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Single-Cell Proteome Profiling of Neuronal Cells

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Single Cell ‘Omics of Neuronal Cells

Part of the book series: Neuromethods ((NM,volume 184))

Abstract

Characterizing neuronal heterogeneity at the single-cell level can improve our understanding of developmental and disease processes and lead to novel therapeutic strategies. While single-cell genomic and transcriptomic measurements have matured in recent years, mass spectrometry-based proteome profiling has lagged due to sensitivity limitations. However, recent progress in miniaturizing sample processing and separations have made it possible to profile >1000 proteins from single mammalian cells including neurons. Here, we present a detailed procedure that has provided the greatest depth of coverage for proteome profiling of single neurons to date. Single motor neurons and interneurons were isolated from human spinal tissue sections by laser capture microdissection, processed for proteomic analysis within nanoliter volumes and analyzed by ultralow-flow nanoLC-MS/MS. The protocol should be adaptable to a variety of applications and measurement platforms to further neuronal research.

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References

  1. Cadwell CR, Palasantza A, Jiang XL, Berens P, Deng QL, Yilmaz M, Reimer J, Shen S, Bethge M, Tolias KF, Sandberg R, Tolias AS (2016) Electrophysiological, transcriptomic and morphologic profiling of single neurons using patch-seq. Nat Biotechnol 34(2):199–203. https://doi.org/10.1038/nbt.3445

    Article  CAS  PubMed  Google Scholar 

  2. Bean BP (2007) The action potential in mammalian central neurons. Nat Rev Neurosci 8(6):451–465. https://doi.org/10.1038/nrn2148

    Article  CAS  PubMed  Google Scholar 

  3. Ko H, Hofer SB, Pichler B, Buchanan KA, Sjostrom PJ, Mrsic-Flogel TD (2011) Functional specificity of local synaptic connections in neocortical networks. Nature 473(7345):87–91. https://doi.org/10.1038/nature09880

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Kitchen RR, Rozowsky JS, Gerstein MB, Nairn AC (2014) Decoding neuroproteomics: integrating the genome, translatome and functional anatomy. Nat Neurosci 17(11):1491–1499. https://doi.org/10.1038/nn.3829

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Choi SB, Lombard-Banek C, Munoz LP, Manzini MC, Nemes P (2018) Enhanced peptide detection toward single-neuron proteomics by reversed-phase fractionation capillary electrophoresis mass spectrometry. J Am Soc Mass Spectrom 29(5):913–922. https://doi.org/10.1007/s13361-017-1838-1

    Article  CAS  PubMed  Google Scholar 

  6. Johnson MB, Wang PP, Atabay KD, Murphy EA, Doan RN, Hecht JL, Walsh CA (2015) Single-cell analysis reveals transcriptional heterogeneity of neural progenitors in human cortex. Nat Neurosci 18(5):637–646. https://doi.org/10.1038/nn.3980

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Chappell L, Russell AJC, Voet T (2018) Single-cell (multi)omics technologies. Annu Rev Genomics Hum Genet 19:15–41. https://doi.org/10.1146/annurev-genom-091416-035324

    Article  CAS  PubMed  Google Scholar 

  8. Saikia M, Burnham P, Keshavjee SH, Wang MFZ, Heyang M, Moral-Lopez P, Hinchman MM, Danko CG, Parker JSL, De Vlaminck I (2019) Simultaneous multiplexed amplicon sequencing and transcriptome profiling in single cells. Nat Methods 16(1):59–62. https://doi.org/10.1038/s41592-018-0259-9

    Article  CAS  PubMed  Google Scholar 

  9. Angel TE, Aryal UK, Hengel SM, Baker ES, Kelly RT, Robinson EW, Smith RD (2012) Mass spectrometry-based proteomics: existing capabilities and future directions. Chem Soc Rev 41(10):3912–3928. https://doi.org/10.1039/c2cs15331a

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Blackstock WP, Weir MP (1999) Proteomics: quantitative and physical mapping of cellular proteins. Trends Biotechnol 17(3):121–127. https://doi.org/10.1016/s0167-7799(98)01245-1

    Article  CAS  PubMed  Google Scholar 

  11. Bludau I, Aebersold R (2020) Proteomic and interactomic insights into the molecular basis of cell functional diversity (vol 21, pg 327, 2020). Nat Rev Mol Cell Bio 21(6):353–353. https://doi.org/10.1038/s41580-020-0249-5

    Article  CAS  Google Scholar 

  12. Kelly RT (2020) Single-cell proteomics: progress and prospects. Mol Cell Proteomics. https://doi.org/10.1074/mcp.R120.002234

  13. Kelly RT, Zhu Y (2019) Ultrasmall sample biochemical analysis. Anal Bioanal Chem 411(21):5349–5350. https://doi.org/10.1007/s00216-019-01957-1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Shao X, Wang X, Guan S, Lin H, Yan G, Gao M, Deng C, Zhang X (2018) Integrated proteome analysis device for fast single-cell protein profiling. Anal Chem 90(23):14003–14010. https://doi.org/10.1021/acs.analchem.8b03692

