A Novel Experimental Scheme to Fully Separate Permethylated Glycans and Allow Online RP-LC-MS with Simplified ESI-MS Analysis

Permethylation of glycans is one of the steps used in glycoprotein analysis. Typical LC-MS configurations involve reverse-phase chromatography coupled online to ESI-MS systems. Historically, reverse phase chromatography has been unsuccessful in separating permethylated glycans. The interactions between the hydrophobic stationary phase and the glycans, which have similar monosaccharide compositions, are not distinct enough to enable their resolved separation. Therefore, ion chromatography is commonly used to separated these modified glycans. In this work the authors have discovered a method that for the first time enables separation of permethylated glycans using reverse-phase media and mass spectrometry compatible mobile phases. The method, which involves heat activation in conjunction with use of small-particle size columns, has been successful in resolving both isomeric and structurally similar glycans. The results of this new separation technique are a significant improvement over ion chromatography and allow the LC to be directly coupled to the mass spectrometer for MS and MS/MS sequence analysis. With electrospray ionization glycans and other large organic molecules greater than 2000 Da. are typically exhibited as charged ions within an ion series. In addition glycans can form multiple adducts resulting in complex spectra for just a single glycan. We observed that glycans separated with this new approach exhibited a very unusual behavior during electrospray ionization resulting in singly charged ions for all glycans even those with masses greater than 4000 Da. The presence of a single ion for each glycan greatly simplifies analysis of the mass spectra. In addition the lack of multiple-charged species results in improved mass accuracy by obviating the need to perform deisotoping calculations that can propagate mass errors. These results and observations will be presented in the poster. In addition this initial discovery has spurred other research which builds on this technique using alternate reverse-phase media for quantitation of glycans.