A Protocol for Robust Discovery Proteomics Using Nano Liquid Chromatography Using Pillar-Array Column Technology and...

Area of Science:

• Proteomics
• Analytical Chemistry
• Biotechnology

Background:

• Nano-liquid chromatography-mass spectrometry (LC-MS/MS) is crucial for shotgun proteomics, offering high separation power and sensitivity.
• Establishing robust and reliable LC-MS/MS workflows for large-scale proteomics remains challenging.

Purpose of the Study:

• To provide a detailed protocol for establishing nano-LC-MS/MS workflows for comprehensive, untargeted deep proteome profiling.
• To optimize a data-independent acquisition (DIA)-parallel accumulation-serial fragmentation (PASEF) method for high-throughput proteomics.
• To demonstrate the robustness and reproducibility of the developed workflow.

Main Methods:

• Utilized state-of-the-art second-generation micropillar-array column technology for enhanced chromatographic separation.
• Developed a straightforward workflow for data-independent acquisition (DIA)-parallel accumulation-serial fragmentation (PASEF) analysis, including ESI source optimization.
• Explored segmented gradients and MS-compatible surfactants for improved analytical performance.

Main Results:

• Demonstrated a trade-off between analysis time and chromatographic resolution impacting peptide and protein identification.
• Achieved consistent identification of 7558 protein groups with high repeatability (CV = 0.3%).
• Maintained high reproducibility in peptide retention times (mean CV = 0.2%) and system pressure (CV = 0.4%) over 21 analyses.

Conclusions:

• The developed nano-LC-MS/MS workflow provides a robust and reproducible method for deep proteome profiling.
• This protocol facilitates reliable, large-scale proteomics studies in life sciences.
• The optimized DIA-PASEF method enhances throughput and data quality in proteomic analyses.