Anal. Chem. 2002, 74, 734-740
“Internal Residue Loss”: Rearrangements Occurring during the Fragmentation of Carbohydrates Derivatized at the Reducing Terminus David J. Harvey,* Taj S. Mattu,† Mark R. Wormald, Louise Royle, Raymond A. Dwek, and Pauline M. Rudd
Oxford Glycobiology Institute, Department of Biochemistry, South Parks Road, Oxford, OX1 3QU, U.K.
Rearrangement reactions involving migration of fucose and, occasionally, other residues have been found in the CID spectra of [M + H]+ and [M + 2H]2+ ions, but not [M + Na]+ ions, generated from several O-linked carbohydrates and milk sugars derivatized at their reducing termini with aromatic amines such as 2-aminobenzamide. Such rearrangements, which are similar to those reported by other investigators from several underivatized carbohydrates and glycosides, cause an apparent loss of sugar residues from within a carbohydrate chain and can produce ambiguous results during spectral interpretation. A mechanism, involving initial protonation of the amine nitrogen atom of the derivative, is proposed to account for the formation of the observed ions. Carbohydrates show two main types of fragment ions, glycosidic fragments that are formed by cleavage between the sugar rings and those formed by cleavage across the sugar rings.1 The former ions usually dominate the spectra and provide information on sugar sequence and branching, whereas the latter ions, which are generally much more abundant in the spectra of [M + Na]+ than the spectra of [M + H]+ ions,2 give additional linkage information. Frequently, fragment ions are formed by the loss of sugar residues from two or more sites producing what are known as “internal fragments”. These latter ions can often arise from several sites; therefore, their utility for structural determination is limited. Recently, it has become evident that certain carbohydrates can also undergo rearrangement reactions with losses of residues from inside the parent molecule rather than from the ends of the sugar chains.3-5 These reactions, which are the products of “internal residue loss”, should not be confused with the internal fragments * Corresponding author: (tel) (44) 1865 275750; (fax) (44) 1865 275216; (email)
[email protected]. † Current address: Jefferson Centre, Thomas Jefferson University, 700 E. Butler Ave., Doylestown, PA 18901. (1) Reinhold: V. N.; Reinhold: B. B.; Costello, C. E. Anal. Chem. 1995, 67, 1772-1784. (2) Orlando, R.; Bush, C. A.; Fenselau, C. Biomed. Environ. Mass Spectrom. 1990, 19, 747-754. (3) Kova´cik, V.; Hirsch, J.; Kova´c, P.; Heerma, W.; Thomas-Oates, J.; Haverkamp, J. J. Mass Spectrom. 1995, 30, 949-958. (4) Bru ¨ ll, L. P.; Heerma, W.; Thomas-Oates, J.; Haverkamp, J.; Kova´cik, V.; Kova´c, P. J. Am. Soc. Mass Spectrom. 1997, 8, 43-49.
