Differential Fragmentation of Mobility-Selected Glycans via Ultraviolet Photodissociation and Ion Mobility-Mass Spectrometry
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The alternative dissociation pathways initiated by ultraviolet photodissociation (UVPD) compared with collision-induced dissociation (CID) may provide useful diagnostic fragments for biomolecule identification, including glycans. However, underivatized glycans do not commonly demonstrate strong UV absorbance, resulting in low fragmentation yields for UVPD spectra. In contrast to UVPD experiments that leverage covalent modification of glycans, we detail the capacity of metal adduction to yield comparatively rich UVPD fragmentation patterns and enhance separation factors for an isomeric glycan set in a drift tube ion mobility system. Ion mobility and UVPD-MS spectra for two N-acetyl glycan isomers were examined, each adducted with sodium or cobalt cations, with the latter providing fragment yield gains of an order of magnitude versus sodium adducts. Furthermore, our glycan analysis incorporated front-end ion mobility separation such that the structural glycan isomers could still be identified even as a mixture and not simply composite spectra of isomeric standards. Cobalt adduction proved influential in the glycan separation by yielding an isomer resolution of 0.78 when analyzed simultaneously versus no discernable separation obtained with the sodium adducts. It is the combined enhancement of both isomeric drift time separation and isomer distinction with improved UVPD fragment ion yields that further bolster multivalent metal adduction for advancing glycan IM-MS experiments.
KeywordsIon mobility Ultraviolet photofragmentation Isomer separations Glycans
Glycans occupy primary roles in a number of vital biological processes, including but not limited to immune response, blood type determination, cancer cell differentiation, and cell recognition [1, 2, 3, 4]. Currently, the combination of data from nuclear magentic resonance (NMR) and tandem mass spectrometric (MS) analyses are necessary to obtain an accurate assessment of the glycan structures present in a given sample . Development of comprehensive MS analytical methods alone would be ideal, but the large structural variety of glycans  and stereochemical blindness of mass spectrometry necessitates the incorporation of secondary separation schemes to infer carbohydrate identity even with tandem fragmentation approaches.
When MS is used for glycan determination, tandem MS remains essential for determining glycan composition. Fragmentation techniques for glycans often include collision-induced dissociation (CID), electron capture dissociation (ECD), and electron transfer dissociation (ETD), however, a priori knowledge of the glycan system is often needed for conclusive identification. Ultraviolet photodissociation is no exception to this trend; however, it has demonstrated the capacity to generate an abundance of informative fragments such as cross-ring cleavages [7, 8, 9, 10, 11, 12].
Along with using alternative fragmentation methods to adjust fragment yield, the use of metal adduction has also been found to modify fragmentation variety. Konig and Leary found the use of cobalt adduction to aid in pentasaccharide linkage determination when complemented with CID-MS of the deprotonated species . Vartanian et al. investigated the use of alkaline earth and transition metal divalent cations as adducts with tetracycline antibiotics for IRMPD-MS analysis; however, when compared to the protonated species, these metal-tetracycline adducts yielded equivalent or poorer quality spectra than the protonated species’ IRMPD spectra . By contrast, later work from Adamson and Hakansson, and Zhou and Hakansson, applied divalent metal ions as charge carriers for glycan analysis with ECD-MS and IRMPD-MS with the conclusion that IRMPD worked well as a supplemental technique for glycan characterization with ECD-MS [15, 16]. These results suggest that the fragmentation of divalent metal adducts alone is not adequte to determine glycan composition. However, given the limited assessments made to date for such systems, it seems imprudent to assume that the technique should be relegated to obscurity. Rather, the work by Adamson and Hakansson, and Zhou and Hakansson, indicate the potential for the application of divalent metal adduction to glycan analysis using photon-based fragmentation [15, 16], To the best of our knowledge, the use of UV photons for the fragmentation of divalent metal-glycan adducts is absent from the published literature. This information gap and a desire to maximize informative fragment yields prompted the pursuit of UVPD-MS of metal-glycan adducts.
While UVPD has been applied to increase fragmentation variety for a range of chemical systems, there are relatively few reports integrating this approach with post ionization separations such as ion mobility spectrometry. Limited reporting for this instrumental combination may, in large part, be due to challenges coupling drift tube separation with ion trapping systems. However, notable developments in this domain now enable such experiments [11, 12, 17, 18, 19, 20]. Selection of fragmentation mass spectra from a given drift time window reveals the fragments specific to a precursor of a particular drift time, excluding isomers of differing mobility. This drift time separation method was used by Zucker et al. with vacuum UVPD in comparison with CID for analysis of tetrasaccharide and trisaccharide isomeric pairs, with vacuum UVPD yielding more informative spectra overall . Similarly, Lee et al. used vacuum UVPD on seven isobaric disaccharides after dual-gate ion mobility separation, also finding improved fragment yield over CID, but with low fragment abundances characteristic of sodiated glycan photodissociation .
