Lan
Jin
a,
Perdita E.
Barran
a,
Jon A.
Deakin
b,
Malcolm
Lyon
b and
Dušan
Uhrín
*a
aUniversity of Edinburgh, School of Chemistry, Joseph Black Building, West Mains Rd., Edinburgh, UK EH9 3JJ
bCancer Research UK Department of Medical Oncology, University of Manchester, Christie Hospital NHS Trust, Wilmslow Road, Manchester, UK M20 4BX
First published on 22nd August 2005
We have performed conformational analyses of heparin-derived oligosaccharide ions in the gas phase using a combination of ion-mobility mass spectrometry and molecular modelling. Negative mode electrospray ionisation was used to generate singly (disaccharide, [C12H15NO19S3Na3]−) and doubly charged (tetrasaccharides, [C24H30N2O38S6Na6]2− and [C24H31N2O35S5Na5]2−) ions containing three and six Na+ ions, respectively. Good agreement was obtained between the experimental and theoretical cross sections. The latter were obtained using modelled structures generated by the AMBER-based force field. Analysis of the conformations of the oligosaccharide ions shows that sodium cations play a major role in stabilizing these ions in the gas phase. This was seen in the formation of oligomers of the disaccharide ion and “compact” structures of tetrasaccharide ions. Interestingly, the gas phase conformations of the three tetrasaccharide ions with different primary structures were significantly different.
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Fig. 1 (a) Monosaccharide units of heparin/HS. (b) Structures of the heparin-derived disaccharide (I) and three tetrasaccharides (II–IV) used in this study. ΔUA(2S)-GlcNS(6S)-Na3 (I), ΔUA(2S)-GlcNS(6S)-IdoA(2S)-GlcNS(6S)-Na6 (II), ΔUA(2S)-GlcNS(6S)-IdoA(2S)-GlcNS-Na6 (III) and ΔUA(2S)-GlcNS(6S)-GlcA-GlcNS(6S)-Na5 (IV). Letters A–D are used to label individual rings starting from the reducing end (A) and progressing to the unsaturated non-reducing end (D) of the oligosaccharide. |
More than a hundred proteins are known to bind to heparin in vitro6 nevertheless, the physiologically relevant ligand in most cases will be HS, which is far more widely distributed. Information about the nature of these complexes comes from a limited number of X-ray structures.7 Almost exclusively, these studies exploited fully sulfated heparin-derived oligosaccharides which form helical structures similar to those found in heparin fibres, as revealed by X-ray diffraction studies,8 and in solution, as shown by NMR.9 Nevertheless, models of HS–protein complexes, supported by biophysical and biochemical data, are emerging that involve longer glycan segments.10,11 Inevitably, deviation from helical conformations of fully sulfated heparin must occur in order to accommodate such modes of binding. It is widely expected that this increased flexibility will be associated with less sulfated regions of HS,12 though little experimental evidence is available to support this supposition. Methodological advances in mapping the conformational space available to HS are therefore of considerable interest. Towards this end we have employed ion-mobility mass spectrometry (IM-MS) in combination with molecular modelling as a novel approach to probe the conformation of a series of highly sulfated HS/heparin oligosaccharides (Fig. 1b).
Ions drifting under the influence of a weak electrostatic field are retarded by collisions with a low pressure buffer gas. As a consequence, species with different overall shapes, or more specifically different rotationally averaged collision cross sections, are separated in time. This forms the basis for the conformational analysis by IM-MS, which, when used in conjunction with electrospray ionisation, has been applied to the study of various biomolecules, including peptides, proteins, and DNA.13,14 Work of this nature does invariably meet opposition from an audience more used to solution-based structural studies. However, recent studies have indicated that, under certain conditions, solution conformations can be retained in the gas phase. This may be attributable to the gentle native electrospray ionisation conditions,15 or may be a feature of the dominance of electrostatic interactions in a solution structure, which are then strengthened in the gas phase. This latter consequence has been illustrated by Gidden et al.,16 who have used IM-MS in conjunction with molecular modelling to explore the persistence of DNA helices in the gas-phase.
