Intact-protein trapping columns for proteomic analysis in capillary high-performance liquid chromatography

Area of Science:

• Analytical Chemistry
• Materials Science
• Biochemistry

Background:

• On-line capillary liquid chromatography (LC) requires efficient sample enrichment and preconcentration steps.
• Traditional trapping methods often lack mechanical stability or sufficient loading capacity for intact proteins.
• Developing robust and high-performance trapping columns is crucial for advanced proteomic analysis.

Purpose of the Study:

• To develop and characterize novel monolithic trapping columns with enhanced mechanical strength for intact protein analysis.
• To evaluate the performance of these columns in terms of stability, recovery, loading capacity, and reproducibility.
• To demonstrate the applicability of the developed trapping columns in an on-line capillary LC system for proteomic applications.

Main Methods:

• Fabrication of monolithic trapping columns using a thin-layer sol-gel coating method with methyltriethoxysilane.
• Entrapment of C8 reversed-phase particles within a sol-gel network inside capillary columns.
• Evaluation of column stability up to 300 bar and performance metrics (recovery, loading capacity, reproducibility) using protein mixtures.
• Application of the columns for trapping intact mouse liver proteins in an on-line capillary LC setup.

Main Results:

• The monolithic trapping columns exhibited high mechanical strength and long-term stability up to 300 bar.
• Average recovery for four protein mixtures was 99.3%, with a loading capacity of 30 μg for the 5 mm × 320 μm i.d. columns.
• Day-to-day relative standard deviation (RSD) for recoveries ranged from 2.34% to 5.87%, and column-to-column RSD values were between 3.01% and 6.81%.
• Successful trapping of intact mouse liver proteins demonstrated the columns' efficiency for proteomic analysis.

Conclusions:

• The developed C8 monolithic trapping columns possess high mechanical strength and excellent stability for on-line protein analysis.
• These columns offer high recovery, loading capacity, and good reproducibility, making them suitable for complex biological samples.
• The monolithic trapping columns represent a significant advancement for intact protein enrichment in capillary liquid chromatography-based proteomic studies.