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mentioned above whose formation involves loss of two or more terminal residues. Internal residue losses have also been observed in the spectra of flavonoids6,7 and anthracyclines8 containing a disaccharide moiety where the “internal” saccharide ring directly attached to the aglycon was eliminated. The reaction does not appear to be linkage-specific or to be confined to the elimination of monosaccharide residues. Ernst et al.,5 for example, reported the elimination of the disaccharide Galβ1 f 4GlcNAc from Lewisa and Lewisx tetrasaccharides with bond formation between the fucose and sialic acid residues. These internal residue losses have been observed mainly from the [M + H]+ ions generated from small oligosaccharides and do not appear to occur from carbohydrates ionized by sodium addition.9 They have, however, been reported from [M + NH4]+ ions.5 Such ions are problematical because they give misleading sequence information or suggest that a sample is impure. We recently noted the presence of such ions in the spectra of derivatized (reducing terminal) O-linked glycans10 and report here on ions that are apparently formed by internal migrations of fucose and, to a lesser extent, by hexose residues in the low-energy CID spectra of [M + H]+ ions from carbohydrates derivatized at the reducing terminal with several aromatic amines. The structures of the compounds that were examined are shown in Figure 1. EXPERIMENTAL SECTION Materials. The milk sugars, 2′-FL, LNT, LNFPI, LNFPII, LNFPIII, LNDFHI, Lewis-A, and Lewis-X, were obtained from Oxford GlycoSciences Ltd (Abingdon, U.K.). 2-Aminobenzamide (2-AB), 3-aminoquinoline (3-AQ), and butyl-4-aminobenzoate (ABBE) were from Aldrich Chemical Co. Ltd. (Poole, Dorset, U.K.). All other reagents were from Aldrich. (5) Ernst, B.; Muller, D. R.; Richter, W. J. Int. J. Mass Spectrom. Ion Processes 1997, 160, 283-290. (6) Li, Q. M.; Claeys, M. Biol. Mass Spectrom. 1994, 23, 406-416. (7) Ma, Y.-L.; Vedernikova, I.; Van den Heuvel, H.; Claeys, M. J. Am. Soc. Mass Spectrom. 2000, 11, 136-144. (8) Warrack, B. M.; Hail, M. E.; Triolo, A.; Animati, F.; Seraglia, R.; Traldi, P. J. Am. Soc. Mass Spectrom. 1998, 9, 710-715. (9) Bru ¨ ll, L. P.; Kova´cik, V.; Thomas-Oates, J. E.; Heerma, W.; Haverkamp, J. Rapid Commun. Mass Spectrom. 1998, 12, 1520-1532. (10) Mattu, T. S.; Royle, L.; Langridge, J.; Wormald, M. R.; Van den Steen, P. E.; Van Damme, J.; Opdenakker, G.; Harvey, D. J.; Dwek, R. A.; Rudd, P. M. Biochemistry 2000, 39, 15695-15704. 10.1021/ac0109321 CCC: $22.00
© 2002 American Chemical Society Published on Web 01/09/2002
Figure 1. Structures of the compounds examined in this paper. Key to symbols: (0) glucose, (]) galactose, (diamond with dot) fucose, (9) GlcNAc, ([) GalNAc, (f) sialic acid. Linkages are represented by the angles of the bonds between monosaccharide residues (| ) 2, / ) 3, and - ) 4).
Preparation of Reducing-Terminal Derivatives. These were prepared essentially as described earlier.11,12 Briefly, the carbohydrate was dissolved in a mixture of dry dimethyl sulfoxide (DMSO) (6 µL) and acetic acid (2 µL) and an excess of 2-AB, 3-AQ, or ABBE was added. The solution was heated briefly at 65 °C, cooled, and an excess of sodium cyanoborohydride was added. The mixture was then heated at 65 °C for 2 h, cooled, and spotted onto a strip of Whatman 3MM chromatography paper. When all of the solvent had evaporated, the excess of reagent was removed by ascending paper chromatography in acetonitrile. After being dried, the area of paper containing the original spot was cut out and the derivatized sugars were eluted with water. The glycans were extracted with a C-18 ZipTip and reconstituted in a mixture of methanol/water (1:1 by volume). Electrospray Mass Spectrometry. Electrospray mass spectra were recorded with a Micromass Q-Tof mass spectrometer (Micromass (UK) Ltd., Wythenshawe, Manchester, U.K.). The electrospray needle voltage was maintained at 3 kV, the source temperature was 100 °C, and the cone voltage was set at 60 V for recording the spectra of the [M + H]+ ions and 200 V for the [M + Na]+ ions. Argon was used as the collision gas, and the collision cell voltage was adjusted to give an even spread of fragment ions over the mass range (range 15-40 V depending on the mass). The parent ion selection window was set to ∼3 mass units. Samples were dissolved in methanol/water (1:1 by volume) containing 0.1% formic acid to a concentration of 50 pmol/µL and infused with the nanoflow probe at 200 nL/min. Electrospray MSn spectra were acquired with a Thermo Finnigan LCQ ion trap mass (11) Bigge, J. C.; Patel, T. P.; Bruce, J. A.; Goulding, P. N.; Charles, S. M.; Parekh, R. B. Anal. Biochem. 1995, 230, 229-238. (12) Harvey, D. J. J. Am. Soc. Mass Spectrom. 2000, 11, 900-915.
