Adduction of multivalent metal ions to glycans has been shown in recent years to produce altered tandem mass spectra with collision-induced dissociation, electron transfer techniques, and photon-based fragmentation approaches. However, these approaches assume the presence of a well-characterized precursor ion population and do not fully account for the possibility of multimeric species for select glycan-metal complexes. With the use of ion mobility separations prior to mass analysis, doubly charged dimers are not necessarily problematic for tandem MS experiments given that monomer and dimer drift times are sufficiently different. However, multistage mass spectrometric experiments performed on glycans adducted to multivalent metals without mobility separation can yield chimeric fragmentation spectra that are essentially a superposition of the fragments from both the monomeric and dimeric adducts. For homodimeric adducts, where the dimer contains two of the same glycan species, this is less of a concern but if heterodimers can form, there exists the potential for erroneous and misleading fragment ions to appear if a heterodimer containing two different isomers is fragmented along with a targeted monomer. We present an assessment of heterodimer formation between a series of six tetrasaccharides, of which three are isomers, adducted with cobalt(II) and a monodeuterated tetrasaccharide. Using ion mobility separations prior to single-stage and tandem mass analysis, the data shown demonstrate that heterodimeric species can indeed form, and that ion mobility separations are highly necessary prior to using tandem techniques on metal-glycan adducts.
The utility of hybrid instruments combining ion mobility spectrometry (IMS) and mass spectrometry continues to expand as the additional separation dimension affords a higher degree of separation for complex chemical systems [1,2,3,4]. Much the same way that the reproducibility of electron impact ionization enabled the universality of gas chromatography-mass spectrometry, hyphenation of IMS with mass spectrometry of biomolecules has created new possibilities for probing analytically difficult systems. As a post-ionization separation technique that adds a size dimension when combined with MS information, IMS not only has the potential to separate isomeric systems, but also serves an integral tool to probe protein conformers [5, 6], isomeric lipids [7,8,9], and glycan isomer mixtures [10,11,12,13,14,15].
Glycans in general are situated as one of the most challenging class of biological macromolecules to characterize given their predominantly homogenous elemental compositions, many possible combinations of residue branching, and possibility of labile chemical modifications. Although knowledge of the originating cell type and present glycosyltransferases can inform the range of glycan species formed, the presence of isomers can greatly complicate analyses [16,17,18]. Furthermore, isomer-specific analyses are important given that the sum of factors contributing to a specific glycan’s structure all hold a role in determining its biological functionality. A key component for identifying actionable disease biomarkers is to ascertain what carbohydrate structure and composition give rise to a certain biochemical response, thereby requiring that structural isomers be adequately differentiated. The separation of monomeric glycan isomer species has long been a frequent target of ion mobility studies [11,12,13,14,15, 19, 20], and optimizing these separations remains a valuable area of research. Subtle linkage and structural variants between glycan isomers make this a difficult challenge.
The many electron-rich oxygen and nitrogen atoms present in glycans readily participate in metal coordination [21, 22]; in the context of glycan ion mobility and mass spectrometry analyses, monitoring metal adducts has been routine [23,24,25,26,27,28]. As the glycomics community has adopted ion mobility separations, select metal adduction has served to demonstrate the benefits of coordination to shift observed mobility values [11, 29]. Because metal cations can reconfigure the gas-phase shape of target glycans, the possibility that larger aggregated combinations of metal ions and glycans can form must be considered. These multimeric aggregate complexes can encompass several forms, such as adducts with one glycan and two metals , two glycans and one metal , and two glycans with two metals [14, 15]. Depending upon the charge of metal cation, the presence of adducts can certainly hinder the identification of the parent species within lower resolution mass spectra. Notably, the last adduct species listed—the cluster with two glycans and two metals—may appear at the same m/z as a corresponding singly charged, monomeric adduct of one glycan and one metal if the overall dimeric adduct has a 2+ charge state. Should a population of monomers and dimers be present when performing tandem MS experiments with unit mass isolation windows (i.e., almost all commercial mass spectrometers), the resulting spectra will contain chimeric fragment ion abundance ratios. At best, the spectra would still contain relevant fragment m/z information provided the overlaid doubly charged dimer is composed of two identical glycans, rendering such an adduct a homodimeric species. However, in the event that a two isomer metal-glycan heterodimer with a 2+ charge state is mistakenly fragmented along with a target monomeric metal-glycan 1+ species, the resultant tandem mass spectra cannot be separated into which component isomer produced a given fragment. Although issues with dimeric adduct formation may be partially mitigated by lowering analyte concentrations or using harsher ionization conditions, it has been our observation that to the extent that a glycan adducted with an added multivalent metal cation appears within mass spectra, even as low as 1 μM glycan and metal each, the characteristic 2+ charge state 0.5 mass unit spacing is still apparent (data not shown) .
Of the six different glycans explored in this effort (Fig. 1), the four isomeric tetrasaccharide alditols—cellotetraitol, maltotetraitol, nigeran tetraitol, and isomaltotetraitol—have been assessed previously as adducts with a total of seven different metal cations. As indicated in our prior publication, cobalt adduction produced the widest degree of separation for the metals studied . The N-acetal tetraitol has also been observed as a metal-glycan adduct previously, but only with sodium and cobalt . Past assessments of these five alditols with cobalt targeted the behavior of the monomeric species, but here the dimeric species remain the primary focus. The final glycan used here, stachyose, has been investigated as a cobalt adduct by Schaller-Duke et al. in tandem mass spectrometry experiments . However, no mobility data has been presented in the literature for the stachyose-cobalt ion adduct, so some time will be spent discussing observations about both the monomer and the dimer species. By investigating a range of isomeric glycans as homodimers and heterodimers, including their deuterated analogs, we detail the challenges that may arise when probing isomeric mixtures of catonized glycans using IMS and tandem mass spectrometry.
