Differentiation of Diastereomeric N

Anal. Chem. 1999, 71, 1997-2002

Differentiation of Diastereomeric N-Acetylhexosamine Monosaccharides Using Ion Trap Tandem Mass Spectrometry Heather Desaire and Julie A. Leary*

College of Chemistry, University of California, Berkeley, California 94720-1460

A quadrupole ion trap mass spectrometer equipped with electrospray ionization was used to distinguish three diastereomeric monosaccharides, N-acetylglucosamine, N-acetylgalactosamine, and N-acetylmannosamine. The saccharides were derivatized to form the metal complex [CoIII(DAP)2HexNAc]Cl3 which, when collisionally activated, produced dramatically different product ion spectra. The product ion spectra generated for the three monosaccharide diastereomers were then used to confirm the stereochemistry of N-acetylhexosamines from a hydrolyzed oligosaccharide. Finally, the origin of each product ion was determined through isotopic labeling studies, and mechanisms were proposed which explain each resulting dissociation. Carbohydrate chemistry is explicitly involved in a host of biological functions, including cell-cell recognition, cell-cell interaction, and molecular targeting.1 Furthermore, structural elucidation of the carbohydrates involved is the first step to understanding how and why they assist in these functions. There are many aspects to structural elucidation of carbohydrates. The stereochemistry of the monosaccharides must be identified, the sequence of these monosaccharides in the oligosaccharide must be determined, and the linkage and anomericity between each monosaccharide must be determined. To obtain this information, researchers have used methods such as NMR spectroscopy, periodate oxidation, and enzymatic reduction.2 Unfortunately, these techniques are time and labor intensive; they require consumption of large quantities of material, and their results are often difficult to interpret. Because mass spectrometry requires very limited sample consumption, and very short analysis times, many researchers interested in oligosaccharide structural determination have begun to use MS as their preferred analytical technique.3 For example, mass spectrometry and tandem mass spectrometry (MSn) have been used to determine linkage type and sequence information for many oligosaccharides.4-14 Recently, Gaucher and Leary have * Corresponding author: (phone) (510) 643-6499; (fax) (510) 642-9295; (email) [email protected]. (1) Stryer, L. Biochemistry; W. H. Freeman: New York, 1988; pp 331-348. (2) Chaplin, M. F., Kennedy, J. F., Eds. Carbohydrate Analysis; Oxford: New York, 1994. (3) Geyer, H.; Geyer, R. Acta Anat. 1998, 161, 18. (4) Egge, H.; Peter-Katalinic, J. Mass Spectrom. Rev. 1988, 6, 331. (5) Zhou, Z.; Ogden, S.; Leary, J. A. J. Org. Chem. Soc. 1990, 55, 5444. (6) Hofmeister, G. E.; Zhou, Z.; Leary, J. A. J. Am. Chem. Soc. 1991, 113, 5964. (7) Staempfli, A.; Zhou, Z.; Leary, J. A. J. Org. Chem. 1992, 57, 3590. 10.1021/ac981052y CCC: $18.00 Published on Web 04/13/1999