Several other researchers have investigated the structures of HS species with mass spectrometry, but focusing on primary structure determination.17–21 Gas-phase fragmentation, used in concert with enzymatic degradation and even 2H and 18O labelling, has successfully been employed to illustrate a mass spectrometry-based identification strategy for very complex mixtures of what are often isobaric species.22–24 In contrast, we have exploited a different methodology here, which aims to determine the three-dimensional structure of GAG ions in the gas phase. If used in conjunction with higher-resolution IM-MS instrumentation,25–26 it is possible that our approach could be used, in an analogous fashion to liquid chromatography separation, to distinguish the isomers present in a mixture, but in this case by virtue of their differing collision cross sections. This approach determines conformations using relatively low amounts of sample, and also could be used for analysis of mixtures, which is attractive given the practical difficulties associated with isolating pure HS species.
Mass spectra were obtained for each of the oligosacharides, which were present in singly and doubly charged forms as partially sodiated species. The experimental and predicted masses for the major species observed for each oligosaccharide are listed in Table 1.
Compound | Observed | Calculated | Assigned species |
---|---|---|---|
I | 642.7 | 642.4 | [C12H15NO19S3Na3]− |
631.3 | 630.9 | [C24H31N2O38S6Na5]2− | |
620.6 | 619.9 | [C12H16NO19S3Na2]− | |
II | 642.8 | 642.4 | [C24H30N2O38S6Na6]2− |
631.6 | 630.9 | [C24H31N2O38S6Na5]2− | |
621.1 | 620.4 | [C24H32N2O38S6Na4]2− | |
III | 592.3 | 591.4 | [C24H31N2O35S5Na5]2− |
581.2 | 580.4 | [C24H32N2O35S5Na4]2− | |
570.1 | 569.4 | [C24H33N2O35S5Na3]2− | |
IV | 592.4 | 591.4 | [C24H31N2O35S5Na5]2− |
581.1 | 580.4 | [C24H32N2O35S5Na4]2− | |
570.1 | 569.4 | [C24H33N2O35S5Na3]2− |
If an ion is pulsed into the cell, the quadrapole is then ‘parked’ on an ion of interest and its arrival time at the detector, relative to the start time of the pulse, is recorded. Arrival time distributions (ATDs) can then be collected at several drift voltages (90, 60, 45, 30 and 20 V) to obtain the reduced mobility (K0) of the ion, via a plot of arrival times versus the pressure of helium divided by the drift voltage. This mobility is used to determine the experimental collision cross section of the ion (σ) according to eqn (1) below.29
![]() | (1) |
Experimental cross sections are then compared to those obtained from candidate geometries modelled with the GLYCAM force field (see below).
(a) Fully sulfated disaccharide (I) | ||||||
---|---|---|---|---|---|---|
Pressure/bar | 4.965 | 4.939 | 4.957 | 4.943 | 4.953 | |
Voltage/V | 91.39 | 61.20 | 45.69 | 30.70 | 20.13 | |
P/V | 0.054 | 0.081 | 0.108 | 0.161 | 0.246 | |
ATD/μs | Monomer | 385 | 490 | 600 | 810 | 1150 |
Dimer | 410 | 530 | 653 | 890 | 1280 | |
Trimer | 485 | 645 | 805 | 1120 | 1625 | |
K 0/cm2 V−1 s−1 | Monomer | Dimer | Trimer | |||
4.024 | 5.274 | 5.992 | ||||
σ (Å2) | 132.1 | 201.6 | 266.8 |
(b) | Compound | |||||
---|---|---|---|---|---|---|
I | II | III | IV | |||
σ (Å2) | Monomer | Dimer | Trimer | |||
Experimental | 132.1 | 201.6 | 266.8 | 198.4 | 190.4 | 191.6 |
Calculated | 134.8 | 191.7 | 250.2 | 204.1 | 190.3 | 191.8 |
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Fig. 2 (a) Arrival time distribution for the 642.4 m/z ion of disaccharide I. An electric gradient of 60 V was used. The peaks labelled a–c correspond to a monomer, dimer and trimer of I, respectively. (b) Determination of experimental ion mobility, K, for three arrival time distributions observed for disaccharide monomer (◆), dimer (■) and trimer (▲) of I. (Data from Table 2(a)) |
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Fig. 3 Theoretical cross sections of 100 candidate structures of (a) disaccharide I and (b) tetrasaccharide II ions as a function of the potential energy. |
The ions generated using tetrasaccharides II–IV were principally monomeric and corresponded to the following species: [C24H30N2O38S6Na6]2− (II, 642.4 m/z) and [C24H31N2O35S5Na5]2− (III, IV, 591.4 m/z) (Table 1). Their arrival time distributions were analyzed as explained above for the disaccharide, and the resulting experimental cross-sectional areas are summarized in Table 2.
