo, C. & Dekker, C. Paving the way to single-molecule protein sequencing. Nat. Nanotechol. 13, 786–796 (2018).
Howard, M. & Rutenberg, A. D. Pattern formation inside bacteria: fluctuations due to the low copy number of proteins. Phys. Rev. Lett. 90, 128102 (2003).
Patel, A. P. et al. Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma. Science 344, 1396–1401 (2014).
Navin, N. et al. Tumour evolution inferred by single-cell sequencing. Nature 472, 90–U119 (2011).
Tang, F. C. et al. mRNA-Seq whole-transcriptome analysis of a single cell. Nat. Methods 6, 377–U86 (2009).
Klein, A. M. et al. Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells. Cell 161, 1187–1201 (2015).
Liu, J., Yin, D. Y., Wang, S. S., Chen, H. Y. & Liu, Z. Probing low-copy-number proteins in a single living cell. Angew. Chem. Int. Ed. 55, 13215–13218 (2016).
Wen, Y. R., Liu, J., He, H., Li, S. S. C. & Liu, Z. Single cell analysis of signaling proteins provides insights into pro-apoptotic properties of anti-cancer drugs. Anal. Chem. 92, 12498–12508 (2020).
Valaskovic, G. A., Kelleher, N. L. & McLafferty, F. W. Attomole protein characterization by capillary electrophoresis mass spectrometry. Science 273, 1199–1202 (1996).
Zhang, L. W. & Vertes, A. Single-cell mass spectrometry approaches to explore cellular heterogeneity. Angew. Chem. Int. Ed. 57, 4466–4477 (2018).
Rubakhin, S. S. & Sweedler, J. V. Characterizing peptides in individual mammalian cells using mass spectrometry. Nat. Protoc. 2, 1987–1997 (2007).
Irish, J. M. et al. Single cell profiling of potentiated phospho-protein networks in cancer cells. Cell 118, 217–228 (2004).
Newman, J. R. S. et al. Single-cell proteomic analysis of S. cerevisiae reveals the architecture of biological noise. Nature 441, 840–846 (2006).
Hughes, A. J. et al. Single-cell western blotting. Nat. Methods 11, 749–U94 (2014).
Cai, L., Friedman, N. & Xie, X. S. Stochastic protein expression in individual cells at the single molecule level. Nature 440, 358–362 (2006).
Huang, B. et al. Counting low-copy number proteins in a single cell. Science 315, 81–84 (2007).
Taniguchi, Y. et al. Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single cells. Science 329, 533–538 (2010).
Yu, J., Xiao, J., Ren, X. J., Lao, K. Q. & Xie, X. S. Probing gene expression in live cells, one protein molecule at a time. Science 311, 1600–1603 (2006).
Tu, X. Y. et al. Molecularly imprinted polymer-based plasmonic immunosandwich assay for fast and ultrasensitive determination of trace glycoproteins in complex samples. Anal. Chem. 88, 12363–12370 (2016).
Muhammad, P., Tu, X. Y., Liu, J., Wang, Y. J. & Liu, Z. Molecularly imprinted plasmonic substrates for specific and ultrasensitive immunoassay of trace glycoproteins in biological samples. ACS Appl. Mater. Inter. 9, 12082–12091 (2017).
Li, W. et al. Controllably prepared aptamer-molecularly imprinted polymer hybrid for high-specificity and high-affinity recognition of target proteins. Anal. Chem. 91, 4831–4837 (2019).
Xing, R. R. et al. Dual molecularly imprinted polymer-based plasmonic immunosandwich assay for specific and sensitive detection of protein biomarkers. Anal. Chem. 91, 9993–10000 (2019).
Zhou, L. L. et al. Orthogonal dual molecularly imprinted polymer-based plasmonic immunosandwich assay: a double characteristic recognition strategy for specific detection of glycoproteins. Biosens. Bioelectron. 145, 111729 (2019).
Muhammad, P. et al. Fast probing of glucose and fructose in plant tissues via plasmonic affinity sandwich assay with molecularly-imprinted extraction microprobes. Anal. Chim. Acta 995, 34–42 (2017).
