Flowing on-line preparation of deglycosylation, labeling and purification for N-glycan analysis

The current in-solution analysis of N-glycans suffers from several disadvantages including tedious de-glycosylation time and multi-step pre-treatment procedures. Here, an ultra-simple flowing on-line analysis of labeled N-glycans for high-performance liquid chromatography with fluorescence detection (HPLC-FLD) was developed for eliminating the deficiencies. This on-line analysis consisted of an immobilized enzyme reactor (IMER) of PNGase F for efficient release of N-glycans, labeling of released N-glycans and following purification of derivatives on microfluidic chip. Notably, efficient preparations for all type of N-glycans were completed within ∼30 min. To our best knowledge, this is the first time to integrated the whole preparation of N-glycan deglycosylation, labeling and purification only by a simple fluidic flow with our developed device. Good reproducibility and stability were achieved with the relative standard deviation (RSD) less than 10%. Furthermore, the glycome studies with human serum revealed a good adaptability for biological samples. Our work provides an efficient N-glycomic strategy that can be applied to further multilayered clinical analysis.