High-efficiency on-line solid-phase extraction coupling to 15-150-microm-i.d. column liquid chromatography for...

Area of Science:

• Proteomics
• Analytical Chemistry
• Chromatography

Background:

• Effective manipulation of small proteomic samples is crucial for liquid chromatography-mass spectrometry (LC-MS).
• Low flow rates in LC-MS can be negatively impacted by extra column volume, affecting separation quality.
• On-line coupling of solid-phase extraction (SPE) with capillary LC offers a solution to these challenges.

Purpose of the Study:

• To develop and evaluate an on-line solid-phase extraction (SPE) system coupled with capillary liquid chromatography (LC).
• To assess the system's performance for high-efficiency proteomic analyses using mass spectrometry (MS).
• To demonstrate the system's capability for automated and robust proteomic sample processing.

Main Methods:

• Utilized commercial switching valves for on-line coupling of capillary LC columns (150-microm channels) with SPE columns.
• Optimized column connections, switching modes, and SPE column dimensions for high-efficiency separations.
• Employed capillary LC operated at 10,000 psi with integrated on-line SPE for sample enrichment and separation.

Main Results:

• Achieved high-efficiency on-line SPE-capillary LC separations with peak capacities of approximately 1000.
• Increased sample processing capacity by ~400-fold for sample volume and ~10-fold for sample mass.
• Identified 1100-1500 unique peptides in 5-hour analyses of tryptic digests, covering ~98% of the theoretical peptide range.

Conclusions:

• The on-line SPE-capillary LC system provides a robust platform for automated proteomic analyses.
• The method significantly enhances sample processing capacity and separation efficiency for low-flow LC-MS.
• This approach enables comprehensive proteomic analysis of both soluble and membrane proteins.