Liu, J., Wen, Y. R., He, H., Chen, H. Y. & Liu, Z. Probing cytoplasmic and nuclear microRNAs in single living cells via plasmonic affinity sandwich assay. Chem. Sci. 9, 7241–7246 (2018).
Zhang, Q. et al. Gold nanoparticle-decorated Ag@SiO2 nanocomposite-based plasmonic affinity sandwich assay of circulating microRNAs in human serum. ACS Appl. Nano Mater. 2, 3960–3970 (2019).
Wulff, G. & Sarhan, A. Use of polymers with enzyme-analogous structures for resolution of racemates. Angew. Chem. Int. Ed. 11, 341–342 (1972).
Vlatakis, G., Andersson, L. I., Muller, R. & Mosbach, K. Drug assay using antibody mimics made by molecular imprinting. Nature 361, 645–647 (1993).
Ye, L. & Mosbach, K. Molecular imprinting: synthetic materials as substitutes for biological antibodies and receptors. Chem. Mater. 20, 859–868 (2008).
Nishino, H., Huang, C. S. & Shea, K. J. Selective protein capture by epitope imprinting. Angew. Chem. Int. Ed. 45, 2392–2396 (2006).
Sibrian-Vazquez, M. & Spivak, D. A. Molecular imprinting made easy. J. Am. Soc. Chem. 126, 7827–7833 (2004).
Li, L., Lu, Y., Bie, Z. J., Chen, H. Y. & Liu, Z. Photolithographic boronate affinity molecular imprinting: a general and facile approach for glycoprotein imprinting. Angew. Chem. Int. Ed. 52, 7451–7454 (2013).
Wang, S. S., Ye, J., Bie, Z. J. & Liu, Z. Affinity-tunable specific recognition of glycoproteins via boronate affinity-based controllable oriented surface imprinting. Chem. Sci. 5, 1135–1140 (2014).
Lee, P. C. & Meisel, D. Adsorption and surface-enhanced Raman of dyes on silver and gold sols. J. Phy. Chem. 86, 3391–3395 (1982).
Ye, J., Chen, Y. & Liu, Z. A boronate affinity sandwich assay: an appealing alternative to immunoassays for the determination of glycoproteins. Angew. Chem. Int. Ed. 53, 10386–10389 (2014).
Bie, Z. J., Chen, Y., Ye, J., Wang, S. S. & Liu, Z. Boronate-affinity glycan-oriented surface imprinting: a new strategy to mimic lectins for the recognition of an intact glycoprotein and its characteristic fragments. Angew. Chem. Int. Ed. 54, 10211–10215 (2015).
Delean, A., Munson, P. J. & Rodbard, D. Simultaneous analysis of families of sigmoidal curves-application to bioassay, radioligand assay, and physiological dose-response curves. Am. J. Physiol. 235, E97–E102 (1978).
Acknowledgements
This work is supported by Key Scientific Instrumentation Grant (21627810) from the National Natural Science Foundation of China, and Excellent Research Program of Nanjing University (ZYJH004).
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J.L., R.Y.W. and Z.L. developed the protocol. J.L., H.H. and Z.L. wrote the paper. All authors have discussed the results and approved the final version of the manuscript.
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Key references using this protocol
Liu, J. et al. Angew. Chem. Int. Ed. 55, 13215–13218 (2016): https://onlinelibrary.wiley.com/doi/full/10.1002/anie.201608237
Liu, J. et al. Chem. Sci. 9, 7241–7246 (2018): https://pubs.rsc.org/en/content/articlepdf/2018/sc/c8sc02533a
Wen, Y. R. et al. Anal. Chem. 92, 12498–12508 (2020): https://pubs.acs.org/doi/10.1021/acs.analchem.0c02344
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Raw data for Fig. 5e and 5f.
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Liu, J., He, H., Xie, D. et al. Probing low-copy-number proteins in single living cells using single-cell plasmonic immunosandwich assays. Nat Protoc 16, 3522–3546 (2021). https://doi.org/10.1038/s41596-021-00547-9
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DOI: https://doi.org/10.1038/s41596-021-00547-9
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