© 1999 American Chemical Society

shown that MSn can also be used for determination of the anomericity of the glycosidic bonds.15 One key shortcoming of MSn techniques has been the inability to determine the stereochemistry of the monosaccharide units.3 Gaucher and Leary have also demonstrated that the stereochemistry of the hexose monosaccharides can be determined using MSn by first derivatizing the monosaccharides with Zn(diethylenetriamine).16 In data presented here, a system used to differentiate the N-acetylhexosamine diastereomers is revealed. Furthermore, we will show that this technique is readily applicable to oligosaccharide samples. We have also used 2H- and 18O-labeling studies to assist in identifying the origin of the neutral losses observed, and we are developing mechanistic rationales explaining why these dissociations are stereoselective. Finally, we will show in a forthcoming publication that this methodology can be used to quantitatively determine the stereochemistry of N-acetylhexosamines in an oligosaccharide where both N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) diastereomers are present. EXPERIMENTAL SECTION General Synthesis of N-Acetylhexosamine Complexes. CoCl2‚6H2O, 375 µg (1.57 µmol), 456 µg of diaminopropane (HCl salt) (3.14 µmol), 345 µg of the monosaccharide (1.57 µmol), and 1.1 µL of triethylamine (7.5 µmol) were dissolved in 70 µL of H2O and 75 µL of MeOH. The reaction mixture was then heated at 60 °C for 15 min. Isotopically Labeled Products. (a) 18O Labeling. The saccharide (345 µg, 1.57 µmol) was heated in H218O (70 µL) for 45 min at 60 °C. The other reagents were added (as described above) and the solution was heated for an additional 15 min at 60 °C. (b) 2H Labeling. CoCl2‚6H2O, 375 µg (1.57 µmol), 456 µg of diaminopropane (HCl salt) (3.14 µmol), 345 µg of the monosaccharide (1.57 µmol), and 1.1 µL of triethylamine (7.5 µmol) were (8) Garozzo, D.; Impallomeni, G.; Montaudo, G.; Spina, E. Rapid Commun. Mass Spectrom. 1992, 6, 550. (9) Garozzo, D.; Giuffrida, M.; Impallomeni, G.; Ballistreri, A.; Montaudo, G. Anal. Chem. 1993, 62, 279. (10) Fura, A.; Leary, J. A. Anal. Chem. 1993, 65, 2805. (11) Hayes, G. R.; Williams, A.; Costello, C. E.; Enns, C. A.; et al. Glycobiology 1995, 5, 227. (12) Reinhold: V.; Reinhold: B. B.; Costello, C. E. Anal. Chem. 1995, 67, 1772. (13) Cancilla, M. T.; Penn, S. G.; Carroll, J. A.; Lebrilla, C. B. J. Am. Chem. Soc. 1996, 118, 6736. (14) Sible, E. M.; Brimmer, S. P.; Leary, J. A. J. Am. Soc. Mass Spectrom. 1997, 8, 32. (15) Gaucher, S. P.; Leary, J. A. J. Am. Soc. Mass Spectrom 1999, 10, 269. (16) Gaucher, S. P.; Leary, J. A. Anal. Chem. 1998, 70, 3009.

Analytical Chemistry, Vol. 71, No. 10, May 15, 1999 1997

Figure 1. Isomeric monosaccharides.

dissolved in 70 µL of 2H2O and 75 µL of MeO2H. The reaction mixture was then heated at 60 °C for 15 min. The synthetic procedure was altered slightly for the hydrolyzed oligosaccharide in order to reduce sample consumption. The complex [CoCl2(DAP)2]Cl was prepared by standard methods.17 Methanolic solutions of [CoCl2(DAP)2]Cl (0.02 M), N(CH2CH3)3 (0.02 M), and an aqueous solution of the monosaccharide (0.02 M) were prepared. Then 5 µL of each solution plus 5 µL of MeOH were combined and heated at 60 °C for 25 min. Total consumption of monosaccharide is 100 nmol. The GlcNAc, GalNAc, and N-acetylmannosamine (ManNAc) monosaccharides were reanalyzed with this synthetic procedure, and the resulting mass spectra were identical to those obtained from the previous synthetic route. All samples were run on a quadrupole ion trap mass spectrometer fitted with an electrospray ionization source (Finnigan LCQ purchased from Finnigan-Mat, San Jose, CA). A portion of the reaction solution was diluted with HPLC grade methanol in order to achieve a final solution concentration of 50 pmol/µL. (For 2H labeling experiments, CH O2H was used to dilute the samples.) 3 All samples were injected into the spectrometer via direct infusion from a syringe pump. The flow rate was 5 uL/min. The capillary was heated to 150 °C, and the spray voltage was maintained at 5.2 kV. Optimization of the signal from the ion of interest was completed by using the automatic tune program on the instrument. To avoid space charge effects, the number of ions in the trap was regulated by the automatic gain control, which was set to 5 × 107 ions for MS1 scans. Helium gas was used as the collision and bath gas, and the pressure was maintained at 1 × 10-3 Torr. Each spectrum consisted of 20-25 “individual” scans, where each individual scan was composed of three “microscans”. All scans were acquired in the positive ion mode. The MS/MS conditions were as follows: rf voltage, 0.6 V; isolation width, 5 mass units; and excitation time, 30 ms. The MS3 conditions were as follows: rf voltage, 0.25 V; isolation width, 2.2 mass units; and excitation time, 1 s. Each diastereomeric precursor ion was subjected to identical acquisition parameters. Determination for a presence or absence of product ions is based on a 3% relative abundance criterion, as it was established that ions present below this abundance are not reproducible.18 RESULTS AND DISCUSSION Stereoselective Dissociations. Structural differences among the three biologically relevant N-acetylhexosamines occur at the C2 and C4 positions (Figure 1). GlcNAc has its hydroxyl groups (17) Bailar, J. C.; Work, J. B. J. Am. Chem. Soc. 1946, 68, 232. (18) Koenig, S.; Leary, J. A. J. Am. Soc. Mass Spectrom. 1998, 9, 1125.

