Bridging Simplicity and Depth in Single-Cell Proteomics: A Cost-Effective Workflow and an Expanded Framework for Data...

Area of Science:

• Biochemistry
• Proteomics
• Cell Biology

Background:

• Single-cell proteomics (SCP) provides functional insights into cellular heterogeneity.
• Current SCP methods often require specialized instrumentation and lack comprehensive quality evaluation.
• Existing evaluation metrics focus on identification counts, not biological interpretability.

Purpose of the Study:

• To develop an accessible, label-free single-cell proteomics workflow.
• To optimize sample preparation for enhanced data quality and reproducibility.
• To introduce a robust data quality framework for more interpretable SCP analysis.

Main Methods:

• Utilized standard laboratory equipment including a single-cell dispenser and multiwell plates.
• Employed trapped ion mobility spectrometry-time-of-flight mass spectrometry (timsTOF) for analysis.
• Systematically optimized sample preparation variables (e.g., trypsin concentration, incubation time, digestion conditions).

Main Results:

• Developed and validated an accessible, label-free SCP workflow.
• Optimized protocols maximized data quality and reproducibility.
• Introduced a data quality framework assessing quantitative consistency and biological interpretability.

Conclusions:

• The developed workflow lowers technical barriers for single-cell proteomics adoption.
• The new framework enables more rigorous, interpretable, and scalable SCP analysis.
• This approach facilitates broader application of SCP across research fields.