Self-packed 300 µm capillary columns enable high peak capacity separations and deep proteome analysis

Area of Science:

• Proteomics
• Analytical Chemistry
• Chromatography

Background:

• Nanoflow chromatography is standard for proteomics.
• Capillary flow chromatography (capLC) is limited by column availability, restricting applications.

Purpose of the Study:

• To develop a method for packing long (e.g., >15 cm), 300 µm inner diameter capLC columns.
• To optimize capLC parameters for enhanced proteomic analysis.
• To evaluate column performance against commercial standards.

Main Methods:

• Packing 300 µm inner diameter columns of various lengths (30 cm, 55 cm, 1 m) with different bead sizes (1.7 µm, 2.5 µm, 3.5 µm).
• Utilizing CSH chemistry for column packing.
• Systematic optimization of gradient lengths (30 min to 10 h) and sample loading (0.5–8 µg).

Main Results:

• Demonstrated outstanding performance with 30 cm columns packed with 1.7 µm beads, achieving a peak capacity of 530 on a 2-hour gradient.
• Showcased high loading capacity (0.5–8 µg) without compromising separation.
• Quantified nearly 9000 proteins and 124,000 unique peptides from 4 µg HeLa digest using a 3-hour gradient on an Exploris 240 system.

Conclusions:

• The developed methodology enables the creation of high-performance capLC columns of any length.
• These columns match or surpass the efficiency of commercially available options.
• The optimized capLC workflow significantly enhances proteomic quantification capabilities.