Simultaneous In-Depth Single-Cell Proteomic and Metabolomic Analysis

Area of Science:

• Biochemistry
• Cell Biology
• Analytical Chemistry

Background:

• Simultaneous proteomic and metabolomic profiling at the single-cell level is essential for understanding cellular heterogeneity and disease mechanisms.
• Achieving high-depth dual-omics analysis in the same single cell remains a significant technical challenge.
• Existing methods often lack the depth or integration required for comprehensive single-cell multi-omics.

Purpose of the Study:

• To develop a unified strategy for one-shot hybrid-mode single-cell proteome and metabolome analysis (hybrid-scPMA).
• To establish an improved workflow integrating automated capture, simplified pretreatment, and hybrid-mode mass spectrometry detection.
• To enable deep proteomic and metabolomic profiling in individual cells for enhanced biological insights.

Main Methods:

• Utilized a hybrid-mode mass spectrometry (MS) approach combining data-independent acquisition (DIA) for proteomics and data-dependent acquisition (DDA) for metabolomics within a single liquid chromatography-mass spectrometry (LC-MS) run.
• Developed an integrated workflow featuring automated single-cell capture, streamlined sample preparation, and LC separation.
• Employed DIA-DDA hybrid-mode MS detection for simultaneous proteomic and metabolomic analysis.

Main Results:

• Achieved an average identification of 3510 protein groups and 255 metabolites per single HepG2 cell, significantly increasing depth compared to previous methods.
• Demonstrated time-resolved proteomic and metabolomic profiling of HepG2 single cells under sorafenib treatment.
• Resolved drug response characteristics at the single-cell level, offering multi-omics insights into drug mechanisms.

Conclusions:

• The proposed hybrid-scPMA strategy and workflow enable unprecedented depth in single-cell proteo-metabolomic analysis.
• This approach provides valuable multi-omics insights into cellular responses to drug interventions at the single-cell level.
• Hybrid-scPMA is a powerful tool for deciphering cellular heterogeneity and elucidating complex disease mechanisms.