Online multidimensional separation with biphasic monolithic capillary column for shotgun proteome analysis.

Area of Science:

• Analytical Chemistry
• Chromatography
• Proteomics

Background:

• Multidimensional chromatography is crucial for complex sample analysis.
• Developing integrated columns enhances separation efficiency and reduces analysis time.
• Capillary-based systems offer high sensitivity for proteomic studies.

Purpose of the Study:

• To develop and evaluate a biphasic monolithic capillary column for multidimensional liquid chromatography.
• To assess the column's performance in protein identification using mass spectrometry.
• To demonstrate the utility of orthogonal separation mechanisms in a single column.

Main Methods:

• Fabrication of a biphasic monolithic capillary column integrating strong-cation exchange (SCX) and reversed-phase (RP) segments.
• Online multidimensional liquid chromatography (MDLC) coupled with tandem mass spectrometry (LC-MS/MS).
• Analysis of yeast protein tryptic digest (10 µg) over five separation cycles.

Main Results:

• Successful preparation of a monolithic column with distinct SCX and RP segments within a 100 µm i.d. capillary.
• Achieved a comprehensive proteomic analysis identifying 780 distinct proteins and 2953 unique peptides with <1% false discovery rate.
• Demonstrated efficient separation in 12 hours at ~900 psi, highlighting the column's high performance.

Conclusions:

• The biphasic monolithic column provides excellent orthogonality between SCX and RP modes for effective multidimensional separations.
• This integrated column design significantly enhances proteomic analysis efficiency and depth.
• The developed column represents a valuable tool for high-performance liquid chromatography in proteomics.