Separation of disaccharide epimers, anomers and connectivity isomers by high resolution differential ion mobility mass spectrometry.

Glycans are ubiquitous, structurally diverse molecules that have specific and general roles involving metabolism, structure, and cell-to-cell signaling. Functional specificity depends strongly on the complexity of structures that polysaccharides can adopt based on their subunit composition, length, extent of branching, glycosidic bond connectivity and anomeric configuration. However, a rapid and comprehensive characterization of glycan isomers can be challenging owing to limitations associated with their separation. Here, ten composition, anomeric and connectivity disaccharide isomers were separated and detected using high-resolution differential ion mobility-mass spectrometry (DMS-MS, also known as FAIMS). Focus was primarily directed to compositional isomers corresponding to epimers that differ by the axial or equatorial position of a single hydroxyl group. DMS resolving power was enhanced 14-fold primarily by increasing the fraction of helium in the ion carrier gas and lowering the flow rate. At relatively high disaccharide concentrations, DMS-MS of each disaccharide resulted in complex and unique multi-peak spectra with up to ten fully and partially resolved peaks for ß-1,4-mannobiose (Man-1,4ß-Man), which can be attributed to the DMS separation and subsequent dissociation of ionic non-covalently bound oligomers into monomer ions. Each DMS spectrum has at least one differentiating peak that is not in the other spectra, indicating that DMS can be used to fully or partially resolve composition, configuration and connectivity isomers. At relatively low disaccharide concentrations, mixtures of disaccharide epimers can also be readily separated by DMS. The integration of high-resolution, ambient pressure DMS with complementary reduced-pressure ion mobility and MS-based glycomics and glycoproteomics workflows may be useful for improving the characterization of glycans and glycosylated biomolecules.

Separation of Isobaric Mono- and Dimethylated RGG-Repeat Peptides by Differential Ion Mobility-Mass Spectrometry.

Methylation of arginine residues in proteins, an enzyme-mediated post-translational modification (PTM), is important for mRNA processing and transport and for the regulation of many protein-protein interactions. However, proteolytic peptides resulting from alternative sites of post-translational methylation have identical masses and cannot be readily separated by standard liquid chromatography-mass spectrometry. Unlike acetylation or phosphorylation, methylation of arginine does not strongly affect the charge states of peptide ions, multiple instances of methylation can occur on a single amino acid residue, and the relative mass of the modification is <1% that of the typical proteolytic peptide. High field asymmetric waveform ion mobility spectrometry (FAIMS) is an orthogonal separation method to liquid chromatography that can rapidly separate gaseous ions prior to detection by mass spectrometry. Here, we report that FAIMS can be used to separate arginine-methylated peptides that differ by the position of a single methyl group for both mono- and dimethylated variants. Although the resolution of separation for these arginine-methylated peptides improved with increasing amounts of helium in the FAIMS carrier gas as expected, we found that the site of methylation can strongly affect the dependence of the electric field used for ion transmission on the extent of helium in the carrier gas. Thus, certain isobaric peptides can be cotransmitted at high helium concentrations whereas lower concentrations can be used for successful separations of such peptide mixtures. The capability to rapidly resolve isobaric arginine-methylated peptides should be useful in the future for the detailed analysis of protein arginine methylation in biological samples.