The number of Na atoms lost during the ionization process (one for the disaccharide and two for tetrasaccharides) corresponds exactly to the number of carboxyl groups, implying a stronger affinity of sulfate and sulfamate groups for sodium compared with carboxyl groups.
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Fig. 4 Lowest energy structures of the [C12H15NO19S3Na3]− disaccharide I ion. (a) A typical structure of I. Na+ is shown as spheres. (b) Overlay of the ten lowest energy structures. The structures were generated using MolMol43 and are oriented as shown in Fig. 1. |
The agreement between the experimental and theoretical cross-sections is very good. For monomers the differences were <2.1%, while for oligomers they were <6.2%
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Fig. 5 Representative structures of (a) [C12H15NO19S3Na3]22− and (b) C12H15NO19S3Na3]33− ions corresponding to the dimer and trimer of I. The structures were generated with VMD.42 Individual monomers are highlighted using van der Waals radii of their atoms. |
The ΔUA ring (D) was always found in the 1H2 conformation while the glucosamine residue (ring A) invariably occupied the 4C1 conformation. Two Na+ ions were coordinated by two sulfate groups, while the third was positioned between the sulfamate and carboxyl groups. As a general feature of all structures we simulated, the Na+ ions were found between 2.2 and 2.9 Å from oxygen atoms of at least two sulfate, sulfamate or carboxyl groups.
Although the prevailing conformations of rings D and A in the dimer and the trimer were the same as observed for the monomer disaccharide (1H2 and 4C1), other forms (B3,O for ring D and 1S5, 5S3 for ring A) were also found. We attribute the existence of these forms to the presence of Coulombic interactions between the Na+ ions and negatively charged groups causing distortions of the ring conformations. In all dimers the Na+ cations were simultaneously coordinated to both monomer units. In the trimers all Na+ ions were coordinated by at least two monomers, while some showed simultaneous interactions with all three monomers. In addition, several hydrogen bonds were observed between some monomers using the criterion for the distance between the atom, D, carrying hydrogen donor and oxygen acceptor (A) was <3 Å and the angle |D–H–A| was <20° (Fig. 5).
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Fig. 6 Fully sulfated tetrasaccharide, II. (a) Representative structures of C24H30N2O38S6Na62− ion. (b) overlay of the five lowest energy structures (c) Crystal structure of II in complex37 with bFGF. The structures are oriented as shown in Fig. 1. |
An example of the structures of the tetrasaccharide ions is given in Fig. 6, which shows that obtained for the fully sulfated tetrasaccharide, II.
