Robust and Easy-to-Use One-Pot Workflow for Label-Free Single-Cell Proteomics

Anal Chem. 2023 Mar 7;95(9):4435-4445. doi: 10.1021/acs.analchem.2c05022. Epub 2023 Feb 20.


The analysis of ultralow input samples or even individual cells is essential to answering a multitude of biomedical questions, but current proteomic workflows are limited in their sensitivity and reproducibility. Here, we report a comprehensive workflow that includes improved strategies for all steps, from cell lysis to data analysis. Thanks to convenient-to-handle 1 μL sample volume and standardized 384-well plates, the workflow is easy for even novice users to implement. At the same time, it can be performed semi-automatized using CellenONE, which allows for the highest reproducibility. To achieve high throughput, ultrashort gradient lengths down to 5 min were tested using advanced μ-pillar columns. Data-dependent acquisition (DDA), wide-window acquisition (WWA), data-independent acquisition (DIA), and commonly used advanced data analysis algorithms were benchmarked. Using DDA, 1790 proteins covering a dynamic range of four orders of magnitude were identified in a single cell. Using DIA, proteome coverage increased to more than 2200 proteins identified from single-cell level input in a 20 min active gradient. The workflow enabled differentiation of two cell lines, demonstrating its suitability to cellular heterogeneity determination.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Proteome* / analysis
  • Proteomics*
  • Reproducibility of Results
  • Workflow


  • Proteome