spectrometer. Samples (∼50 pmol/µL) were prepared as above and infused with a capillary needle. Molecular Modeling. Molecular modeling was performed on a Silicon Graphics Indigo 2 workstation using InsightII and Discover software (MSI Inc.). Figures were produced using the program Molscript.13 Low-energy structures were built within the InsightII program using typical torsion angles for the glycosidic linkages14 and extended conformations for the Glc-ol and 2-AB residues, followed by energy minimization using the Discover package. Conformations with the 2-AB amine nitrogen close to the fucose ring oxygen were obtained by altering torsion angles within the molecules, followed by energy minimization. RESULTS Ions Formed by Fucose Migration in the Spectra of Milk Sugars. Figure 2a shows the positive ion MS/MS spectrum of the [M + Na]+ ion from the 2-AB derivative of the linear trisaccharide, 2′-FL (1). The only two significant fragment ions arose from successive eliminations of fucose (m/z 485.2) and galactose (m/z 323.1). Fragments are annotated according to the scheme proposed by Domon and Costello.15 The corresponding spectrum of the [M + H]+ ion (Figure 2b) showed an additional ion at m/z 447.2 representing loss of 162 mass units corresponding to loss of a hexose (galactose) residue from within the carbohydrate chain and implying a fucose migration to the derivatized glucose residue. Ions appearing at lower mass than the [M - Fuc - Gal + H]+ ion at m/z 301.1 originated from the 2-AB derivative (13) Kraulis, P. J. J. Appl. Crystallogr. 1991, 24, 945-949. (14) Petrescu, A. J.; Petrescu, S. M.; Dwek, R. A.; Wormald, M. R. Glycobiology 1999, 9, 343-352. (15) Domon, B.; Costello, C. E. Glycoconjugate J. 1988, 5, 397-409.
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Figure 2. Positive ion MS/MS spectra of (a) the [M + Na]+ ion and (b) the [M + H]+ ion from the 2-AB derivative of the linear trisaccharide, 2′-FL (1).
Figure 3. Relative ion abundance plotted against collision cell voltage for the molecular (m/z 609.1) and major fragment ions in the MS/MS spectrum of the 2-AB derivative of 2′-FL (1).
(see below). The ion involving the fucose migration also appeared at the corresponding mass in the MS/MS spectrum of the [M + H]+ ion from the 3-AQ derivative (data not shown), although to a lesser extent (9% of the relative abundance of the [M - fucose]+ ion compared with 22% in the spectrum of the 2-AB derivative), confirming that it contained the derivatized terminal glucose residue and the fucose. The extent of formation of the fucose rearrangement ion showed the same dependence on the collision cell voltage as the ion formed by direct loss of the fucose residue (m/z 463.1) (Figure 3), suggesting that similar mechanisms were involved. The positive ion MS/MS spectra, acquired on the Q-TOF instrument, of the 2-AB, 3-AQ, or ABBE derivatives of [M + H]+ ions from three isomers of the monofucosylated tetrasaccharides LNFPI, -II, and -III (2-4) and the difucosylated compound LNDFHI (5), all of which contained fucose on one or both of the two nonreducing-terminal residues, also gave fragment ions that could not be explained by simple glycosidic or cross-ring cleavages. Figure 4 shows the MS/MS spectra of the [M + H]+ ions from the monofucosylated glycans 2-4. Fragments are rational736 Analytical Chemistry, Vol. 74, No. 4, February 15, 2002
ized according to the scheme shown in the figure. Peaks marked with an asterisk did not have analogous peaks in the CID spectra of the corresponding [M + Na]+ ions and appeared not to be the products of simple cleavages. The ions at m/z 609.2 and 447.2 in the spectra of the 2-AB derivatives (Figure 4) contained the reducing-terminal derivative (as shown by their mass shifts in the spectra of the corresponding 3-AQ derivatives) together with fucose and either two (m/z 609.2) or one (m/z 447.2) hexose residue. As no fucose was attached to the two hexoses at the reducing terminal of the parent compound, it must have migrated from the nonreducing terminal residues. Selection