1998 Analytical Chemistry, Vol. 71, No. 10, May 15, 1999

Figure 2. MS of a typical reaction mixture. Ion of interest, m/z 426, is [Co(DAP)2(GlcNAc)]+.

Figure 3. Proposed structures of the Co(DAP)(GlcNAc) complexes: m/z 426 and 352.

and N-acetyl group equatorial, while GalNAc has an axial hydroxyl group at C4, and ManNAc has an axial N-acetyl group at C2. Each monomer is reacted with 1 equiv of CoII(Cl2) and 2 equiv of diaminopropane (DAP) to give the air-oxidized complex [CoIII(DAP)2HexNAc]Cl3.19 When the reaction solution is subjected to electrospray ionization, the ion at m/z 426 [CoIII(DAP)2(HexNAc-2H)]+ is detected as the major product of the reaction as shown in Figure 2. We propose that the ion m/z 426 represents the octahedral complex illustrated in Figure 3, which depicts the GlcNAc diastereomer. While this complex has not been fully characterized, the structure is consistent with Bunel’s work, which shows that Co(III)(en)2(hexosamine) is an octahedral complex, with the saccharide coordinated to the metal at C1 and C2.20,21 Furthermore, the structure is consistent with the MSn data presented here and all previously isolated compounds from our laboratory.16,22-24 When this complex is allowed to undergo collision-induced dissociation (CID), the MS2 spectra show that the major product is loss of 74 Da, a DAP ligand (Figure 4). It should also be noted that Co(DAP)2ManNAc displays a neutral loss of 120 Da (C4H8O4) in the MS2 spectrum with a relative abundance of about 20% (Figure 4A). The Co(DAP)2GlcNAc product ion spectrum displays this loss at a relative abundance of about 4% (Figure 4B) and also exhibits a unique loss of 131 Da (C5H9NO3). Co(DAP)2GalNAc loses only the DAP ligand (Figure 4C). These MS2 spectra may (19) Cotton, F. A., Wilkinson, G., Eds. Advanced Inorganic Chemistry; Wiley: New York, 1972; p 878. (20) Bunel, S.; Ibarra, C.; Moraga, E.; Andrei, B.; Bunton, C. A. Carbohydr. Res. 1993, 244, 1. (21) Bunel, S.; Ibarra, C.; Moraga, E.; Calvo, V.; et al. Carbohydr. Res. 1993, 239, 185. (22) Smith, G.; Leary, J. A. J. Am. Chem. Soc. 1996, 118, 3293. (23) Smith, G.; Pedersen, S. F.; Leary, J. A. J. Org. Chem. 1997, 62, 2152. (24) Smith, G.; Leary, J. A. J. Am. Chem. Soc. 1998, 120, 13046.

Table 2. Comparison of Dissociations for the Ion m/z 352a

N-acetylglucosamine N-acetylgalactosamine N-acetylmannosamine hydrolysate a

Figure 4. MS/MS of m/z 426: (A) ManNAc; (B) GlcNAc; (C) GalNAc. Table 1. Comparison of Dissociations for the Ion m/z 426a

N-acetylglucosamine N-acetylgalactosamine N-acetylmannosamine hydrolysate a

-74

-120

-131

++ ++ ++ ++

++ ++ ++

++ ++

++ means the dissociation is observed.

