Microproteomics with microfluidic-based cell sorting: Application to 1000 and 100 immune cells.

Area of Science:

• Cell Biology
• Proteomics
• Microfluidics

Background:

• Cellular function studies require understanding molecular mechanisms.
• Cellular heterogeneity necessitates consideration in assay design and data interpretation.
• Single-cell analysis is limited by practical constraints like assay sensitivity.

Purpose of the Study:

• To develop an optimized microproteomics workflow for analyzing limited numbers of cells.
• To enable in-depth proteome exploration from rare or small cell populations.
• To overcome limitations in current proteomics methods for small sample sizes.

Main Methods:

• Developed a microproteomics workflow integrating microfluidic chip-based cell sorting with mass spectrometry (MS)-based proteomics.
• Utilized non-damaging cell sorting to isolate 1000 or 100 THP-1 cells.
• Performed nano-liquid chromatography-MS (LC-MS) analysis on peptide mixtures after enzymatic digestion.

Main Results:

• Achieved reasonable proteome coverage from as few as 100 sorted cells.
• Data from 1000 sorted cells demonstrated proteome comprehensiveness comparable to 1 μg of whole cell lysate.
• The workflow effectively handles limited cell numbers, minimizing sample loss.

Conclusions:

• The developed microproteomics workflow facilitates molecular-level elucidation of cell functions in small cell populations.
• This approach is valuable for studying rare cell subpopulations and limited clinical specimens.
• Further refinement could enhance its utility for diverse biological and clinical applications.