Atmospheric Pressure Dual-Gate Drift Tube Ion Mobility Linear Ion Trap Mass Spectrometer
All experimental drift time and mass spectral data presented here were obtained on an ExcellIMS MA3100 (Acton, MA, USA) dual-gate atmospheric pressure ion mobility spectrometer paired to a linear ion trap mass spectrometer (LIT-MS) (LTQ, Thermo Scientific, San Jose, CA, USA) as described previously [13, 14, 31]. For reference, Fig. S1 (Supporting Information) displays a schematic of the IM-MS system used. The IMS system was constructed with a rectangular drift tube design consisting of metal-plated ceramic electrodes that generate a homogeneous electric field, which was maintained at ~470 V/cm for all experiments. Each experiment was performed with the drift tube temperature maintained at 180 °C. Dried and purified nitrogen was used as the drift gas at a flow rate of 2 L/min. As the drift gas flow is directed into the end of the drift tube immediately in front of the MS inlet capillary, a portion of this flow is directed into the inlet capillary with the remaining flow acting as a countercurrent flow to assist in ion desolvation. The pressure within the atmospheric drift tube was (~690 Torr in Pullman, WA, USA) measured separately for each individual experiment to permit the calculation of reduced mobilities and ion-neutral collision cross sections. Samples were ionized by electrospray ionization (ESI) and introduced directly into the counter-current flow of the drift gas. The ions travel through a ~6.25-cm desolvation region prior to encountering the first of two Bradbury-Nielson style gates (BN-gate). Once past the first BN-gate, ions travel through the 10.56-cm-long drift region and then meet the second BN-gate immediately prior to the LTQ capillary inlet, which finally directs the ions into the LIT-MS for detection. The LIT-MS was operated at the “normal” scan rate for all analyses involving collision-induced dissociation at trapping times of 200 ms, and single-stage MS utilized the “zoom” scan rate and trapping times of 100 ms. All CID experiments occurred with an applied normalized collision energy of 15. A width of ± 1.5 mass units was used to isolate the precursors for each pair of isomeric tetraitols, while the d-isomaltotetraitol-stachyose heterodimer was isolated with a width of ± 3 mass units to also capture the fragmentation and mobility data for the corresponding homodimeric species, as well. Because this particular IM-MS system cannot obtain adequate ion current when performing CID-MS with a “zoom” scan rate and the heterodimer containing d-isomaltotetraitol and the N-acetal tetraitol appears at an m/z differing only marginally from the sodiated N-acetal tetraitol, the d-isomaltotetraitol and N-acetal tetraitol heterodimer was isolated with a window of only ± 1 mass unit. Although the N-acetal and d-isomaltotetraitol-cobalt heterodimer did suffer interference from the Na adduct during ion isolation for CID, the sources of any resulting fragments can be traced back to the appropriate precursors via the precursors’ arrival time distributions. Linear frequency modulation followed by Fourier transformation was used for IMS data sets as described previously [14, 31]. Briefly, both gates were simultaneously pulsed with ± 50 V using external gate triggers produced by a pulse generator (NI BNC-2120; National Instruments, Austin, TX, USA) operated by a function generator interface via a custom waveform script (IGOR Pro; WaveMetrics, Inc., Lake Oswego, OR, USA). The arrival time distributions for each ion within the mass range analyzed are encoded by the application of a linear frequency chirp to the ion gates, which in this case was the frequency range of 5–10,005 Hz over the course of 8 min. This results in ion current decay over time that can undergo Fourier transformation to yield arrival time distributions; an example of the characteristic ion current decay and resulting arrival time distribution can be viewed in Fig. S2 (Supporting Information). Data acquisition of the LIT-MS and the ion mobility gating are both triggered to start concurrently on the trailing edge of a + 5 V, 1-Hz pulse. For the experiments involving CID, the measured drift times for each fragment correspond to the appropriate precursor ion(s) drift time(s), which is because the drift times are encoded by the BN-gates prior to fragmentation within the LIT-MS . The dimeric metal-glycan adducts observed were of the form [2Glycan - 2H + 2Co2+]2+. Each theoretical glycan fragment composition presented here was generated and identified using GlycoWorkbench 2 [32, 33]. To ensure a moderated assessment of potential fragment compositions, the theoretical fragments generated were limited to no more than one cross-ring cleavage and no more than two separate breakage locations per product ion.
