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Multiplexed single-cell proteomics using SCoPE2

Nature Protocols volume 16, pages 5398–5425 (2021)Cite this article

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Abstract

Many biological systems are composed of diverse single cells. This diversity necessitates functional and molecular single-cell analysis. Single-cell protein analysis has long relied on affinity reagents, but emerging mass-spectrometry methods (either label-free or multiplexed) have enabled quantifying >1,000 proteins per cell while simultaneously increasing the specificity of protein quantification. Here we describe the Single Cell ProtEomics (SCoPE2) protocol, which uses an isobaric carrier to enhance peptide sequence identification. Single cells are isolated by FACS or CellenONE into multiwell plates and lysed by Minimal ProteOmic sample Preparation (mPOP), and their peptides labeled by isobaric mass tags (TMT or TMTpro) for multiplexed analysis. SCoPE2 affords a cost-effective single-cell protein quantification that can be fully automated using widely available equipment and scaled to thousands of single cells. SCoPE2 uses inexpensive reagents and is applicable to any sample that can be processed to a single-cell suspension. The SCoPE2 workflow allows analyzing ~200 single cells per 24 h using only standard commercial equipment. We emphasize experimental steps and benchmarks required for achieving quantitative protein analysis.

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Fig. 1: Single-cell proteomics with SCoPE2.
Fig. 2: LC-MS/MS setup for SCoPE2 experiments.
Fig. 3: Evaluating data acquisition and interpretation using diagnostic plot generated by DO-MS.
Fig. 4: Evaluating protein quantification results from SCoPE2 analysis.

Data availability

All data are available at MassIVE MSV000087041 and via the SCoPE2 website: https://scope2.slavovlab.net/mass-spec/protocol. Datasets associated with certain figures have been uploaded to figshare: Fig. 3: Slavov, Nikolai (2021): Evaluating data acquisition and interpretation using diagnostic plots generated by DO-MS (https://doi.org/10.6084/m9.figshare.15060774.v1); Fig. 4: Slavov, Nikolai (2021): Principal Component Analysis (PCA) of quantification results from SCoPE2 analysis (https://doi.org/10.6084/m9.figshare.15060789.v1); Extended Data Fig. 1: Slavov, Nikolai (2021): The ABIRD device may suppress contaminant ions and enhance peptide identification (https://doi.org/10.6084/m9.figshare.15060846.v1) and Slavov, Nikolai (2021): Variable TMT search indicates very low level of over labeled peptides during SCoPE2 analysis (https://doi.org/10.6084/m9.figshare.15145092.v1). Source data are provided with this paper.

Code availability

The SCoPE2 pipeline used here is available at https://github.com/SlavovLab/SCoPE2 and via the SCoPE2 website (https://scope2.slavovlab.net/mass-spec/protocol).

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Acknowledgements

We thank A. Chen, and J. Neveu for assistance, discussions and constructive comments. This work was funded by a New Innovator Award from the NIGMS from the National Institutes of Health to N.S. under Award Number DP2GM123497, an Allen Distinguished Investigator award through The Paul G. Allen Frontiers Group to N.S., a Seed Networks Award from CZI CZF2019-002424 to N.S., through a Merck Exploratory Science Center Fellowship, Merck Sharpe & Dohme Corp. to N.S., and a Thermo Scientific Tandem Mass Tag Research Award to E.E. Funding bodies had no role in data collection, analysis and interpretation.

Author information

Author notes

  1. Edward Emmott

    Present address: Centre for Proteome Research, Department of Biochemistry & Systems Biology, University of Liverpool, Liverpool, UK

  2. David H. Perlman

    Present address: Merck Exploratory Sciences Center, Merck Sharp & Dohme Corp., Cambridge, MA, USA

  3. These authors contributed equally: Aleksandra A. Petelski, Edward Emmott.

Authors and Affiliations

  1. Department of Bioengineering, Northeastern University, Boston, MA, USA

    Aleksandra A. Petelski, Edward Emmott, Andrew Leduc, R. Gray Huffman, Harrison Specht, David H. Perlman & Nikolai Slavov

  2. Barnett Institute, Northeastern University, Boston, MA, USA

    Aleksandra A. Petelski, Edward Emmott, Andrew Leduc, R. Gray Huffman, Harrison Specht & Nikolai Slavov

  3. Department of Biology, Northeastern University, Boston, MA, USA

    Nikolai Slavov

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Contributions

Experimental design: A.P. and N.S. Sorting cells: A.L. and A.P. LC-MS/MS: G.H, E.E., H.S. and D.H.P. Sample preparation: A.P. Funding: N.S, E.E. Data analysis: A.P. and N.S. Supervision: N.S. Writing and editing: E.E., A.P. and N.S.

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Correspondence to Nikolai Slavov.

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Peer review information Nature Protocols thanks M. Arthur Moseley, Wenqing Shui and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

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Related links

Key references using this protocol

Specht, H. et al. Genome Biol. 22, 50 (2021): https://doi.org/10.1186/s13059-021-02267-5

Budnik, B. et al. Genome Biol. 19, 161 (2018): https://genomebiology.biomedcentral.com/articles/10.1186/s13059-018-1547-5

Specht, H. & Slavov, N. J. Proteome Res. 20, 880887 (2021): https://pubs.acs.org/doi/10.1021/acs.jproteome.0c00675

Extended data

Extended Data Fig. 1 The ABIRD may suppress contaminant ions and enhance peptide identification.

ABIRD may suppress contaminant ions and enhance peptide identification. Replicate injections of a 1× standard were analyzed with the ABIRD on or off. a, The replicates with ABIRD on had a reduced number of +1 ions (likely corresponding to contaminants) and an increased number of higher-charge-state ions, which are likely to correspond to peptides. b, With the ABIRD on, the number of identified peptides is increased across all confidence levels (PEP).

Source data

Supplementary information

Supplementary Data 1

XML file for MaxQuant that specifies the parameters for TMTpro 16-plex searches.

Source data

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 1

Statistical source data.

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Petelski, A.A., Emmott, E., Leduc, A. et al. Multiplexed single-cell proteomics using SCoPE2. Nat Protoc 16, 5398–5425 (2021). https://doi.org/10.1038/s41596-021-00616-z

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