Surface-Charged Hybrid Monolithic Column for MS-Compatible Peptide Separation with High Peak Capacity and Its Application in Proteomic Analysis.

For bottom-up proteomics, peptide separation with high peak capacity under MS-compatible conditions is of vital significance to increase proteome coverage. Herein, a surface-charged ethane-bridged hybrid monolithic column was prepared based on the efficient ring-opening reaction of N -methyl- aza -2,2,4-trimethyl-silacyclopentane after C18-functionalization. The existence of secondary amino groups on the surface was beneficial to reduce the secondary interactions of silanol groups and increase peak capacity for peptide separation with MS-compatible mobile phases (e.g., using 0.1% FA as the mobile phase modifier). Such columns offered a 4-fold increase in peak capacity compared with ethane-bridged hybrid monolithic columns without surface charge modification. By a 100 cm length surface-charged ethane-bridged hybrid capillary column, high peak capacity of 700 was achieved within a 240 min gradient for the separation of Hela tryptic peptides with 0.1% FA-containing mobile phases, under the low backpressure of ∼200 bar. On average, 44493 ± 459 peptides corresponding to 5148 ± 47 proteins were identified from 750 ng Hela tryptic digests. Finally, the surface-charged ethane-bridged hybrid monolithic column was successfully applied in the quantitative proteomic analysis of dopaminergic neuron death model of N -methyl-4-phenylpyridinium iodide induced SH-SY5Y cells. These results demonstrated great promise of such surface-charged ethane-bridged hybrid monolithic columns for bottom-up proteomic analysis in complex samples.