Figure 5. MS3 spectra (m/z 426 f 352 f): (A) ManNAc; (B) GalNAc; (C) GlcNAc.

be used to distinguish the three diastereomers on the basis of the presence or absence of the two ions, m/z 306 and 295. (Table 1). However, due to the limited number of differentiating ions, MSn experiments were pursued. It was anticipated that MSn of m/z 352 might provide additional support for differentiation of the three diastereomers. When MS3 experiments were carried out for m/z 426 f 352 f, additional distinctions were indeed visible among the diastereomers (Figure 5). No product ions are observed for the ManNAc complex under the collision energy provided (Figure 5A). The GalNAc (Figure 5B) and GlcNAc spectra (Figure 5C) each display characteristic ions: GlcNAc predominantly undergoes a loss of 90 Da, with a small 60 Da loss, and GalNAc undergoes a unique 30 Da loss as the major product ion. (Table 2). Thus, the diastereomers can be unambiguously

-30

-60

-90

++ -

++ ++ ++

++ ++ ++

++ means the dissociation is observed.

identified on the basis of the presence and absence of ions found in both the MS2 and MS3 spectra. This methodology is readily applicable to oligosaccharide systems. For example, hexa-N-acetylchitohexaose was subjected to a novel acid hydrolysis procedure which released the GlcNAc monomers (Scheme 1).25 The hydrolyzed sample was then derivatized with [Co(DAP)2Cl2]Cl (Scheme 2). The new derivatization procedure allowed for smaller sample consumption (see Experimental Section). The derivatized products were then analyzed as described above. Tables 1 and 2 show the product ions detected in the MS2 and MS3 experiments. As expected, the MS2 spectrum (m/z 426 f) has product ions at m/z 352, 306, and 295. Each of these ions was also present for the GlcNAc monosaccharide (Table 1). Likewise, the MS3 spectrum (m/z 426 f 352 f) also matches the GlcNAc data, with the major product ion at m/z 262 and a less-abundant ion at m/z 292. Thus, by subjecting the oligosaccharide to hydrolysis, and then derivatizing the monosaccharides with the [Co(DAP)2Cl2]Cl complex, the stereochemistry of the N-acetylhexosamines can be determined. While the example provided here demonstrated a case where only one type of N-acetylhexosamine diastereomer was present, the derivatization method can also be used quantitatively for oligosaccharides where two or more different diastereomeric N-acetylhexosamines are present. (Additional manuscript to be published separately.) Labeling Studies. While the data clearly demonstrate that stereochemical information can be determined by using mass spectrometry, a fundamental understanding of the stereoselective dissociation reactions is necessary if one is to apply this methodology to other biologically relevant diastereomers. Thus, isotopic labeling studies were undertaken in order to determine the origin of the dissociation products. All the monomers were labeled with 18O and 2H in two studies, such that (1) all the exchangeable hydrogens were substituted for 2H and (2) the anomeric hydroxyl oxygen was labeled with 18O. In so doing, one effectively obtains information on the possible loss of C1 upon CID by virtue of the anomeric 18O labeling. Similarly, any loss of C6 would be recognized because it is the only carbon with two nonexchangeable protons. Finally, it was assumed that any dissociation product containing the nitrogen on C2 would result in an odd-mass loss (in accordance with the nitrogen rule). Thus, these two isotopic labeling experiments provided a wealth of information, including effective labels at C1, C2, and C6 (Tables 3 and 4). On the basis of the isotopic labeling studies, the origins of all of the dissociation products were deduced. In the MS2 study, the (25) Cancilla, M. T.; Gaucher, S. P.; Desaire, H. R.; Leary, J. A., in preparation.

