Assessing Isomeric Heterogeneity of Carbohydrates by Ion Mobility Mass Spectrometry

Abstract

Ion mobility spectrometry (IMS) coupled to different types of mass spectrometry (MS), which create a powerful analytical tool called ion mobility mass spectrometry (IMMS), were employed to evaluate the isomeric heterogeneity of carbohydrates. Isomer separation was achieved on millisecond timescale for a wide variety of carbohydrate compounds including monosaccharide methyl glycosides, disaccharides, oligosaccharide-alditols isolated from glycoprotein and glycopeptides from glycoprotein digests using drift tube IMS or/and traveling wave IMS coupled to time of flight MS. With IMS coupled to tandem MS, fragmentation patterns for the individual resolved isomeric mobility peaks can be obtained, providing evidence for multiple isomeric precursors as mobility-separated species. The special design of Synapt G2 high definition MS, where a collision dissociation cell is located in front of and after traveling wave IMS, respectively, enables both mobility-resolved precursor ions to be fragmented and mobility-separated product ions to be dissociated. Many isomeric product ions were observed for parent disaccharides and higher oligosaccharide-alditols. And mobility-resolved product ions from mass-selected oligosaccharide-alditol precursors yielded different characteristic mass spectra. A hybrid tandem IMMS instrument, IMMS-IMMS, dual gate drift tube IMS coupled to Synapt G2 MS was established. In addition to its capability in evaluating the structural complexity of precursor ions, ion mobility identity of product ions derived from individual mobility- and mass-selected precursor ions from a mixture of isomers were able to be measured for the first time. This provides a valuable tool to establish a direct precursor-product relationship between mobility-selected precursor ions and specific sets of product ions having unique mobilities. N-linked glycoprotein digests, without deglycosylation, were subjected to IMMS analysis directly. Mixtures of peptides and glycosylated peptides with different charge states were separated on different ion mobility-mass correlation trend lines. Tedious and time-consuming sample preparation was eliminated and the detection of low abundance ions was improved. IMMS analysis is fast, sensitive, universal, is capable of resolving isomers and is able to provide unique mobility identities and valuable MSn data on the basis of mobility resolved species including precursor and product ions. Ultimately, IMMS could serve as a novel tool for high throughput identification of glycoprotein.