and further fragmentation in the ion trap instrument of the ion at m/z 609.2, as a fragment of the molecular ion, gave both m/z 463.2 (loss of fucose) and 447.2 (loss of hexose), but the spectrum did not define the location of the fucose in this ion as a further rearrangement might have occurred during the elimination of the hexose. Finally, both of these ions, when selected independently, gave m/z 301.1 (Glc-2-AB). Corresponding rearrangement ions, involving migration of fucose to the derivatized end of the molecule, were seen in the MS/MS spectrum of the difucosylated glycan (5). An additional ion was present at m/z 812.4 in the spectrum of the 2-AB derivative of LNFPII (3) (Figure 4b) corresponding to loss of a hexose residue. Again, this ion must have been formed from a rearrangement reaction. The MS/MS spectrum of the [M + H]+ ion from the 2-AB derivative of LNFPII (3) (Figure 4b), which contained galactose at the 3-position of the branching GlcNAc residue, showed, in addition to the ion at m/z 609.3, formed by fucose migration, an additional ion at m/z 625.3 which appeared to have a composition of [(Hex)3-2-AB + H]+ indicating the occurrence of a competing reaction involving migration of the reducing-terminal hexose residue and expulsion of GlcNAc-Fuc from the center of the molecule. The relative abundance of the ion formed by fucose migration (m/z 609.3) was also lower than in the spectra of the other compounds, consistent with the occurrence of a competing reaction. Thus, of the two monosaccharides linked to the GlcNAc
Figure 4. MS/MS spectra of the [M + H]+ ions from the 2-AB derivatives of the monofucosylated glycans 2-4 (a-c, respectively).
Figure 5. MS/MS spectrum of the [M + H]+ ion from the 2-AB derivatives of an O-linked glycan 7 from secretory immunoglobulin A.
residue of isomeric LNFPII-2-AB (3) and LNFPIII-2-AB (4), fucose appeared to migrate in preference to galactose but the group (either fucose or galactose) linked at the 3-position was more labile. Galactose was also observed to migrate to a limited extent in the CID spectrum of the 2-AB derivative of LNT (6) where it was 3-linked. Ions Formed by Fucose Migration in the Spectra of O-Linked Glycans. Ions formed by fucose migration were also found in the spectra of O-linked glycans ionized as their [M + H]+ or [M + 2H]2+ ions when examined by LC/MS using ammonium formate as the buffer. Fragmentation of the smaller O-linked glycans produced mainly singly charged ions whereas the larger glycans produced almost exclusively doubly charged ions. Figure 5 shows the CID spectrum of the [M + H]+ ion from an O-linked glycan (7) from secretory immunoglobulin A as its 2-AB derivative. All major fragments were products of B- and particularly Y-type glycosidic cleavages. Two ions appeared to be
rearrangement ions produced by migration of the fucose residue. The ion at m/z 650.3 had a composition indicating loss of HexHexNAc that could only have arisen by rearrangement. Similarly, the ion at m/z 488.2 appeared to have the composition FucHexNAc-2-AB. In the spectrum of the doubly charged ion from the larger O-linked glycan (8) (Figure 6), three ions appeared to be rearrangement products. The ion at m/z 650.3 had a composition of (Hex)1(HexNAc)1(Fuc)1-2-AB, as found above, and the ion at m/z 941.5 had the same composition with the addition of sialic acid. Finally, the weak ion at m/z 1468.8, could only have been formed by elimination of a protonated HexNAc residue, again implying a rearrangement. Full details of the structural elucidation of the O-glycans from secretory immunoglobulin A will be published later. Ions Formed by Fragmentation of the Derivative. The spectra of the 2-AB derivatives were more complex than those of their 3-AQ analogues and showed evidence of elimination reactions Analytical Chemistry, Vol. 74, No. 4, February 15, 2002
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Figure 6. MS/MS spectrum of the doubly charged ion from the 2-AB derivative of the O-linked glycan 8 from secretory immunoglobulin A.