Analytical Chemistry, Vol. 71, No. 10, May 15, 1999

1999

Scheme 1

Scheme 2

Table 3. Isotopic Labeling Results. MS2 Data from the Ion at m/z 426 no label

2H

label

18O

label

Table 4. Isotopic Labeling Results. MS3 Data from m/z 426 f 352 f unlabeled

2H

label

18O

label

A. Loss of C4H8O4. Labeling Data for GlcNAc and ManNAc precursor ion 426 437 428 production 306 314 308 mass difference 120 Da 123 Da 120 Da label present in loss 3 2H no 18O a associated carbon lost? not C2 C6 not C1 origin of loss C3-C6

A. Loss of CH2O. Labeling Data for GalNAc precursor ion 352 359 product ion 322 329 mass difference 30 Da 30 Da label present in loss no 2H associated carbon lost? not C2a C6 origin of loss, C6

B. Loss of C5H9NO3. Labeling Data for GlcNAc precursor ion 426 437 production 295 304 mass difference 131 Da 133 Da label present in loss 2 2H a associated carbon lost? C2 C6 origin of loss C1, C2, C6

B. Loss of C2H4O2. Labeling Data for GlcNAc and GalNAc precursor ion 352 359 354 product ion 292 298 294 mass difference 60 Da 61 Da 60 Da label present in loss 1 2H no 18O associated carbon lost? not C2a C6 not C1 origin of loss, C5-C6

428 295 133 Da 18O C1

a The presence or absence of C2 is determined by the nitrogen rule, since nitrogen is present at C2.

loss of 120 Da (C4H8O4) occurs for both GlcNAc and ManNAc (Table 3A). Because the mass of this neutral loss is even, it is assumed that the nitrogen and C2 are not part of the loss. When labeled with 18O, the complex of interest (m/z 428) produced a product ion m/z 308 for both GlcNAc and ManNAc. Because the 18O label was retained in the product ion (m/z 308) the C1 oxygen (and carbon 1) cannot be included in the 120 Da (C4H8O4) neutral loss. Similarly, acidic protons were exchanged for 2H for both the GlcNAc and ManNAc monomers. The precursor ion of interest for the 2H labeling study is m/z 437 because there are 11 acidic protons in the complex. When this ion dissociates, m/z 437 f 314, a neutral loss of 123 Da is observed for both GlcNAc and ManNAc. Thus, three exchangeable protons are present in the C4H8O4 loss. Furthermore, the 2H data also confirm that C6 is included in the dissociation ion, because of the number of nonacidic hydrogens lost. In summary, the 18O-labeling experiments and the nitrogen rule indicate that neither GlcNAc nor ManNAc loses C1 or C2 in the C4H8O4 neutral loss, so the origin must be C3-C6. 2H labeling, which confirms the loss of C6, supports this assignment. This typeofdissociationincommonlyobservedinrelatedcompounds.16,22-24 In the MS2 study, the loss of 131 Da from m/z 426 appears to correspond to C5H9NO3 and occurs solely for the GlcNAc 2000 Analytical Chemistry, Vol. 71, No. 10, May 15, 1999

354 324 30 Da no 18O not C1

C. Loss of C3H6O3. Labeling Data for GlcNAc and GalNAc precursor ion 352 359 354 product ion 262 267 264 mass difference 90 Da 92 Da 90 Da label present in loss 2 2H no 18O associated carbon lost? not C2a C6 not C1 origin of loss, C4-C6 a The presence or absence of C2 is determined by the nitroen rule, since nitrogen is present at C2.

diastereomer, as indicated in Table 3B. Because of the odd mass, some portion of this loss must contain nitrogen and presumably the acetyl side chain on C2, i.e., C3H5NO. Thus, the remaining portion of the neutral loss, C2H4O2, is a two-carbon unit from the monosaccharide. The origin of these two carbons can again be determined from the results of the isotopic labeling studies. 2Hlabeling studies of GlcNAc show that a total of two acidic protons were lost (m/z 437f 304, Table 3B). One of these protons resides on the C2 nitrogen; this leaves only one acidic proton for the remaining portion of the neutral loss (C2H4O2). Because only one of these four remaining protons is exchangeable, three protons must be nonexchangeable. In order for these three nonexchangeable protons to reside on two carbons, C6 must be lost; as it is the only carbon with two nonexchangeable protons. Finally, when GlcNAc is labeled with 18O, the complex of interest (m/z 428) produced the product ion m/z 295, a loss of 133 Da (Table 3B). In this case, the 18O label was lost, implying that the C1 oxygen (and the anomeric carbon) must be included

