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

Nanoflow chromatography remains the standard workflow for proteomics. Capillary flow chromatography applications are mainly limited to fast acquisitions, likely due to the absence of commercially available long (> 15 cm), 300 µm inner diameter columns. We present a methodology for packing 300 µm diameter columns of any desired length and systematically optimising several capLC parameters. Utilizing CSH chemistry, known for its ability to maintain excellent peak shapes even under high loading conditions, we systematically tested various bead sizes and column lengths. Specifically, we examined 30 cm columns packed with 1.7 µm beads, 55 cm columns with 2.5 µm beads, and 1 m columns using 3.5 µm beads. We also varied the gradient lengths from 30 min to 10 h. Our results demonstrated outstanding performance with the 30 cm columns packed with 1.7 µm beads. For example, on a 2-hour gradient, we achieved a median full width at half maximum of 7.4 s and a peak capacity of 530. We also show that the columns can handle high sample amounts in 0.5-8 µg range without loss of separation power. Finally, we systematically investigated the effect of gradient length and loading and demonstrate that we can quantify close to 9000 proteins and 124 thousand unique peptides on Exploris 240 system, analysing 4 µg of HeLa digest material on a three hour gradient. We demonstrate that the performance of the presented columns equals or surpasses the most efficient commercially available columns.