For the purpose of distinguishing isomers, high resolution ion mobility spectrometry (IMS) offers a tractable approach to isomer separation as it provides a rapid mechanism to distinguish isomers according to the ionized glycans’ mobility down a voltage gradient against a counter-current neutral gas flow. Mobility separation combined with UVPD-MS holds potential as a valuable technique for isomeric mixture analysis. Accordingly, we demonstrate enhanced UVPD fragmentation patterns and isomeric separation using IM-MS for two cobalt cation-adducted isomeric tetrasaccharide N-acetylated alditols.
The two tetrasaccharides in question were initially analyzed as sodium adducts, but the fragment yield was less than ideal at ≤1% of the precursor abundance. Along with evidence from Konig and Leary that cobalt adduction may improve the yield of glycan structural information, the addition of cobalt acetate adducted to glycans was found by Dwivedi et al.  to promote improved drift time separation for a simple mixture of disaccharides. Cobalt acetate was also found to be necessary here to produce two distinct drift time peaks for the isomers together as a mixture. Additionally, metal adduction via the addition of cobalt acetate to the glycan solutions was used to increase UV photon absorption to facilitate fragmentation. The isomeric glycans used here were found to have improved relative fragment abundance to that of the precursor when adducted to cobalt than when assessed as sodiated adducts.
The two isomeric tetrasaccharides used were O-glycans with one N-acetyl group on two residues per glycan, one fucose residue each, and were alditols with fully reduced reducing-ends. Both glycans were purified with high performance liquid chromatography (HPLC) and were characterized by nuclear magnetic resonance (NMR) . Structures of both glycans can be found in Figure 4 and Supplementary Material (Figure S-1). One of the glycans has residues linked in a linear fashion with no true branching, while the second tetrasaccharide does have a branched structure. For fragmentation experiments, solutions of each individual tetrasaccharide were prepared at 25 μM each for the mixture of both, and 50 μM each for individual standard analyses, with cobalt acetate added to each solution at a final concentration of 50 μM to ensure complete complexation. For drift time spectra obtained with single-stage MS, solutions were prepared with 10 μM each of the tetrasaccharides and 50 μM of cobalt acetate. This concentration of added cobalt salts for adduction was found to yield cobalt-glycan adduct ions at abundances that are approximately equal to that of the corresponding sodium adducts.
Results and Discussion
Overall, the branched tetrasaccharide produced a larger variety of fragment ions in comparison to the linear glycan, but with the linear glycan yielding a more distinctive UVPD fragment pattern. As an example of its distinctive spectral pattern, the linear tetrasaccharide produced an easily recognizable cluster of four fragments at the nominal m/z values 425, 443, 466, and 484, with all appearing in roughly similar proportions relative to one another. Although the branched glycan does also produce each of these same four fragment ions, the unique feature of each appearing at nearly equal abundances in the linear tetrasaccharide facilitates distinguishing the two isomers. An interesting feature of the branched tetrasaccharide is the higher abundance of the 774 nominal m/z fragment, which is due to a loss of water, due to CID more so than UVPD, as well as the same fragment ion from the linear glycan having a comparatively muted presence. Additionally, the branched glycan has the 628 and 646 m/z fragments appearing at abundances that are more equivalent than from the linear tetrasaccharide, and the m/z 569 ion is a feature produced solely by the branched glycan, all of which can serve to distinguish the branched isomer from the linear isomer.
While gas-phase mobility separations theoretically possess the capacity to separate isomeric species prior to mass analysis, the feasibility of such approaches are only fundamentally limited by the underlying resolution of the system. When the resolution of an instrumental configuration is near its maximum, alternative approaches to differentially altering the gas-phase conformations of isomers are needed to realize separation prior to mass analysis. For the limited system examined in this work of two isomeric glycans, metal adduction provides a mechanism to realize gas-phase separations of isomers while having the added benefit of enhancing tandem mass spectrometry yields and variety. UVPD is known for providing feature-rich fragmentation spectra, but often at the cost of ion abundance relative to the precursor if unmodified glycans are the target analyte. Our results demonstrate that transition metal adduction may be yet another technique to raise the relative fragment yield in a fashion analogous to chromophore attachment . The distinguishing feature of metal adduction, however, is in the simplicity of the technique as well as avoiding derivatization and subsequent purification protocols. While unknown isomeric glycan mixture determination remains extremely challenging with current technology, the combination of mobility separation with high fragment variety dissociation methods may be a route by which the carbohydrate isomer barrier can be circumvented .
Support for K.A.M. was provided in part by the Defense Threat Reduction Agency under Grant Award Number HDTRA-14-1-0023. The authors acknowledge support from the New Faculty Seed Grant from Washington State University.
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