Open-tubular trap columns: towards simple and robust liquid chromatography separations for single-cell proteomics

Area of Science:

• Analytical Chemistry
• Biochemistry
• Mass Spectrometry

Background:

• Nanoflow liquid chromatography-mass spectrometry is crucial for analyzing trace samples like single cells.
• Narrow-bore columns used in these analyses are prone to clogging, compromising robustness.
• Online solid-phase extraction (SPE) trap columns are vital for removing debris but can also clog.

Purpose of the Study:

• To investigate the use of large-bore porous layer open tubular trap columns (PLOTraps) to prevent clogging in nanoflow LC-MS.
• To evaluate PLOTraps for improved robustness and debris removal in trace sample analysis.
• To optimize PLOTrap stationary phases and diameters for enhanced chromatographic performance.

Main Methods:

• Development and testing of porous layer open tubular trap columns (PLOTraps) with varying stationary phases and diameters.
• Integration of PLOTraps as both sample loops and cleanup devices using a 6-port valve.
• Comparison of PLOTrap performance against conventional packed trap columns in forward and backflush modes.

Main Results:

• PLOTraps effectively removed debris, preventing analytical column clogging.
• Optimized PLOTraps demonstrated comparable chromatographic performance to backflushed packed traps.
• PLOTraps offered superior debris removal, leading to more robust trace sample analysis.

Conclusions:

• Large-bore PLOTraps provide a robust solution to clogging issues in nanoflow LC-MS.
• PLOTraps enable integrated sample cleanup and injection, simplifying workflows for trace samples.
• This approach enhances the reliability of in-depth proteome profiling, particularly for single-cell analyses.