Microseparation techniques coupled to mass spectrometry for a sensitive and unequivocal identification of glycopeptides

Abstract

Capillary electrophoresis (CE) and low-flow liquid chromatography (i.e., micro, capillary, and nanoLC) both coupled to mass spectrometry (MS) are nowadays widely accepted for glycopeptide analysis. However, there are still analytical challenges related to the separation of glycopeptide glycoforms and their unequivocal identification. In the present study, CE-MS and capillary LC-MS (capLC-MS) were evaluated for a robust and high-throughput analysis of glycopeptides. Recombinant human erythropoietin (rhEPO) was selected as model glycoprotein due to its relevance as a biopharmaceutical and its high glycopeptide microheterogeneity. The CE-MS method provided adequate glycopeptide separation for those glycoforms differing in the number of sialic acids. However, the separation of glycoforms with the same sialic acid content but different glycan branching (number of antennas) was not achieved. In contrast, capLC-MS with an appropriate reversed phase C18 column enabled to improve their separation. Furthermore, the analysis of rhEPO glycopeptides with the established microseparation methods coupled to tandem mass spectrometry (CE-MS/MS and capLC-MS/MS) provided specific fragmentation patterns and a more reliable glycoform identification. The capLC-MS/MS method was the most promising as, it was able to detect and unequivocally identify a greater number of glycopeptide glycoforms compared to the CE-MS/MS method, including those with glycolylneuraminic acid, which are necessary to differentiate between endogenous and recombinant hEPO.Data are available via ProteomeXchange with identifier PXD047106.