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

• Published in: Talanta (Oxford) 2018-10, Vol.188, p.499-506
• Main Authors: Lin, Haizhu, Shao, Xi, Lu, Yan, Deng, Chunhui
• Format: Article
• Language: English
• Subjects: Animals; Cattle; Chromatography, Liquid - methods; Glycopeptide; Glycopeptides - blood; Glycoproteins - metabolism; Humans; Hydrophilicity; Hydrophobic and Hydrophilic Interactions; Imino Acids - chemistry; Iminodiacetic acid; Limit of Detection; Microspheres; On-column enrichment; Serum Albumin, Bovine - chemistry; Silanes - chemistry; SiO2; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; Trypsin - chemistry
• ISSN: 0039-9140; 1873-3573
• DOI: 10.1016/j.talanta.2018.06.031

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. Silica microspheres were grafted with hydrophilic iminodiacetic acid (IDA) groups and packed in a capillary column to prepare SiO2-IDA capillary trap column for on-column enrichment of glycopeptides based on hydrophilic interaction liquid chromatography (HILIC). The on-column enrichment method exhibited excellent enrichment performance from digests of both standard proteins and human serum. [Display omitted] •A novel on-column glycopeptide enrichment method was developed.•Superior hydrophilicity brought by IDA groups.•Good column reproducibility of the capillary packed columns.