Chemicals and Reagents
The four isomeric tetrasaccharide alditols were made from reducing sugar tetrasaccharide stocks that were prepared as previously described . Cellotetraitol [β-D-Glcp-(1-4)-β-D-Glcp-(1-4)-β-D-Glcp-(1-4)-D-Glc], maltotetraitol [α-D-Glcp-(1-4)-α-D-Glcp-(1-4)-α-D-Glcp-(1-4)-D-Glc], isomaltotetraitol [α-D-Glcp-(1-6)-α-D-Glcp-(1-6)-α-D-Glcp-(1-6)-D-Glc], and nigeran tetraitol [α-D-Glcp-(1-3)-α-D-Glcp-(1-4)-α-D-Glcp-(1-3)-D-Glc] were all purified by high-performance liquid chromatography (HPLC). For the preparation of the monodeuterated isomaltotetraitol glycan sample, a reductive deuteration was performed on isomaltotetraose via borodeuteride, which was also then purified by HPLC [34, 35]. All five of these tetraitol species were prepared at and characterized by NMR at the University of Colorado Health Sciences Center (Aurora, CO, USA). The N-acetal tetraitol assessed was an O-glycan sourced from bovine submaxillary mucin and was purified by HPLC and NMR-characterized as previously described, also at the University of Colorado Health Sciences Center (Aurora, CO, USA) [36, 37]. These five tetraitols were all received as solids and were then each diluted in LC-MS grade water as concentrated stock solutions to be diluted to the appropriate final concentration. Likewise, a concentrated stachyose solution to be diluted further was prepared in LC-MS grade water from commercially sourced > 98% stachyose hydrate solid (Sigma-Aldrich; St. Louis, MO, USA). Cartoon structures following guidelines by Varki et al.  for each of the six glycans analyzed can be found in Fig. 1. Solutions of each pair of a deuterated and non-deuterated glycan isomer were prepared in LC-MS grade methanol with 0.1% formic acid at a concentration of 18 μM for the deuterated isomaltotetraitol species and 12 μM for the non-deuterated glycans. To ensure greater accuracy in calculating peak centroids by minimizing peak widths due to space-charge effects, arrival time distributions for individual glycan solutions were obtained with 3 μM glycan concentrations, with the only exception being the N-acetal tetraitol, which was analyzed individually at 12 μM due to lower ion current observed . For the added Co2+, cobalt(II) chloride salts (J. T. Baker Chemical Co.; Phillipsburg, NJ, USA ) were dissolved in LC-MS grade water and added to the appropriate glycan solutions at a final concentration of ~ 50 μM. All metal-glycan adducts, individually and as mixture solutions, were analyzed by Fourier transform ion mobility-mass spectrometry with ≥ 3 replicates each to permit the calculation of standard deviations for each ion adduct’s drift times, ion-neutral collision cross sections, and reduced mobilities.
Results and Discussion
Cobalt-Tetrasaccharide Adduct Arrival Time Distributions
The series of tetrasaccharides analyzed here were each capable of self-dimerization when adducted with cobalt(II) cations. As shown in Fig. 2, the drift time ranges for both monomer and dimer adducts are distinguishable, with the dimers having characteristically shorter drift times than monomers as they are doubly charged. It is worth noting that the order of dimer arrival times does not necessarily correlate with the same drift time order for a homodimer species’ corresponding monomer. Indeed, upon calculation of each dimer’s reduced mobility and ion-neutral collision cross section (ion-neutral CCS) in Table 1, the differing drift time orders indicate that the glycans likely are arranged in alternate configurations to coordinate with the cobalt cations as dimers than as monomers, which might alter the adducts’ packing density. For furtherer visualization of these values, the reduced mobilities and ion-neutral CCS values have been assembled into bar graphs in Figs. S3 and S4 (Supporting Information), respectively.
The dimer species involving the isomeric glycan set consisting of cellotetraitol, maltotetraitol, nigeran tetraitol, and d-isomaltotetraitol here all appeared at similar relative abundances akin to what has been previously reported . Maltotetraitol yet again has the largest abundance of metal-glycan dimer relative to the monomer, while nigeran tetraitol and d-isomaltotetraitol also show similar, albeit somewhat low, dimer abundances. Cellotetraitol demonstrated somewhat higher homodimer abundance than previously published, but this may be attributed to different ionization conditions, atmospheric drift tube pressures, and other experimental aspects that are difficult to control.
While a fifth alditol, the N-acetal tetraitol, has also made a prior appearance in the literature as a cobalt adduct , the monomeric adduct was the article’s focus and no mention was made of the dimer species. Furthermore, the mobility separation technique applied to that specific experimental method exhibited a noticeably lower resolving power compared to the Fourier multiplexed separation presented here, and as such the N-acetal tetraitol-cobalt dimer from the previous publication would still differ from the arrival time distribution in Fig. 2. This glycan yielded a distinctive dimer with two peaks, which contrasts with every other homodimer species here having produced only one dimer drift time peak each. Given that the N-acetal tetraitol has the greatest structural and compositional departure from the five other tetrasaccharides, the presence of two distinct dimer species arrangements is fitting to have occurred with the sole nitrogen-containing glycan, which may enable a greater range of binding interactions and resulting adduct structures.
Finally, stachyose is the sole tetrasaccharide presented that heretofore has not previously been subject to cobalt adduct formation for ion mobility measurements. An intriguing result of our initial examination of the stachyose-cobalt adducts was that this glycan produces cobalt adducts that are all but entirely dimeric at the concentrations used here. Because it was desirable to also assess the monomer species for comparison, stachyose analysis at 3 μM was particularly crucial to permit a small amount of the monomer to avoid dimerization.
Mixtures of Isomeric Tetrasaccharides and d-Isomaltotetraitol-Cobalt Adducts
One key segment of glycomics research using mass analysis has been directed to the use of multistage mass spectrometry to distinguish glycans . Divalent metal cationization is a method used to alter the fragmentation pathways of carbohydrates [13, 14, 42,43,44,45,46,47], but when there are potentially dimeric species forming [14, 15], metal adduction becomes a less desirable approach with tandem mass spectrometry. More specifically, if the isomers do not undergo sufficient separation before mass analysis, any heterodimer formation could further confound already convoluted fragmentation spectra. To determine if our isomeric set of glycans could possibly form mixed isomer heterodimers, it was first necessary to obtain drift time-selected dimer CID-MS spectra for individual solutions of each glycan, which are shown in Fig. 3. The non-deuterated cellotetraitol, nigeran tetraitol, and maltotetraitol each have relatively distinctive fragmentation patterns even if they do share some fragments of the same m/z, which is analogous to what has been reported for the cobalt-glycan adducts’ corresponding monomers . While the species chosen for deuterium labeling, isomaltotetraitol, has a noticeably simple fragmentation pattern, it is important to recognize that the two major fragments produced by isomaltotetraitol both contain the reducing end hydroxyl, which is crucial to maintaining the ability to distinguish isomaltotetraitol from the other isomers. At the same time, the more varied dissociation patterns yielded by the other isomers permit the observation of additional fragment ions to verify if any dimer species produce fragment ions from two different isomers. Because performing CID on target ions immediately following multiplexed mobility separation yields arrival time distributions of the precursors producing each fragment, it is possible to exploit two different dimensions of information to differentiate these isomeric glycans and tease out the composition of the dimer species in a mixture solution containing each isomer pair.
