Fritted tip capillary column with negligible dead volume facilitated ultrasensitive and deep proteomics

Insufficient chromatographic performance results in reduced utilization of MS/MS scan capacity of advanced MS instruments. Improvement in peptide separation in liquid chromatography is critical for increasing the sensitivity and quantification performance of LC-MS-based proteomics. However, existing column fabrication methods suffer from slow packing, large dead volume, and band broadening. Herein, we reported that directly pulling emitter tips within short frits after fast packing (termed "filled tip") can minimize the dead volume, improving ionization efficiency and reducing band broadening. Within 10 min, our method can pack over 10 cm for 50 μm I.D. capillary columns under 6-8 MPa and over 50 cm for 75 μm I.D. long capillary columns under 70 MPa. We can identify an average of 3043 protein groups and 33 309 peptide-spectrum matches (PSMs) from 1 ng of HeLa digest using a 50 μm I.D. x 20 cm "filled tip" column, with good reproducibility. The number of protein groups increased by 50% and 96% when compared with a 50 μm I.D. "void tip" column and a 100 μm I.D. column with a manually pulled tip, respectively. We identified an average of 5534 protein groups and 71 769 PSMs from 10 ng of HeLa digest. In addition, using 75 μm I.D. x 50 cm "filled tip" columns, we can identify on average 8829 protein groups and 170 751 PSMs in single-shot data-dependent acquisition analysis from 500 ng of 293T digested peptides. Importantly, good repeatability and reproducibility of "filled tip" method were verified by results from columns fabricated in three batches and by different persons. When compared with conventional columns with "void tips", "filled tip" columns reduced median full peak widths by 19% and alleviated sampling redundancy by 10%. Collectively, we developed an easy-to-use, versatile and robust column fabrication method for both narrow-bore and long capillary columns, which achieved great sensitivity and depth in proteomic analysis.