[Methods and applications of single-cell proteomics analysis based on mass spectrometry]

Area of Science:

• Biochemistry and Molecular Biology
• Proteomics
• Cell Biology

Background:

• Cellular heterogeneity is critical in differentiation, disease, and therapy.
• Proteins are key to cell activity but challenging to study at the single-cell level due to low abundance and complexity.
• Existing methods like fluorescence and electrochemistry have limitations for comprehensive single-cell proteomics.

Purpose of the Study:

• To review and categorize representative mass spectrometry (MS)-based methods for single-cell proteomics.
• To highlight the advantages and limitations of different protein separation techniques prior to MS analysis.
• To discuss the future prospects of single-cell proteomic research.

Main Methods:

• Capillary Electrophoresis-Mass Spectrometry (CE-MS): Utilizes capillaries for sample extraction and separation, offering high efficiency but limited to large cells.
• Liquid Chromatography-Mass Spectrometry (LC-MS), especially nanoLC: Preferred for its reproducibility, low sample loss, and compatibility with MS, enabling identification of over 1000 proteins in single cells.
• Direct Infusion-Mass Spectrometry: Simplifies sample preparation by eliminating enzymatic hydrolysis and separation, offering higher resolution and multi-omics compatibility, though currently limited in protein detection.

Main Results:

• CE-MS is effective for large cells but faces issues with sample loss and reproducibility.
• nanoLC-MS shows significant progress, with reduced sampling volumes and improved protein identification in various cell types.
• Direct infusion MS simplifies workflows but has detected fewer proteins compared to separation-based methods.

Conclusions:

• Each MS-based single-cell proteomics method (CE-MS, LC-MS, direct infusion) has distinct strengths and weaknesses regarding throughput, sensitivity, and protein identification.
• Advancements in LC-MS, particularly nanoLC, are driving progress in identifying a larger number of proteins from single cells.
• Future research should focus on improving sensitivity, throughput, and the number of identified proteins for broader applications in single-cell analysis.