Upon examination of Fig. 4a, the arrival time distributions for the primary characteristic fragments produced by CID are consistent with a mixture of d-isomaltotetraitol and maltotetraitol as cobalt adducts. Fragment ions at m/z 403 and 565 were monitored specifically to track the drift time of species containing the deuterated isomaltotetraitol as both fragments include the reducing end hydroxyl. For maltotetraitol, drift time spectra were extracted for fragments at m/z 402 and 544, which are shown in Fig. 3 to be produced by the maltotetraitol-cobalt dimer. Each arrival time distribution has the dimer, maltotetraitol monomer, and d-isomaltotetraitol monomer locations indicated by a line to the x-axis and labeled accordingly. The dimer centroid for both isomers’ primary fragments suggest that the dimer species was sourced from a precursor appearing at approximately the same drift times, which suggests that the fragment ions were all sourced from the same precursor ion. The mixture arrival time distributions (ATDs) for fragments at m/z 564 and 402 indicate that some non-deuterated isomaltotetraitol—which may have formed resulting from proton/deuterium exchange with the protic solvent—was present. However, the higher ratios of dimer to monomer abundance of the maltotetraitol-cobalt adducts versus isomaltotetraitol (Figs. 2 and 4) indicate that these dimers do contain maltotetraitol. The final piece of evidence for a malto- and d-isomaltotetraitol-cobalt heterodimer can be seen in the drift time shift for the dimer peak; d-isomaltotetraitol has a homodimer with a mean reduced mobility of 1.028 cm2/V s, while the only dimer peak appearing in this particular mixture for all major fragments yielded an average reduced mobility of 1.064 cm2/V s. This heterodimer species demonstrates a far closer mobility to that of the maltotetraitol homodimer than that of the d-isomaltotetraitol homodimer, thereby indicating that the maltotetraitol glycan may have a more pronounced influence on the overall size of the cobalt-glycan heterodimer.
Figure 4b displays the precursor arrival time distributions for the major fragments of d-isomaltotetraitol and cellotetraitol when analyzed together as a mixture. As mentioned in the prior paragraph, d-isomaltotetraitol has its two predominant fragments appearing at m/z 403 and 565, which contain the glycan’s reducing end, and at 544 to a minor extent. Cellotetraitol-cobalt homodimers largely yield fragments of m/z 544, 402, and 382, of which only the fragment of 402 contains the reducing end. However, the relative abundances of the m/z 544 fragment ion from cellotetraitol and from d-isomaltotetraitol are such that the major contributor by far in the mobility spectra is cellotetraitol, as would be expected by the drift time-selected CID-MS data from Fig. 3. Unlike for the prior isomeric tetraitol pair that appeared to yield a heterodimer species with an altered drift time, the mean drift times of dimer species for cellotetraitol and d-isomaltotetraitol analyzed together as cobalt adducts indicate that two different dimer species may be present in the ion swarm. The drift time difference, though slight, corresponds well to the appropriate homodimer fragments for each of the two glycan isomers. Despite this correspondence of all labeled fragments being sourced from the higher mobility dimer and the unlabeled fragments produced by the lower mobility dimer, the two mixture dimer species have reduced mobilities that exceed the mobilities of the homodimers beyond the bounds of centroid determination error based upon the drift time peak widths.
In contrast to the d-isomaltotetraitol heterodimers containing maltotetraitol and cellotetraitol, the d-isomaltotetraitol and nigeran tetraitol dimer did not exhibit appreciable mobility changes (Fig. 4c). This is likely due to the very similar mobilities of the two isomers. However, this does prompt questions regarding the influence of the glycan linkage configurations and positions within a mixed isomer metal-glycan dimer. The isomer combination mentioned in the previous paragraph—nigeran tetraitol and d-isomaltotetraitol—had two dimeric species, both of which had higher reduced mobilities (i.e., smaller ion-neutral CCS) than the two isomer species as individual cobalt-glycan homodimers. Along with raising the question of what kind of isomer combinations will result in more compact adducts, it also would be useful to determine if there are any circumstances under which the heterodimers have increased ion-neutral collision cross sections. Further assessments with a larger combination of differing isomer structures would certainly be merited for finding this potential arrangement, particularly if coupled with corresponding computational simulations. However, given the range of deprotonation and subsequent metal coordination sites, such an undertaking is extremely challenges with the current set of computational tools [48, 49].
Formation of Heterodimers Containing d-Isomaltotetraitol with Non-isomer Tetrasaccharides
Given the lower m/z resolution obtained with ion trap mass spectrometers, one of our concerns was that dimer tandem MS experiments involving a singly deuterated species may not alone be conclusive evidence that dimeric cobalt-glycan adducts will form between two different glycan species. To further affirm heterodimer formation, we pursued the formation of dimeric metal-glycan adducts using non-isomeric glycan pairs, which will exhibit a definitive shift in m/z relative to either of the contributing oligosaccharides. For this purpose, the heterodimeric cobalt-glycan adducts assessed were resulting from mixture solutions pairing d-isomaltotetraitol with the fructose-containing tetrasaccharide stachyose as well as with an O-linked tetraitol containing N-acetal groups . As with the set of isomeric tetraitols, stachyose and the N-acetal tetraitol were both analyzed as individual solutions with cobalt but without any added d-isomaltotetraitol to yield arrival time distributions of their homodimers (Fig. 2).