Figure 7. Mechanism for fucose migration for the 2-AB derivative 2′-FL according to the mechanism proposed by Ma et al.7
involving the derivative. Thus, the ion at m/z 301.1 (Figure 2b), corresponding to [Glc-2-AB + H]+, showed a prominent loss of ammonia to give m/z 284.0 and two further losses of water to give m/z 266.0 and 248.0. Confirmation of the composition of these ions was obtained from the corresponding spectra of the 3-AQ derivatives of Lewisx, where they were present as very minor peaks with the appropriate mass shifts. An MS3 experiment, in which the ion at m/z 301.1 was selected and fragmented, confirmed the losses of ammonia and water. In the spectra of the 2-AB derivatives, ammonia was also seen to be lost from the molecular and [M - Fuc + H]+ ion to a minor extent. 738
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DISCUSSION Several mechanisms have been proposed to account for rearrangement ions of this type in the spectra of carbohydrates,3-5,7 but none appear to be totally satisfactory for explaining the occurrence of migrations in the spectra of the 2-AB derivatives. Both charge-remote3 and charge-induced7 mechanisms have been proposed, but the fact that these rearrangements are not seen in the spectra of sodium-adducted ions suggests that the chargeinduced mechanism catalyzed by protonation of one of the oxygen atoms is the more likely. Ma et al.7 proposed protonation of one of the oxygen atoms within a sugar ring (Figure 7, structure b)
Figure 8. Proposed mechanism for fucose migration during the fragmentation of 2-AB-derivatized 2′-FL.
and cleavage of the adjacent C-O bond to give a carbonium ion at C-1 of the attacked ring. This is an attractive mechanism because a new bond could then form between this carbonium ion and another oxygen atom (Figure 7, structure c). If this oxygen atom was the one linking two sugar residues, one of these residues could be eliminated to account for the internal residue loss (Figure 7, structure d). This, essentially, is the mechanism proposed by Kova´cik et al.3 and Ma et al.7 to account for migration of a residue from the 2-position of the central residue of a trisaccharide. In both cases, the attacked glycosidic bond and migrating residue were attached to the same sugar, as in the case of 2′-FL-2-AB, above. However, molecular models show that the separation between the C-1 atom of the migrating residue and the oxygen of the glycosidic bond is not ideal for bonding in this semirigid system and that such a mechanism may be an oversimplification. In the case of the derivatives of LNFPIII (4), where the fucose migrates from the 3-position of the GlcNAc residue, the system is too rigid to allow this situation to occur and only oxygen atoms located toward the derivatized end of the reduced glucose residue can approach to within bonding distance. The situation is even worse for the migration observed to occur from the 4-position of the GlcNAc residue in the derivatives of LNFPII (3).
