High-Throughput Workflow for Detergent-free Cell-Based Proteomic Characterization.

Area of Science:

• Proteomics
• Biochemistry
• Cell Biology

Background:

• High-throughput (HTP) protein quantification is crucial for large-scale biological studies.
• Current methods can be time-consuming and labor-intensive, limiting scalability.
• Automated workflows are needed to accelerate proteomic analyses.

Purpose of the Study:

• To develop an automated, high-throughput cell-based workflow for protein quantification using liquid chromatography-mass spectrometry (LC-MS).
• To enable large-scale perturbation studies in a 96-well plate format.
• To optimize cell lysis, protein solubilization, and peptide preparation for rapid analysis.

Main Methods:

• Utilized adaptive focused acoustics (AFA) for automated cell lysis and protein solubilization in a 96-well plate format.
• Integrated protein processing into tryptic peptides within 2 hours on an automated liquid handling platform.
• Employed data-independent acquisition mass spectrometry (DIA-MS) for protein quantification.

Main Results:

• Successfully quantified over 5100 unique proteins from AC16 human cardiomyocyte-like cells.
• Achieved high throughput, processing one 96-well plate in approximately 4 hours.
• Demonstrated high reproducibility, with 50% of measured proteins showing a coefficient of variation (CV) under 25% from ~30,000 cells.
• Identified over 30,000 peptides in total.
• An optimized detergent-free buffer yielded comparable results, identifying 5000 proteins with 40% having a CV under 25%.

Conclusions:

• The developed automated workflow significantly enhances throughput for cell-based protein quantification via LC-MS.
• This HTP approach is suitable for large-scale perturbation studies and complex biological investigations.
• The workflow demonstrates robust protein identification and quantification with high reproducibility.