Because stachyose as a cobalt-glycan ion adduct has not yet been reported in the drift time domain, we first will report the assessment of this adduct’s monomer behavior. As can be seen in Fig. 2, two monomer peaks are present for this non-reducing sugar, which was seen for only one other sugar, the N-acetal tetraitol; stachyose also was the only other species besides the N-acetal tetraitol to contain non-glucose residues. The cobalt-stachyose adduct arrival time distribution shows the presence of a single dimer species, which suggests that the one dimerization configuration permits more favorable metal-glycan interactions and arrangement in space in comparison to the monomeric species. This explanation is bolstered by the approximately two orders of magnitude higher abundance of the dimeric stachyose-cobalt species relative to the monomer abundances.
With a cobalt-glycan dimer of the non-isomeric tetrasaccharide pair of stachyose and d-isomaltotetraitol, the larger mass difference between the glycans yields a noticeable shift in m/z that permits clear identification of the new species produced by the mixture. As seen in Fig. 5a, the doubly charged d-isomaltotetraitol and stachyose-cobalt dimer appears at a monoisotopic m/z of 725.7, as opposed to the difficulty of locating the heterodimer in isomer pairs even with one isomer being radiolabeled. A notable contrast of this mixed dimer species with the isomeric pairs is that with stachyose and d-isomaltotetraitol, the cobalt-glycan heterodimer species has a distinctly longer drift time than either of the adduct’s corresponding homodimers (Fig. 5b). In effect, this is one such scenario that does result in a cobalt-glycan heterodimer that has a more expanded structure than the homodimers of stachyose or d-isomaltotetraitol, as opposed to the isomeric pair heterodimers which had higher mobilities—and therefore smaller ion-neutral collision cross sections—than the homodimers.
Examination of the stachyose homodimer via tandem MS yields both singly and doubly charged fragment ions (Fig. 6), but with the majority being doubly charged. This is not surprising given that the primary species of the stachyose-cobalt adducts is the dimer, and in combination with the fragmentation data, it is likely that the structure of stachyose permits coordination of cobalt(II) cations more favorably than the other five glycans assessed here given the greater retention of both glycans in the adduct fragments. In comparison to the d-isomaltotetraitol CID spectra (Fig. 6a), stachyose as a dimeric cobalt adduct follows a multitude of dissociation pathways (Fig. 6b). The theoretical combinations of individual stachyose fragments that could produce these m/z values can be found in Fig. S5 (Supporting Information). When the heterodimer is formed between d-isomaltotetraitol and stachyose with cobalt cations, the resulting MS/MS spectra yields a combination of d-isomaltotetraitol fragments, stachyose fragments, and fragment m/z values that could correspond only to dimer compositions including components from both tetrasaccharides (Fig. 6c). These mixed dimer fragments serve to confirm that the identity of this drift time peak is that of the d-isomaltotetraitol and stachyose-cobalt heterodimer.
The N-acetal tetraitol homodimer is unique compared to the other glycans in that there were two species clearly seen in homodimer arrival time distributions, and “zoom” mass scan mode higher resolution mass spectra of these adducts without mobility separation correspond to a superposition of theoretical spectra generated  of monomeric and dimeric N-acetal glycan-cobalt adducts, rather than to inclusion of a trimeric or higher multimeric species (Fig. S7, Supporting Information). Drift time-selected CID spectra of the two peaks can be seen in Fig. 7a. The two homodimers both produce the same two singly charged fragments, m/z 628 and 646, while the higher mobility dimer also produced two doubly charged fragments at m/z 690.7 and 719. It appears likely that the coordination arrangement in the more compact, higher mobility N-acetal tetraitol and cobalt homodimer involves interactions between the two metal-glycan pairs that are likely stronger relative to that of the other homodimer. In contrast, the lower mobility dimer produced predominantly the singly charged fragments. The same level of collision energy induced what may be different fragmentation pathways, which indicates that the two homodimers may be arising due to two differing interaction arrangements between the two pairs of cobalt and glycan ion adducts. These contrasting metal-glycan configurations are best exemplified by the sparing amount of doubly charged fragments made by the lower mobility dimer; the higher mobility dimer produces these fragment ions at roughly 20 times higher in abundance than the maximum quantity of the doubly charged fragments observed in the lower mobility dimer’s drift time-selected CID spectra. As would be expected, the only drift time peak found in the precursor arrival time distributions for fragment ions m/z 690.7 and 719 corresponds to the higher mobility N-acetal tetraitol-cobalt homodimer given the substantially lower abundance of these fragments produced by the lower mobility homodimer (Fig. 7b).
