Solid Phase Extraction of N‑Linked Glycopeptides Using Hydrazide Tip

Glycoproteome contains valuable information where biomarkers may be discovered for disease diagnosis and monitoring. Nowadays, with the ever-increasing performances of mass spectrometers, the emphasis is shifting to the sample preparation for better throughput and reproducibility. Therefore, to facilitate high throughput N-linked glycopeptide isolation, in this study, a novel hydrazide tip was devised and an integrated workflow of N-linked glycopeptide isolation using hydrazide tips was presented. With the use of bovine fetuin as a standard glycoprotein, the incubation time was determined for each major step of glycopeptide isolation. With the use of commercially available human serum, multiple parallel isolations of glycopeptides were performed using hydrazide tips with a liquid handling robotic system. We demonstrated that, with the hydrazide tips, the processing time was significantly decreased from 3 to 4 days to less than 8 h with excellent reproducibility. The hydrazide pipet tips have great potential in achieving automation of N-linked glycopeptide isolation for high-throughput sample preparation when used in combination with liquid handling robotic systems.