Convenient sample preparation for human serum n-glycan profiling by mass spectrometry: application to congenital disorders of glycosylation (CDG) type I

Abstract

The development of a robust method suitable for medium to high throughput N-glycan profiling for a simple and rapid platform for human serum sample preparation. Congenital disorders of glycosylation (CDG) are multisystem disorders due to defects in N-glycosylation synthesis pathway. This ultimately produced abnormal N-glycan which affected their structure and metabolic function. The method to assess the diversity of the N-linked glycan released from human serum was developed using the filter aided N-glycan sample preparation (FASP) protocol. The samples were boiled in sodium dodecyl sulphate (SDS), removed by urea and exchanged for volatile buffer. Then, PNGase F treatment was applied in the upper chamber of a membrane spin filter to release N-glycan. The released glycan were recovered in the filtrate following centrifugation, and subsequently analysed using MALDI-TOF. Authentic standard glycoprotein is used to assess the sample processing protocol together with human serum and CDG type 1 patient. Non-derivatised N-glycan from glycoprotein standard and human serum were analysed using MALDI-TOF. Bi and tri-antennary sialylated glycans were observed (m/z 2792.5, 3241.8, 3603.0, 3965.7 [M+Na]+) from standard fetuin for validation of sample processing. Additionally, there are differences upon comparing N-glycan profiles between control and CDG type 1 serum. We show that the protocol is suitable and advantageous for N-glycan profiling of human serum since only a small volume of serum is required, which facilitates working with replicate samples. This method was found to work efficiently on different sample matrices and reduced sample preparation time before MALDI-TOF analysis.