Moving on to the final heterodimer, d-isomaltotetraitol and the N-acetal tetraitol combine as a cobalt dimer species at m/z 759.7 (Fig. S6, Supporting Information). As pointed out in Fig. S6a (Supporting Information), the heterodimer appears at a nominal m/z that overlaps with the second isotope of the N-acetal tetraitol sodium adduct. Although the d-isomaltotetraitol and N-acetal tetraitol heterodimer species is visible with the “zoom” mass scan mode, the ion current was too low with the higher resolution scan rate to also perform CID. As a consequence, the mass isolation for tandem MS captures both the sodium adduct and cobalt-glycan heterodimer species (Fig. S6b, Supporting Information). This situation exemplifies where drift time selection of the tandem MS spectra is particularly important; these species have markedly different drift times, which permits distinguishing fragment ion sources based upon the precursors’ drift times (Fig. S6b, Supporting Information). When compared to the d-isomaltotetraitol homodimer and two N-acetal tetraitol homodimers, the d-isomaltotetraitol and N-acetal tetraitol heterodimer drift time aligns well with what was expected, appearing roughly between the drift times of the corresponding homodimer species, thereby permitting both m/z and drift time separation of the heterodimer from any homodimer species (Fig. 8a). In Fig. 8b, a drift time-selected MS spectrum of the primary fragments produced by the heterodimer reveals fragment ions sourced clearly from each of the component glycans, which resulted from a precursor of the same drift time (Fig. 8c). The predominant fragment is at m/z 727, which is yielded from the two metal-glycan pairs dissociating. Although the singly charged N-acetal tetraitol-cobalt adduct, m/z 792, is also produced by the heterodimer, this fragment species appears at a substantially lower abundance, indicating that the d-isomaltotetraitol glycan more readily retains the cobalt ion upon fragmentation as compared to the N-acetal tetraitol. As was seen with the previous non-isomeric glycan heterodimer, a doubly charged fragment ion at m/z 686.7 in this spectrum was found to correspond to a combination of two specific fragments from both d-isomaltotetraitol and the N-acetal tetraitol to confirm the identity of this species as the heterodimer (Fig. S8, Supporting Information).
The primary observations of the present experiments are (1) metal-glycan heterodimers involving two different glycans can and do form under reasonable operating conditions, (2) the presence of these species could easily confound fragmentation patterns produced without ion mobility separation, and (3) variable fragmentation ratios are observed for different conformations of the cationized dimers.
In exploring the speciation phenomena occurring with divalent cobalt adducted to glycans, it has become apparent that glycan MS analysis of metal adducts need be performed with the appropriate separation techniques to ensure that isomeric species are not assessed under circumstances that permit heterodimer formation. Although the divalent metal adduction technique is not particularly common for glycan analysis, the idea that heterodimers can confound mass spectra is readily applicable as a caution to other areas of biomolecule analysis, notably for sodiated glycan dimers  and steroid dimers  as recent examples.
For those attempting to use multistage dissociation mass spectrometry or energy-resolved MS patterns alone to distinguish isomeric carbohydrate species, it is important to bear in mind that these techniques cannot be applied to even a simple mixture of two isomers without some form of front-end separation. Even in a scenario where isomers produce different fragment ratios or m/z values, the overlapped MS/MS spectra cannot be deconvoluted; therefore, only with sufficient mobility or chromatographic separation can isomer mixtures be successfully analyzed. Chromatographic separations of increasingly similar isomers can be time-consuming and require specialty columns, so the significant separation power in a shorter timespan obtained with IM-MS makes ion mobility an attractive option to get around having confounded MS/MS spectra.
The data and discussion presented illustrate that ion mobility separations provide valuable separation enhancement that cannot be simply classified as a lower resolution “alternative” to chromatography. Instead, ion mobility spectrometry should be considered an additional separation method that can provide standalone separations or is added along with chromatography, not something that can be replaced by chromatography, due to the different molecular features that are targeted to provide mobility separation. Even under circumstances in which dimerization occurs only to a limited extent, the utility of ion mobility separations for isomeric species distinction is appealing for wide application.
Armenta, S., Alcala, M., Blanco, M.: A review of recent, unconventional applications of ion mobility spectrometry (IMS). Anal. Chim. Acta. 703, 114–123 (2011)
Baumbach, J.I.: Process analysis using ion mobility spectrometry. Anal. Bioanal. Chem. 384, 1059–1070 (2006)
Fenn, L.S., McLean, J.A.: Biomolecular structural separations by ion mobility-mass spectrometry. Anal. Bioanal. Chem. 391, 905–909 (2008)
