Differential mobility spectrometry analysis of glycans and glycopeptides using SelexION® Technology
J. Larry Campbell1, Catherine S. Lane2, Sarah A. Flowers3, Chang Liu1, J.C. Yves LeBlanc1
1SCIEX, Canada; 2SCIEX, UK; 3Georgetown University, Washington, DC, USA
Glycans and glycosylated biomolecules perform numerous roles through their interactions in cellular environments, with more than half of human proteins being glycosylated.1 The characterization of glycosylation in biopharmaceuticals is essential for the determination of function and efficacy.2 Thus, glycan analysis is gaining great interest. However, carbohydrates are highly variable in structure owing to differences in their anomeric configurations, monomer stereochemistry and inter-residue linkage positions. The separation of carbohydrates has been a great challenge for current analytical techniques.3-4 Differential mobility spectrometry (DMS) is a technology that can separate gas-phase ions prior to analysis by mass spectrometry (MS)5. It has been used in the separation of various isomeric species, including stereoisomers6, structural isomers7, and tautomers8. In this study, DMS-MS was employed for the structural analysis of glycans and glycopeptides.
The separation of the isomeric glycan and glycopeptide species studied here was accomplished because of differences in the DMS behavior between each isomer.9 The ions bear subtle structural differences that allow the DMS to separate them based on their different mobilities during the high- and low-field portions of the asymmetric waveform applied across the DMS cell (Separation Voltage, SV). As such, each isomer requires a different DC compensation voltage (CV) to bring their trajectories on-axis for successful sampling by the MS. Besides the differences between the isomers' structures, differences in how these species bind to added volatile chemical modifiers in the DMS cell makes their DMS behavior differences more prominent, yielding increased selectivity and peak capacity for the DMS experiments.
Sample preparation: Glycans were purchased from Sigma-Aldrich (Oakville ON, Canada) and from Dextra Laboratories (Reading, UK). For work in positive ionization mode, glycans were diluted to 1 µg/mL in methanol and water (50/50, v/v) containing 50 µM sodium chloride. For negative mode work, glycans were diluted to 1-5 µg/mL with acetonitrile and water (20/80, v/v) containing 10 mM ammonium bicarbonate. Two glycopeptides, supplied by Anaspec (Fremont, CA), were diluted to 10 µg/mL with acetonitrile and water (50/50, v/v) containing 0.1% formic acid.
DMS-MS conditions: The SelexION Device was mounted in the atmospheric region between the sampling orifice and a Turbo V™ Source on either the SCIEX QTRAP® 5500 or 6500+ System (Figure 2). The temperature of the DMS cell was maintained at 150 ºC, and the nitrogen curtain gas was operated at 30 psi. Chemical modifiers (water, methanol or acetonitrile) were added into the nitrogen curtain gas flow at 1.5% (mole ratio). The fundamentals of the DMS device have been described elsewhere5. In this study, both separation voltage (SV) and compensation voltage (CV) were scanned using Analyst® Software 1.6. As the SV was stepped from 0 to 4000 V, CV was scanned from -40 V to +20 V. These data were plotted as dispersion plots, with SV as the x-axis, and the optimal CV for ion transmission as the y-axis.
Data analysis: Data was analyzed using PeakView® Software or Explore in SCIEX OS Software.
Separation of isomeric O-linked glycopeptides
Finally, the separation of a pair of isomeric glycopeptides (MUC5AC-3 and MUC5AC-13) was demonstrated (Figure 1). These two glycopeptides are different only in the site of the O-linked GalNAc modification. Diagnostic y and b ions, that retained the O-linked GalNAc modification, were observed in the MS/MS spectra of the separated glycopeptides, and allowed confirmation of DMS separation.
Separating disaccharides and trisaccharides
SelexION Technology was employed to analyze the sodiated or deprotonated molecular ions of disaccharides and trisaccharides. Maltose and melibiose have the same mass, however differ structurally based on the sugar linkage. To assess the ability of DMS to separate these very similar sugars, the compensation voltage (CV) was ramped across a range of separation voltages (SV) and the CV optimum was determined at each step. These CV curves were then plotted for each sugar to visualize separation potential (Figure 3).
When these carbohydrates were analyzed without chemical modifiers, molecular ions could not be fully separated (Figure 3, left). The controlled addition of water or methanol vapor in the DMS cell induced different shifts in optimal CVs at the higher separation voltages, enhancing the separation of isomeric carbohydrates. The ability to add different chemical modifiers is a unique feature of the planar differential mobility cell and provides an additional orthogonal level of separation that can be explored for different molecule types.
In addition to maltotriose and raffinose, two additional isomeric trisaccharides were studied (Figure 4). As well as using water as the chemical modifier, the separation was explored using methanol. In this case, good separation was obtained for 3 out of 4 sugars (Figure 4). The final set of sugars tested were deprotonated Lewis x and Lewis a trisaccharides. In this case, methanol provided the best separation by DMS (Figure 5).
Differentiation by sialic acid linkage
Another structural feature of glycans that makes them challenging to study is the attachment of sialic acid residues to the ends of the glycan chains. Understanding this linkage is important as it impacts biological function.
Here, two pairs of glycans with α2-3- and α2-6-linked sialic acid were studied to determine whether DMS could resolve this type of structural difference. As shown in Figure 6 and 7, in the presence of methanol as a chemical modifier, baseline separation of these isomers could be achieved.
In this study, SelexION Technology was used to analyze biologically relevant glycan and glycopeptide isomers, to determine whether differential mobility separation would be useful in the study of glycan and glycopeptide structural isomers. With the addition of gas-phase chemical modifiers to the DMS, various isomeric species were successfully separated including:
- Sodiated and deprotonated glycans,
- Deprotonated glycans containing α2-3 or α2-6 linked sialic acid,
- Protonated glycopeptides.
Coupled with QTRAP System technology, high sensitivity MS/MS data with CID fragmentation can also be collected, to identify diagnostic fragments for monitoring separation (such as the GalNAc modification fragments for the O-linked glycopeptide, Figure 1) or to confirm identity of glycans after separation. SelexION Technology coupled to QTRAP systems provides a fast and convenient approach for isomeric glycan and glycopeptide analyses.