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Fig. 7 Φ,Ψ energy maps calculated as detailed in the Material and Methods section for disaccharide segments of oligosaccharides I–IV. The following conformations of individual rings were considered: DC: 1H2-4C1; CB and BA: 4C1-4C1. Ten isoenergy contours are drawn by interpolation of 1 kcal mol−1 above the global minimum. The conformation found for disaccharide ions are marked by + (monomer) and * (dimer and trimer). Conformations found for tetrasaccharide ions II, III and IV are labelled using ▲, ● and ■, respectively. The dihedral angles found in the solution structures of the tetrasaccharide II are marked by x and s, respectively. (a) CD disaccharide segments. The energy minima labelled as 1 and 2 are separated by 4 kcal mol−1. (b) Rings C and B in tetrasaccharide ions II, III. The energy minima 1 and 2 are separated by 3 kcal mol−1. (c) Rings C and B in the tetrasaccharide ion IV. (d) Rings B and A in tetrasaccharide ion II. The energy minima marked as 1, 2 and 3 are separated by 1 kcal mol−1. (e) Rings B and A in tetrasaccharide ion III. The energy minima 1 and 2, and 1 and 3 are separated by 1 and 3 kcal mol−1, respectively. (f) Rings B and A in tetrasaccharide ion IV. |
The overlay of the lowest energy structures of the tetrasaccharide ions showed some heterogeneity in the conformation of individual monosaccharide rings. In particular, both 1H2 and 2H1 forms were present in ring D. Ring A also showed, in addition to a 4C1 conformation, both boat and skew boat forms. The IdoA (ring B) consistently occupied a 4C1 conformation, although the solution structure of II indicates the prevalence of the 2So form.35 Calculations performed on this tetrasaccharide with the Born Solvent model36 resulted in interconversion between the usual 1C4 and 2So forms for the IdoA ring. This indicates that the 4C1 is specific to the gas phase rather than the AMBER force-field we have used. For a monosaccharide our IM-MS instrumentation will not distinguish between the three forms of IdoA monosaccharide, as their theoretical cross sections differ by <2% (data not shown). Nevertheless, in the context of a larger molecule different conformations of IdoA monosaccharide will influence the overall shape of the molecule and therefore be indirectly traceable via molecular modelling.
A notable feature of the lowest energy structures of the tetrasaccharide ions is their “compact” shape (Fig. 6b), which differs from the more extended conformation observed in the X-ray structure35 of II complexed with basic fibroblast growth factor (bFGF) (Fig. 6c). The theoretical cross section of the X-ray structure (231.8 Å2) is substantially larger than that of the corresponding low energy structures in the gas phase (204.1 Å2). Clearly, the value obtained from our calculated structures is much closer to the experimental value of 198 Å2. Even better agreement between the theoretical and experimental cross sections was observed for tetrasaccharide ions III and IV (Table 2), indicating that our model structures are a true representation of their gas phase conformation. The reasons why the gas-phase conformations are more compact are explained in the discussion.
The overall shape of an oligosaccharide molecule is, to a large extent, determined by the conformation of the glycosidic linkage. In the following we therefore discuss the conformations of the glycosidic linkages of the gas phase oligosaccharide ions I–IV. We also turn our attention to Na+ ions and their effect on the gas phase conformation of heparin-derived oligosaccharide ions.
For these calculations we have used conformations of individual rings corresponding to the most populated species found in our modelled gas phase structures in the presence of Na+.
Starting from the non-reducing end of the molecule we first examine the DC disaccharide segment. The dihedral angles observed for the monomeric disaccharide ion [C12H15NO19S3Na3]− deviate up to +30° on both angles from the deepest minimum of the Φ, Ψ map. Deviations in the opposite direction, and of up to −45o in the ΨDC angle, were observed for dimeric and trimeric disaccharide ions. The largest differences were, however, observed for the DC linkages in the tetrasaccharide ions II and III, corresponding to the existence of anti conformers (symbols ▲ and ● in Fig. 7a). This region of the Φ, Ψ map was not particularly energetically favoured in the absence of the sodium, but presumably became stabilized by the Na+ cations interacting with both D and A rings. The corresponding dihedral angles observed in the X-ray and solution structures were close to the minimum of the Φ, Ψ map.
