Bridging Simplicity and Depth in Single-Cell Proteomics: A Cost-Effective Workflow and an Expanded Framework for Data Evaluation
Year: 2026
Type: article
Abstract: Single-cell proteomics (SCP) offers direct insight into functional protein states that drive cellular heterogeneity, complementing genomic and transcriptomic analyses. Although recent reports have demonstrated improved proteome coverage, their reliance on specialized instrumentation limits the broader adoption. Additionally, current evaluation practices remain largely centered on protein and peptide identification counts, which alone do not fully reflect data quality or biological interpretability. Here, we describe an accessible, label-free SCP workflow that implements easily accessible laboratory equipment: a single-cell dispenser, conventional multiwell plates, and an incubator with water-bath-based humidity control. Using trapped ion mobility spectrometry─time-of-flight mass spectrometry (timsTOF), we systematically optimized key sample preparation variables, including trypsin concentration, incubation time, reduction/alkylation, digestion conditions, and plate types, which together maximize data quality and reproducibility. We further introduce a data quality framework that moves beyond identification counts, emphasizing quantitative consistency and biological interpretability via individual protein coverage completeness across cells, coefficients of variation across technical replicates, peptide-to-protein ratios, and single-cell-to-bulk correlations. Collectively, our approach lowers technical barriers to accessing SCPs while enabling more rigorous, interpretable, and scalable SCP analysis across diverse research contexts.
Source: Journal of Proteome Research
Language: English
Subfield: Spectroscopy
Field: Chemistry
Domain: Physical Sciences
Open Access status: green
Funder: Oregon State University
OpenAlex ID: W7161636447