An Automated High-Throughput Sample Preparation Protocol for LC-MS/MS Analysis of Glycopeptides

Current sample preparation workflows for glycopeptide analyses have low levels of automation and low sample throughput. In this study, we have developed an automated glycopeptide preparation workflow for a 96-well plate liquid handling robotic system. The protocol is based on the filter-aided capture and elution method. The glycopeptides bound to lectins were trapped on the filter, and the filters were then incubated with PNGaseF to release the glycopeptides specifically. The experimental conditions were optimized for lectin: peptide ratios using a standard glycoprotein and colon tissue proteomes. Duplicates of ten samples were processed in parallel and subjected to LC-MS/MS analysis. The results of LC-MS/MS analysis showed that 96 samples can be processed in parallel in 6.5 hours.