Liquid Chromatographic and Mass Spectrometric Methods for Quantitative Proteomic Analysis from Single-Cell and...

Area of Science:

• Proteomics
• Analytical Chemistry
• Biotechnology

Background:

• Liquid chromatography (LC) and mass spectrometry (MS) are essential for protein analysis.
• Advances in LC-MS enhance sensitivity for identifying and quantifying low-abundance proteins.
• Single-cell proteomics presents unique challenges due to limited sample amounts.

Purpose of the Study:

• To optimize LC-MS methods for sensitive protein identification and quantification in single-cell proteomics.
• To evaluate the sensitivity, reproducibility, and robustness of data-independent acquisition (DIA) for low-level protein analysis.
• To apply optimized methods for analyzing proteome changes in single cells.

Main Methods:

• Exploration of various LC conditions and MS platforms.
• Utilized data-independent acquisition (DIA) for comprehensive proteomic analysis.
• Applied optimized LC-MS/DIA methods to single HeLa and PC3 cells.

Main Results:

• Achieved identification and quantification of over 6300 proteins at the single-cell level with <20% coefficient of variation (CV).
• Detected up to 5000 proteins from isolated single HeLa cell samples.
• Revealed proteome alterations in docetaxel-treated versus non-treated PC3 cells at the single-cell level.

Conclusions:

• The optimized LC-MS/DIA approach provides a robust and sensitive platform for single-cell proteomics.
• This methodology enables deep proteome profiling from picogram-to-nanogram levels of proteins.
• The study offers a comprehensive technical evaluation for advancing single-cell proteomic applications.