Woods, A.S., Ugarov, M., Egan, T., Koomen, J., Gillig, K.J., Fuhrer, K., Gonin, M., Schultz, J.A.: Lipid/peptide/nucleotide separation with MALDI-ion mobility-TOF MS. Anal. Chem. 76, 2187–2195 (2004)
Bohrer, B.C., Merenbloom, S.I., Koeniger, S.L., Hilderbrand, A.E., Clemmer, D.E.: Biomolecule analysis by ion mobility spectrometry. Annu. Rev. Anal. Chem. 1, 293–327 (2008)
McLean, J.A., Ruotolo, B.T., Gillig, K.J., Russell, D.H.: Ion mobility-mass spectrometry: a new paradigm for proteomics. Int. J. Mass Spectrom. 240, 301–315 (2005)
Zheng, X., Smith, R.D., Baker, E.S.: Recent advances in lipid separations and structural elucidation using mass spectrometry combined with ion mobility spectrometry, ion-molecule reactions and fragmentation approaches. Curr. Opin. Chem. Biol. 42, 111–118 (2018)
Hinz, C., Liggi, S., Griffin, J.L.: The potential of ion mobility mass spectrometry for high-throughput and high-resolution lipidomics. Curr. Opin. Chem. Biol. 42, 42–50 (2018)
Kliman, M., May, J.C., McLean, J.A.: Lipid analysis and lipidomics by structurally selective ion mobility-mass spectrometry. Biochim. Biophys. Acta - Mol. Cell Biol. Lipids. 1811, 935–945 (2011)
Lee, S., Valentine, S.J., Reilly, J.P., Clemmer, D.E.: Analyzing a mixture of disaccharides by IMS-VUVPD-MS. Int. J. Mass Spectrom. 309, 161–167 (2012)
Dwivedi, P., Bendiak, B., Clowers, B.H., Hill, H.H.: Rapid resolution of carbohydrate isomers by electrospray ionization ambient pressure ion mobility spectrometry-time-of-flight mass spectrometry (ESI-APIMS-TOFMS). J. Am. Soc. Mass Spectrom. 18, 1163–1175 (2007)
Clowers, B.H., Dwivedi, P., Steiner, W.E., Hill, H.H., Bendiak, B.: Separation of sodiated isobaric disaccharides and trisaccharides using electrospray ionization-atmospheric pressure ion mobility-time of flight mass spectrometry. J. Am. Soc. Mass Spectrom. 16, 660–669 (2005)
Morrison, K.A., Clowers, B.H.: Differential fragmentation of mobility-selected glycans via ultraviolet photodissociation and ion mobility-mass spectrometry. J. Am. Soc. Mass Spectrom. 28, 1236–1241 (2017)
Morrison, K.A., Bendiak, B.K., Clowers, B.H.: Enhanced mixture separations of metal adducted tetrasaccharides using frequency encoded ion mobility separations and tandem mass spectrometry. J. Am. Soc. Mass Spectrom. 28, 664–677 (2017)
Williams, J.P., Grabenauer, M., Holland, R.J., Carpenter, C.J., Wormald, M.R., Giles, K., Harvey, D.J., Bateman, R.H., Scrivens, J.H., Bowers, M.T.: Characterization of simple isomeric oligosaccharides and the rapid separation of glycan mixtures by ion mobility mass spectrometry. Int. J. Mass Spectrom. 298, 119–127 (2010)
Cummings, R.D., Pierce, J.M.: The challenge and promise of glycomics. Chem. Biol. 21, 1–15 (2014)
Lairson, L.L., Henrissat, B., Davies, G.J., Withers, S.G.: Glycosyltransferases: structures, functions, and mechanisms. Annu. Rev. Biochem. 77, 521–555 (2008)
Solís, D., Bovin, N.V., Davis, A.P., Jiménez-Barbero, J., Romero, A., Roy, R., Smetana, K., Gabius, H.J.: A guide into glycosciences: how chemistry, biochemistry and biology cooperate to crack the sugar code. Biochim. Biophys. Acta - Gen. Subj. 1850, 186–235 (2015)
Zheng, X., Zhang, X., Schocker, N.S., Renslow, R.S., Orton, D.J., Khamsi, J., Ashmus, R.A., Almeida, I.C., Tang, K., Costello, C.E., Smith, R.D., Michael, K., Baker, E.S.: Enhancing glycan isomer separations with metal ions and positive and negative polarity ion mobility spectrometry-mass spectrometry analyses. Anal. Bioanal. Chem. 467–476 (2016)
Plasencia, M.D., Isailovic, D., Merenbloom, S.I., Mechref, Y., Clemmer, D.E.: Resolving and assigning N-linked glycan structural isomers from ovalbumin by IMS-MS. J. Am. Soc. Mass Spectrom. 19, 1706–1715 (2008)
Briggs, J., Finch, P., Matulewicz, M., Weigel, H.: Complexes of copper (II), calcium, and other metal ions with carbohydrates: thin-layer ligand-exchange chromatography and determination of relative stabilities of. Carbohydr. Res. 97, 181–188 (1981)
Gyurcsik, B., Nagy, L.: Carbohydrates as ligands: coordination equilibria and structure of the metal complexes. Coord. Chem. Rev. 203, 81–149 (2000)
Cancilla, M.T., Penn, S.G., Carroll, J.A., Lebrilla, C.B.: Coordination of alkali metals to oligosaccharides dictates fragmentation behavior in matrix assisted laser desorption ionization Fourier transform mass spectrometry. J. Am. Chem. Soc. 118, 6736–6745 (1996)
Kohler, M., Leary, J.A.: LC/MS/MS of carbohydrates with postcolumn addition of metal chlorides using triaxial electrospray probe. Anal. Chem. 67, 3501–3508 (1995)
Konig, S., Leary, J.A.: Evidence for linkage position determination in cobalt coordinated pentasaccharides using ion trap mass spectrometry. J. Am. Soc. Mass Spectrom. 9, 1125–1134 (1998)
Sible, E.M., Brimmer, S.P., Leary, J.A.: Interaction of first row transition metals with α1-3, α1-6 mannotriose and conserved trimannosyl core oligosaccharides: a comparative electrospray ionization study of doubly and singly charged complexes. J. Am. Soc. Mass Spectrom. 8, 32–42 (1997)
Smith, G., Kaffashan, A., Leary, J.A.: Influence of coordination number and ligand size on the dissociation mechanisms of transition metal monosaccharide complexes. Int. J. Mass Spectrom. 183, 299–310 (1999)
Harvey, D.J.: Ionization and collision-induced fragmentation of N-linked and related carbohydrates using divalent cations. 12, 926–937 (2001)