    Article  CAS  PubMed  Google Scholar 

  15. Li ZY, Huang M, Wang XK, Zhu Y, Li JS, Wong CCL, Fang Q (2018) Nanoliter-scale oil-air-droplet Chip-based single cell proteomic analysis. Anal Chem 90(8):5430–5438. https://doi.org/10.1021/acs.analchem.8b00661

    Article  CAS  PubMed  Google Scholar 

  16. Zhu Y, Piehowski PD, Zhao R, Chen J, Shen Y, Moore RJ, Shukla AK, Petyuk VA, Campbell-Thompson M, Mathews CE, Smith RD, Qian WJ, Kelly RT (2018) Nanodroplet processing platform for deep and quantitative proteome profiling of 10-100 mammalian cells. Nat Commun 9(1):882. https://doi.org/10.1038/s41467-018-03367-w

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Zhu Y, Clair G, Chrisler WB, Shen Y, Zhao R, Shukla AK, Moore RJ, Misra RS, Pryhuber GS, Smith RD, Ansong C, Kelly RT (2018) Proteomic analysis of single mammalian cells enabled by microfluidic Nanodroplet sample preparation and ultrasensitive NanoLC-MS. Angew Chem Int Ed Engl 57(38):12370–12374. https://doi.org/10.1002/anie.201802843

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Zhu Y, Dou M, Piehowski PD, Liang Y, Wang F, Chu RK, Chrisler WB, Smith JN, Schwarz KC, Shen Y, Shukla AK, Moore RJ, Smith RD, Qian WJ, Kelly RT (2018) Spatially resolved proteome mapping of laser capture microdissected tissue with automated sample transfer to Nanodroplets. Mol Cell Proteomics 17(9):1864–1874. https://doi.org/10.1074/mcp.TIR118.000686

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Cong YZ, Liang YR, Motamedchaboki K, Huguet R, Truong T, Zhao R, Shen YF, Lopez-Ferrer D, Zhu Y, Kelly RT (2020) Improved single-cell proteome coverage using narrow-bore packed NanoLC columns and ultrasensitive mass spectrometry. Anal Chem 92(3):2665–2671. https://doi.org/10.1021/acs.analchem.9b04631

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Budnik B, Levy E, Harmange G, Slavov N (2018) SCoPE-MS: mass spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation. Genome Biol 19(1):161. https://doi.org/10.1186/s13059-018-1547-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Dou MW, Clair G, Tsai CF, Xu KR, Chrisler WB, Sontag RL, Zhao R, Moore RJ, Liu T, Pasa-Tolic L, Smith RD, Shi TJ, Adkins JN, Qian WJ, Kelly RT, Ansong C, Zhu Y (2019) High-throughput single cell proteomics enabled by multiplex isobaric labeling in a Nanodroplet sample preparation platform. Anal Chem 91(20):13119–13127. https://doi.org/10.1021/acs.analchem.9b03349

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Tsai CF, Zhao R, Williams SM, Moore RJ, Schultz K, Chrisler WB, Pasa-Tolic L, Rodland KD, Smith RD, Shi T, Zhu Y, Liu T (2020) An improved boosting to amplify signal with isobaric labeling (iBASIL) strategy for precise quantitative single-cell proteomics. Mol Cell Proteomics 19(5):828–838. https://doi.org/10.1074/mcp.RA119.001857

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Cong Y, Motamedchaboki K, Misal SA, Liang Y, Guise AJ, Truong T, Huguet R, Plowey ED, Zhu Y, Lopez-Ferrer D, Kelly RT (2020) Ultrasensitive single-cell proteomics workflow identifies >1000 protein groups per mammalian cell. Biorxiv. https://doi.org/10.1101/2020.06.03.132449

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Acknowledgments

This work was supported by the National Institutes of Health under award numbers R33 CA225248 and R01 GM138931, and through a sponsored research agreement from Biogen, Inc.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT, USA

    Santosh A. Misal & Ryan T. Kelly

Authors
  1. Santosh A. Misal
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  2. Ryan T. Kelly
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Corresponding author

Correspondence to Ryan T. Kelly .

Editor information

Editors and Affiliations

  1. The Beckman Institute, University of Illinois Urbana-Champaign, Urbana, IL, USA

    Jonathan V. Sweedler

  2. Dept Pharmacology, 38 John Morgan Bldg, University of Pennsylvania, Philadelphia, PA, USA

    James Eberwine

  3. Bridge Institute, University of Southern California, Los Angeles, CA, USA

    Scott E. Fraser

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© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Misal, S.A., Kelly, R.T. (2022). Single-Cell Proteome Profiling of Neuronal Cells. In: Sweedler, J.V., Eberwine, J., Fraser, S.E. (eds) Single Cell ‘Omics of Neuronal Cells. Neuromethods, vol 184. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2525-5_3

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  • DOI: https://doi.org/10.1007/978-1-0716-2525-5_3

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2524-8

  • Online ISBN: 978-1-0716-2525-5

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