Ma et al., in their mechanism, proposed that the initial protonation occurred, not at a sugar residue but on the flavonone residue and that this proton was later transferred to a sugar ring. It would appear that a similar mechanism is probably occurring in the derivatized carbohydrates reported in this paper. One would expect that the initial protonation accompanying ionization would occur at the basic nitrogen linking the derivative to the carbohydrate (Figure 8, structure f). This proposal is supported by the appearance of much more abundant protonated ions in the spectra of these derivatives than in those of the underivatized carbohydrates and the observation that the relative abundance of the rearrangement ions is a function of the basicity of the aromatic amine. Molecular models (Figure 9) confirm that this proton can then approach the oxygen atom of the fucose ring with only small conformational changes from the lowest energy state. For FL-2AB and the O-linked glycan 8, this only involved changing torsion angles within the Glc-ol and GalNAc-ol residues, respectively, and the 2-AB residue from the linear conformation having the lowest energy. For LNFPI-2-AB, LNFPII-2-AB, and LNFPIII-2-AB, it was also necessary to change significantly the GlcNAc-Gal linkage torsion angles but not any of the other glycosidic linkages. For FL-2-AB, LNFPI-2-AB, and the O-linked glycan 8, there are many conformations that bring the 2-AB amine nitrogen very close to Analytical Chemistry, Vol. 74, No. 4, February 15, 2002
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Once the atoms are in close proximity, transfer of the proton from the nitrogen atom to the ring oxygen atom (Figure 8, structure g), followed by an electron shift would produce a carbonium ion at C-1 of the fucose residue, which could then form a bond to the nitrogen (Figure 8, structure h), preserving its quaternary nature and completing the fucose migration. “Internal residue loss” could then occur by the normal mechanism of charge-remote glycosidic cleavages (Figure 8, structures i and j). The mechanism is particularly attractive because there would be expected to be a hydrophobic attraction between the aromatic ring of the 2-AB derivative and the hydrophobic face or methyl group of the fucose residue that would hold the reacting atoms in a suitable orientation for the reaction to occur and promote the adoption of a “bent” conformation, as in Figure 9, in the first place. In addition, it was observed that the most abundant rearrangement ions occur by bond cleavage adjacent to HexNAc residues, analogous to the situation in fragmentations not preceded by rearrangement. Furthermore, formation of the subsequent glycosidic cleavage ions required the same collision energy as those fragments formed directly by glycosidic cleavage without prior rearrangement.
Figure 9. Molecular models of (a) FL-2-AB (1), (b) LNFPI-2-AB (2), (c) LNFPII-2-AB (3), (d) LNFPIII-2-AB (4), and (e) O-linked glycan2-AB (8) in conformations that bring the amine nitrogen of 2-AB close to the fucose ring (see text for details). The fucose residues are shown in light gray and the 2-AB amine nitrogen and the fucose ring oxygen atom are shown as larger spheres in dark gray. Hydrogen atoms have been removed for clarity.
the fucose ring oxygen, but for LNFPII-2-AB and LNFPIII-2-AB, there are fewer conformations that bring these two atoms close together. Many more conformations that bring these two atoms into close proximity are possible if one invokes the involvement of higher energy states such as boat conformations for one or more sugar rings. There is precedence for the formation of boat conformations from the mass spectra of derivatized steroids produced by electron impact where some rearrangements could only be explained on the assumption that chair:boat isomerism occurred.16 (16) Sloan, S.; Harvey, D. J.; Vouros, P. Org. Mass Spectrom. 1971, 789-799.
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CONCLUSIONS The [M + H]+ ions of several derivatized carbohydrates appear to undergo rearrangement reactions with migration of fucose and, occasionally, other residues, toward the derivatized end of the molecule. The reaction was also seen in the spectra of doubly charged ions. A mechanism involving migration of fucose to the derivative itself appears to account for the rearrangements. The occurrence of these rearrangement ions in the MS/MS spectra of [M + H]+ ions have important implications for the use of these ions to determine the structures of unknown molecules as their presence can lead to the prediction of erroneous structures or to the conclusion that more than one isomeric compound is present. Use of the [M + Na]+ or [M + other metal]+ ion avoids this problem and also gives spectra in which the relative abundance of cross-ring cleavage ions is often enhanced. ACKNOWLEDGMENT We thank the Biotechnology and Biological Sciences Research Council for funding the mass spectrometer. We also thank Julian Phillips and Stan Evans of Thermo Finnigan for access to the ion trap mass spectrometer. Received for review August 21, 2001. Accepted September 28, 2001. AC0109321
