Preparation of iminodiacetic acid functionalized silica capillary trap column for on-column selective enrichment of N-linked glycopeptides.

In this work, SiO2-IDA microspheres were obtained via a previous method by grafting iminodiacetic acid (IDA) groups onto silica microspheres, then the SiO2-IDA microspheres were packed in capillary columns to prepare SiO2-IDA capillary trap columns for the first time. Based on the trap columns, a novel on-column enrichment method toward glycopeptides was developed as a sample pretreatment step prior to matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) or nano-LC-MS/MS analysis. The preparation procedures of the trap columns are well reproducible. Thanks to outstanding hydrophilicity brought by the grafted zwitterionic IDA groups, the trap columns showed excellent on-column enrichment performance toward glycopeptides by hydrophilic interaction liquid chromatography (HILIC). Experimental results showed that the on-column enrichment method could reach a low detection limit (50 fmol HRP digest) and a high selectivity (molar ratio of HRP digest and BSA digest 1:100). Finally, it was applied to on-column enrichment of glycopeptides from digests of human serum followed by nano-LC-MS/MS analysis. In total, 207 glycopeptides assigned to 78 glycoproteins were identified from 2 μL human serum.