Clowers, B.H., Hill, H.H.: Influence of cation adduction on the separation characteristics of flavonoid diglycoside isomers using dual gate-ion mobility-quadrupole ion trap mass spectrometry. J. Mass Spectrom. 41, 339–351 (2006)
Schaller-Duke, R.M., Bogala, M.R., Cassady, C.J.: Electron transfer dissociation and collision-induced dissociation of underivatized metallated oligosaccharides. J. Am. Soc. Mass Spectrom. (2018)
Morrison, K.A., Siems, W.F., Clowers, B.H.: Augmenting ion trap mass spectrometers using a frequency modulated drift tube ion mobility spectrometer. Anal. Chem. 88, 3121–3129 (2016)
Ceroni, A., Maass, K., Geyer, H., Geyer, R., Dell, A., Haslam, S.M.: GlycoWorkbench: a tool for the computer-assisted annotation of mass spectra of glycans. J. Proteome Res. 7, 1650–1659 (2008)
Damerell, D., Ceroni, A., Maass, K., Ranzinger, R., Dell, A., Haslam, S.M.: The GlycanBuilder and GlycoWorkbench glycoinformatics tools: updates and new developments. Biol. Chem. 393, 1357–1362 (2012)
Hellerqvist, C.G., Lindberg, B., Pilotti, Å., Lindberg, A.A.: Structural studies of the O-specific side-chains of the cell-wall lipopolysaccharide from Salmonella senftenberg. Carbohydr. Res. 16, 297–302 (1971)
Cumming, D.A., Hellerqvist, C.G., Harris-Brandts, M., Michnick, S.W., Carver, J.P., Bendiak, B.: Structures of asparagine-linked oligosaccharides of the glycoprotein fetuin having sialic acid linked to N-acetylglucosamine. Biochemistry. 28, 6500–6512 (1989)
Li, H., Bendiak, B., Kaplan, K., Davis, E., Siems, W.F., Hill, H.H.: Evaluation of ion mobility-mass spectrometry for determining the isomeric heterogeneity of oligosaccharide-alditols derived from bovine submaxillary mucin. Int. J. Mass Spectrom. 352, 9–18 (2013)
Li, H., Bendiak, B., Siems, W.F., Gang, D.R., Hill, H.H.: Determining the isomeric heterogeneity of neutral oligosaccharide-alditols of bovine submaxillary mucin using negative ion traveling wave ion mobility mass spectrometry. Anal. Chem. 87, 2228–2235 (2015)
Varki, A., Cummings, R.D., Aebi, M., Packer, N.H., Seeberger, P.H., Esko, J.D., Stanley, P., Hart, G., Darvill, A., Kinoshita, T., Prestegard, J.J., Schnaar, R.L., Freeze, H.H., Marth, J.D., Bertozzi, C.R., Etzler, M.E., Frank, M., Vliegenthart, J.F.G., Lutteke, T., Perez, S., Bolton, E., Rudd, P., Paulson, J., Kanehisa, M., Toukach, P., Aoki-Kinoshita, K.F., Dell, A., Narimatsu, H., York, W., Taniguchi, N., Kornfeld, S.: Symbol nomenclature for graphical representations of glycans. Glycobiology. 25, 1323–1324 (2015)
Tolmachev, A., Clowers, B., Belov, M., Smith, R.: Coulombic effects in ion mobility spectrometry. Anal. Chem. 81, 4778–4787 (2009)
Siems, W.F., Wu, C., Tarver, E.E., Hill, H.H., Larsen, P.R., Mcminn, D.: Measuring the resolving power of ion mobility spectrometers. 66, 4195–4201 (1994)
Zaia, J.: Mass spectrometry of oligosaccharides. Mass Spectrom. Rev. 23, 161–227 (2004)
Adamson, J.T., Håkansson, K.: Electron capture dissociation of oligosaccharides ionized with alkali, alkaline earth, and transition metals. Anal. Chem. 79, 2901–2910 (2007)
Zhou, W., Håkansson, K.: Electron capture dissociation of divalent metal-adducted sulfated N-glycans released from bovine thyroid stimulating hormone. J. Am. Soc. Mass Spectrom. 24, 1798–1806 (2013)
Huang, Y., Dodds, E.D.: Ion-neutral collisional cross sections of carbohydrate isomers as divalent cation adducts and their electron transfer products. Analyst. 140, 6912–6921 (2015)
Huang, Y., Dodds, E.D.: Discrimination of isomeric carbohydrates as the electron transfer products of group II cation adducts by ion mobility spectrometry and tandem mass spectrometry. Anal. Chem. 87, 5664–5668 (2015)
Huang, Y., Dodds, E.D.: Ion mobility studies of carbohydrates as group I adducts: isomer specific collisional cross section dependence on metal ion radius. Anal. Chem. 85, 9728–9735 (2013)
Morrison, K.A., Clowers, B.H.: Contemporary glycomic approaches using ion mobility–mass spectrometry. Curr. Opin. Chem. Biol. 42, 119–129 (2018)
Rabus, J.M., Abutokaikah, M.T., Ross, R.T., Bythell, B.J.: Sodium-cationized carbohydrate gas-phase fragmentation chemistry: influence of glycosidic linkage position. Phys. Chem. Chem. Phys. 19, 25643–25652 (2017)
Bythell, B.J., Abutokaikah, M.T., Wagoner, A.R., Guan, S., Rabus, J.M.: Cationized carbohydrate gas-phase fragmentation. J. Am. Soc. Mass Spectrom. 28, 688–703 (2017)
Loos, M., Gerber, C., Corona, F., Hollender, J., Singer, H.: Accelerated isotope fine structure calculation using pruned transition trees. Anal. Chem. 87, 5738–5744 (2015)
Chouinard, C.D., Cruzeiro, V.W.D., Roitberg, A.E., Yost, R.A.: Experimental and theoretical investigation of sodiated multimers of steroid epimers with ion mobility-mass spectrometry. J. Am. Soc. Mass Spectrom. 28, 323–331 (2017)
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 received support from the New Faculty Seed Grant from Washington State University.
Electronic supplementary material
About this article
Cite this article
Morrison, K.A., Bendiak, B.K. & Clowers, B.H. Assessment of Dimeric Metal-Glycan Adducts via Isotopic Labeling and Ion Mobility-Mass Spectrometry. J. Am. Soc. Mass Spectrom. 29, 1638–1649 (2018). https://doi.org/10.1007/s13361-018-1982-2
- Ion mobility
- Mass spectrometry
- Metal adduction
- Metal-glycan adducts
- Glycomics and IMS
- Transition metal-glycan dimers