Functionalization of hybrid monolithic columns via thiol-ene click reaction for proteomics analysis

•Vinyl-functionalized monolithic columns with 75 and 150 μm i.d. were prepared.•The monolithic columns were facilely modified via thiol-ene click reaction.•The effect of flow rate on cLC-MS/MS performance was investigated.•The SCX and RP monolithic columns were applied in two-dimensional separation.

The vinyl-functionalized hybrid monolithic columns (75 and 150 μm i.d.) were prepared via sol-gel chemistry of tetramethoxysilane (TMOS) and vinyltrimethoxysilane (VTMS). The content of accessible vinyl groups was further improved after the monolithic column was post-treated with vinyldimethylethoxysilane (VDMES). The surface properties of monolithic columns were tailored via thiol-ene click reaction by using 1-octadecanethiol, sodium 3-mercapto-1-propanesulfonate and 2,2′-(ethylenedioxy)diethanethiol/vinylphosphonic acid, respectively. The preparing octadecyl-functionalized monolithic columns were adopted for proteomics analysis in cLC-MS/MS. A 37-cm-long × 75-μm-i.d. monolithic column could identify 3918 unique peptides and 1067 unique proteins in the tryptic digest of proteins from HeLa cells. When a 90-cm-long × 75-μm-i.d. monolithic column was used, the numbers of unique peptides and proteins were increased by 82% and 32%, respectively. Furthermore, strong cation exchange (SCX) monolithic columns (4 cm in length × 150 μm i.d.) were also prepared and coupled with the 37-cm-long × 75-μm-i.d. octadecyl-functionalized monolithic column for two-dimensional SCX-RPLC-MS/MS analysis, which could identify 17114 unique peptides and 3211 unique proteins.