The next two disaccharide segments can only be examined in the context of the tetrasaccharides. Considering tetrasaccharide ions II and III first, we note that the CB segments are structurally identical in these two compounds and that the dihedral angles of the tetrasaccharide ion II are close to the minimum of the Φ, Ψ map (Fig. 7b). However, in compound III the ΦCB angle differs by 45°. It is possible that this difference is a consequence of a different conformation found in the BA disaccharide segment of ions II and III (see Table 4 and Fig. 7d,e), and it is therefore not possible to attribute this difference solely to effects of the Na+ ions. On the other hand, a large difference in the dihedral angle ΦCB (80°) was observed between the sodium-free disaccharide segment and the gas phase conformation of tetrasaccharide ion IV (Fig. 7c). The latter clearly occupies a high energy region of the Φ, Ψ map.
Because of the differences in the primary sequence of tetrasaccharides II–IV, three separate cases must be considered when analysing the BA disaccharide segment. In compound II the gas phase ion structure is not found in the global minimum (Φ, Ψ) = (180°, 10°), but occupies a secondary minimum (+2 kcal mol−1) with coordinates (26, −79). This conformation also differs by ∼105° in the ΨAB angle from the X-ray and solution structures. Ions III and IV on the other hand show conformations coinciding with the global minima of the sodium-free disaccharide segments (Fig. 7e,f).
In summary, the analysis presented above showed that considerable differences exist between the structures calculated in the presence or absence of Na+. It appears that sodium atoms play an important role in stabilising the gas phase conformation of ions, leading to the appearance of “compact” structures of tetrasaccharide ions. In these structures rings A and D are brought close to each other and bridged by sodium cations. In order to accommodate such conformations, some glycosidic linkages adopt conformations that differ from those calculated for the sodium free disaccharide segments, or those observed in the solution or X-ray structures of tetrasaccharide II.
Let us now take a closer look at the conformations of the gas phase structures of the three tetrasaccharides II–IV as described by the dihedral angles along individual glycosidic linkages (Table 4). It can be seen that a small change in the primary structure can lead to different conformations. For example, tetrasaccharides II and III, which differ only in one sulfate at position A6, show markedly different conformations along the CB and BA glycosidic linkages. Similarly, GlcA/IdoA isomerism (compounds II and IV) resulted in very different conformations. This is an interesting result showing that, at least in the gas phase, differences in the primary structures of HS/heparin sequences can lead to different conformations. It is also possible that the conformations revealed by IM-MS exist transiently in solution generating short-lived “kinks and turns” in otherwise regular, helical structures of HS/heparin.9
Na+ ions interact differently with the negatively charged groups of glycosaminoglycans in solution and in the gas phase. In solution the hydrated sodium counterions are delocalized in the volume around the polyanion,40 while in the gas phase the Na+ cations are localized near several negatively charged groups as seen in this study. This effect will lessen the Coulombic repulsion that isolated sulfate groups would impart on each other, and has the effect of ‘tightening’ the structures. This explains the lower cross section obtained in the gas-phase compared with that from X-ray data. By using the ionization conditions as described by Zai and Costello41 it may be possible to obtain Na+-free ions of heparin-derived oligosaccharides for ion-mobility studies that would bear a closer resemblance to conditions seen in solution. In particular, this may be possible for low sulfated HS species which so far have received much less attention compared with highly sulfated domains. Although the low sulfated species are unlikely to be involved in protein recognition, they may well be the source of flexibility in HS/heparin polymers and this could be probed by MS-IM. A significant advantage of IM-MS over other biophysical methods applicable to conformational studies of biomolecules is that the ion-mobility experiments can be performed at near liquid nitrogen temperatures. Under these conditions different conformers can potentially be separated providing they exhibit distinct cross sectional areas. Other methods, such as NMR, can only report on the time-averaged structures.
Footnote |
† Electronic supplementary information (ESI) available: Preparation of heparin-derived oligosaccharides. Colour images of Figures 4-6 are available in the HTML version. See http://dx.doi.org/10.1039/b508644b |
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