Optimization of trap column properties and loading conditions for proteome profiling of single-cell-level sample...

Area of Science:

• Proteomics
• Analytical Chemistry
• Biotechnology

Background:

• Nanoflow liquid chromatography-mass spectrometry (NLC-MS) is crucial for analyzing limited biological samples.
• Sample loading efficiency is a key limitation in NLC-MS, impacting sensitivity, robustness, and throughput.
• Trap-and-elute workflows are used to enhance loading but require systematic evaluation.

Purpose of the Study:

• To systematically evaluate how trap column properties and loading parameters affect proteome coverage in NLC-MS.
• To assess the impact of trap column configurations under low-input and high-throughput conditions.
• To provide guidelines for optimizing trap-and-elute workflows in proteomics.

Main Methods:

• Investigated trap column inner diameter, particle size, packing material, loading flow rate, and sample concentration.
• Utilized both bulk-prepared digests and single-cell samples.
• Assessed proteome coverage, chromatographic quality, and peak characteristics.

Main Results:

• Trap columns significantly reduce performance losses from large-volume sample loading.
• Minimal impact on peak width, peak area, and identification depth was observed.
• Trap column geometry and loading speed showed minor influence on chromatographic quality and proteome depth.
• Trap-and-elute workflows offer substantial flexibility in LC system design.

Conclusions:

• Trap-and-elute strategies are highly suitable for high-sensitivity, high-throughput proteomics.
• Practical guidelines for optimizing trap column configurations can be established.
• These workflows enhance the analysis of limited and heterogeneous biological samples.