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BioengineeringGenome biology
Peer-reviewed journal article

Mass-spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation

Bogdan BudnikiD, Ezra LevyiD, Guillaume HarmangeiD, Nikolai SlavoviD

Abstract

Cellular heterogeneity is important to biological processes, including cancer and development. However, proteome heterogeneity is largely unexplored because of the limitations of existing methods for quantifying protein levels in single cells. To alleviate these limitations, we developed Single Cell ProtEomics by Mass Spectrometry (SCoPE-MS), and validated its ability to identify distinct human cancer cell types based on their proteomes. We used SCoPE-MS to quantify over a thousand proteins in differentiating mouse embryonic stem (ES) cells. The single-cell proteomes enabled us to deconstruct cell populations and infer protein abundance relationships. Comparison between single-cell proteomes and transcriptomes indicated coordinated mRNA and protein covariation. Yet many genes exhibited functionally concerted and distinct regulatory patterns at the mRNA and the protein levels, suggesting that post-transcriptional regulatory mechanisms contribute to proteome remodeling during lineage specification, especially for developmental genes. SCoPE-MS is broadly applicable to measuring proteome configurations of single cells and linking them to functional phenotypes, such as cell type and differentiation potentials.

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✦ Multi-agent verified full-text synthesis

Claude Opus 5.5 · xhigh effort · 5 Nov 2026 · from the full text of version 4 (25 Jun 2018) · CC BY-NC-ND 4.0

SCoPE-MS combines acoustic cell lysis with isobaric tandem-mass-tag carrier channels to quantify over a thousand proteins in single mammalian cells, resolving cancer cell types and stem cell differentiation.

Single-cell protein quantification in mammalian cells has been limited to antibody and fluorescence assays measuring a few dozen proteins, while bulk LC-MS/MS loses low-input samples during cleanup and MS2 fragmentation. Introducing a 100-200 cell TMT-labeled carrier channel alongside mechanically sonicated single cells minimizes peptide losses and supplies fragment ions for peptide sequencing.

Key results

  1. ShownSCoPE-MS achieves over 10-fold signal-to-noise ratio in single mammalian cells and separates human Jurkat and U-937 blood cancer cells independently of carrier channel composition.

    In single-cell 10-plex TMT sets (mean single-cell reporter ion intensity ~500 vs near zero in empty channels 126-130C), PCA of 767 quantified proteins across 3 label-swapped sets of single Jurkat and U-937 cells (11 um diameter) separated cells by type alongside 2,500-cell bulk samples using 200 Jurkat, U-937, or HEK-293 carrier cells, identifying 107 cell-type-specific proteins at FDR < 2%. · Results; Figure legends

  2. ShownRelative single-cell protein quantification by SCoPE-MS agrees with orthogonal FACS fluorescence measurements and demonstrates reproducible fold-change estimation.

    In single FACS-sorted MDA-MB-231 adenocarcinoma cells expressing mCherry and LifeAct-iRFP670, SCoPE-MS relative abundances correlated with FACS fluorescence at Spearman rho > 0.7 for both proteins; split-sample technical replicates (1/2 cell each) showed reliability between 60% and 85% for larger fold changes, and inter-tag Jurkat/U-937 peptide ratios correlated at mean rho > 0.5. · Results; Figure legends

  3. ShownSCoPE-MS quantifies over a thousand proteins across 190 differentiating mouse embryonic stem cells, resolving time-dependent trajectories and co-regulated protein complexes.

    Across 190 single mouse ES and embryoid body cells sampled at days 3, 5, and 8 after LIF withdrawal, SCoPE-MS quantified 1,526 proteins at FDR <= 1% (primarily abundant proteins at >= 10^5 copies/cell); pairwise protein-protein correlations clustered ribosomal (60S and 40S), proteasome, electron transport chain, neuronal, blood, and muscle proteins, and the first three PCs correlated with differentiation day. · Protein covariation across differentiating ES…; Principal component analysis of differentiating…; Figure legends

  4. ShownComparing SCoPE-MS single-cell proteomes with inDrop single-cell transcriptomes reveals coordinated mRNA-protein covariation with higher concordance for ribosomal proteins than developmental genes.

    Hierarchical clustering of pairwise mRNA-mRNA (inDrop) and protein-protein (SCoPE-MS) correlations in day 7 and 8 differentiating EB cells yielded 3 matching clusters (Jaccard similarity 55% for the largest cluster and 33% for the medium cluster), with correlation-vector concordance significantly higher for 60S RPL and 40S RPS ribosomal proteins (p < 10^-20) and lower for developmental genes (p < 10^-3). · Coordinated mRNA and protein covariation in…; Figure legends

Methods, data and reproducibility

Individually micropipetted live cells were lysed by Covaris S220 sonication in glass microtubes, heat-denatured at 90 C, trypsin-digested, labeled with 10-plex TMT (channel 130N empty; channel 131 with 100-200 carrier cells), and analyzed by nano-LC-MS/MS on an Orbitrap Elite (150-250 ms MS2). Spectra were searched in MaxQuant 1.5.7.0, normalized, kNN-imputed (k = 1), and compared to FACS and inDrop scRNA-seq.

  • SCoPE-MS 10-plex TMT proteomics of single human Jurkat and U-937 cells and 2,500-cell bulk controls with Jurkat, U-937, or HEK-293 carrier channels · human · n = 3 label-swapped sets, 3 carrier-control sets, 6 bulk samples (767 proteins) · MSV000082077
  • FACS fluorescence and SCoPE-MS quantification of single MDA-MB-231 adenocarcinoma cells expressing mCherry and LifeAct-iRFP670 · human · n = Single cells sorted into 96-well / PCR strip tubes · PXD008985
  • SCoPE-MS single-cell proteomics of differentiating mouse E14 embryonic stem cells and embryoid bodies at days 3, 5, and 8 post-LIF withdrawal · mouse · n = 190 single cells (1,526 quantified proteins at FDR <= 1%) · MSV000082077
  • Published inDrop single-cell RNA-seq dataset of differentiating mouse embryonic stem cells at days 7 and 8 (from Klein et al.) · mouse
Methods & tools
SCoPE-MS, MaxQuant (v1.5.7.0), SwissProt, Covaris S220, LTQ Orbitrap Elite, TMT10-plex
Data & accessions
http://northeastern.edu/slavovlab/2016_SCoPE-MS/
Reagents & environment
MaxQuant 1.5.7.0 with human and mouse SwissProt databases (and reduced custom databases of 4,961 human and 5,267 mouse proteins)
Reproducibility gaps
Custom scripts for reporter-ion normalization, k-nearest-neighbors imputation, and mRNA-protein covariation analysis are not linked via a code repository URL in the text.
Limitations (3)
  • Shotgun SCoPE-MS primarily quantifies abundant proteins (>= 10^5 copies/cell and a few at >= 10^4 copies/cell), missing low-abundance regulatory proteins such as transcription factors without targeted MS and reduced lysis volumes. · stated by the authors
  • Single-cell SCoPE-MS quantification is noisier than bulk LC-MS/MS and contains missing values across cells that partially correlate with principal component loadings. · stated by the authors
  • Manual micropipetting of single cells and sonication in glass Covaris tubes limits throughput and costs $15-$30 per cell unless tubes are washed/reused and sample preparation is automated. · stated by the authors

Who should read:Read this if you develop or apply single-cell mass spectrometry proteomics, study post-transcriptional regulation during stem cell differentiation, or integrate single-cell proteome and transcriptome covariation.

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