bioRxivSystems BiologyPreprint
Automated sample preparation for high-throughput single-cell proteomics
Harrison SpechtiD, Guillaume HarmangeiD, David H. PerlmaniD, Edward EmmottiD, Zachary NiziolekiD, Bogdan BudnikiD, Nikolai SlavoviD
Abstract
A major limitation to applying quantitative LC-MS/MS proteomics to small samples, such as single cells, are the losses incured during sample cleanup. To relieve this limitation, we developed a Minimal ProteOmic sample Preparation (mPOP) method for culture-grown mammalian cells. mPOP obviates cleanup and thus eliminates cleanup-related losses while expediting sample preparation and simplifying its automation. Bulk SILAC samples processed by mPOP or by conventional urea-based methods indicated that mPOP results in complete cell lysis and accurate relative quantification. We integrated mPOP lysis with the Single Cell ProtEomics by Mass Spectrometry (SCoPE-MS) sample preparation, and benchmarked the quantification of such samples on a Q-exactive instrument. The results demonstrate low noise and high technical reproducibility. Then, we FACS sorted single U-937, HEK-293, and mouse ES cells into 96-well plates and analyzed them by automated mPOP and SCoPE-MS. The quantified proteins enabled separating the single cells by cell-type and cell-division-cycle phase.
- organism: mouse
- organism: human
- assay: proteomics
- research focus: cell type annotation
- research focus: cell-cell communication
- approach: experimental assay
Abstract summary
Claude Opus 5.5 · xhigh effort · 2 Nov 2026 · from the abstract · CC BY-NC-ND 4.0
Minimal ProteOmic sample Preparation (mPOP) eliminates cleanup losses in single-cell mass spectrometry proteomics, enabling automated SCoPE-MS profiling that separates mammalian cells by cell type and cell-division-cycle phase.
Sample losses during cleanup steps hinder quantitative LC-MS/MS proteomics on low-input specimens like single cells. Compared with conventional urea-based sample preparation, Minimal ProteOmic sample Preparation (mPOP) removes the cleanup step altogether, preventing cleanup-related losses and simplifying automation.
Key results
ShownAutomated mPOP integrated with SCoPE-MS resolves single mammalian cells by both cell identity and cell-division-cycle phase.
FACS-sorted single U-937, HEK-293, and mouse ES cells prepared in 96-well plates via automated mPOP and SCoPE-MS yielded protein quantifications that separated cells by cell type and cell-division-cycle phase (no numerical metrics reported in the abstract). · Abstract
ShownmPOP achieves complete cell lysis, accurate relative quantification, low noise, and high technical reproducibility.
Bulk SILAC samples showed complete lysis and accurate quantification relative to conventional urea-based methods, and mPOP integrated with SCoPE-MS benchmarked on a Q-exactive instrument showed low noise and high reproducibility (specific values are not reported in the abstract). · Abstract
Methods, data and reproducibility
The authors developed Minimal ProteOmic sample Preparation (mPOP) to lyse culture-grown mammalian cells without sample cleanup, comparing it against conventional urea-based processing on bulk SILAC samples. They then integrated automated mPOP lysis in 96-well plates with Single Cell ProtEomics by Mass Spectrometry (SCoPE-MS) on a Q-exactive instrument to profile FACS-sorted single cells.
- Bulk SILAC culture-grown mammalian cell samples
- FACS-sorted single U-937 and HEK-293 cells in 96-well plates · human (inferred from U-937 and HEK-293 cell lines)
- FACS-sorted single mouse ES cells in 96-well plates · mouse
- Methods & tools
- Minimal ProteOmic sample Preparation (mPOP), Single Cell ProtEomics by Mass Spectrometry (SCoPE-MS), LC-MS/MS, SILAC, Q-exactive, FACS
- Reproducibility gaps
- The abstract does not provide data accessions, code repositories, or protocol links.
Limitations (2)
- mPOP is specifically described for culture-grown mammalian cells, and its applicability to primary tissues or non-mammalian samples is not addressed in the abstract. · noted in this summary
- The abstract provides no quantitative figures for protein counts, cell numbers, noise levels, or technical reproducibility. · noted in this summary
Read this if you perform single-cell or low-input LC-MS/MS proteomics and want an automated, cleanup-free cell lysis protocol compatible with SCoPE-MS in 96-well plates.
- Citations
- 77
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