in the C5H9NO3 neutral loss. In summary, the C5H9NO3 neutral loss must consist of C2 and the N-acetyl side chain, C1, and C6. Labeling studies were also used to determine the origins of each of the ions obtained in the MS3 experiment, m/z 426 f 352f, for GalNAc and GlcNAc. (MS3 of ManNAc provided no product ions.) The loss of CH2O (present only for GalNAc) must occur exclusively from the elimination of C6. The C1 oxygen cannot be part of the loss, as was confirmed by 18O labeling of GalNAc and subsequent CID (m/z 428 f 354 f 324). In this experiment, the 18O label was retained in the product ion (Table 4A). Furthermore, no exchangeable protons were present in the CH2O neutral loss. This is confirmed by the 2H-labeling experiment (m/z 437 f 359 f 329), which shows the mass of the neutral loss (30 Da) did not shift when 2H was incorporated into the complex. Because the loss of CH2O contained no exchangeable protons, it must contain the two nonexchangeable protons found at C6. Labeling studies confirm that C6 is present in the 60 Da (C2H4O2) loss for both GlcNAc and GalNAc (Table 4B). In each case, MS3 of the deuterated complex (m/z 437 f 359 f) provided an ion at m/z 298 which corresponded to a loss of C2H32HO2, confirming that one acidic proton and three nonacidic protons were present in the neutral loss. Again, because the number of nonacidic protons is greater than the number of carbons, C6 must be lost. Incorporation of 18O into the two diastereomeric complexes confirmed that the C1 oxygen was not part of the neutral loss, as the 18O label was not lost in the MS3 experiment, m/z 428 f 354 f 294. Finally, because the neutral loss is even, the presence of the N-acetyl side chain at C2 is highly unlikely. Thus, C1 and C2 are not part of the 60 Da (C2H4O2) neutral loss; the loss must consist of C6 and one other carbon. Extensive labeling studies from previous work show that when C6 is involved in a 60 Da loss (as is the case here), the remaining portion of the neutral loss always contains C5 and the C5 oxygen.16,22-24 Precluding any complex rearrangement, loss of C5 and C6, and the accompanying oxygens, leads to the most logical dissociation product ion. Finally, labeling studies confirmed that C6 is present in the 90 Da (C3H6O3) neutral loss as well, but both C1 and C2 remain complexed to the metal. The 18O label was not lost, suggesting that C1 remains complexed, and the C2 nitrogen must be retained in accordance with the nitrogen rule (Table 4C). Based on this information and the assumption that the carbon backbone does not undergo major rearrangement, the loss of 90 Da (C3H6O3) is most likely the loss of carbons 4, 5, and 6. This conclusion is supported by studies on very similar compounds subjected to collisional dissociation in an ion trap.16 In summary, then, each of the dissociations involves C6, and the carbons involved in each dissociation are depicted in Figure 6. Dissociation Mechanisms. Based on the origins of the neutral losses, mechanisms can be postulated for each dissociation. Because Co has an oxidation state of +3 in these complexes, there must be two sites of deprotonation in the m/z 426 precursor ion. Rational mechanisms, which explain how each carbon unit dissociates, must begin at these sites of deprotonation.16,22-24 The two most likely sites of deprotonation on the diastereomers are on the C1 oxygen and the C2 nitrogen of the monosaccharide (Figure 3). These sites of deprotonation drive each of the dissociations.

Figure 6. Origins of dissociations. Note: The dissociation corresponding to -131 Da (not shown) corresponds to the loss of C1, C2, and C6 from the octahedral complex.

Figure 7. Mechanism of dissociation for loss of 120 Da (C4H8O4).

Figure 8. Mechanism of dissociation for loss of 131 Da (C5H8NO3). R ) Co(DAP)2.

(a) Loss of C4H8O4 from Precursor Ion m/z 426 (Figure 7). Labeling studies presented here confirm that C1 and C2 are not part of this dissociation. Thus, the neutral loss must contain carbons 3-6. This neutral loss has been observed in previous work, and a similar dissociation mechanism as proposed previously is depicted in Figure 7.16 The negative charge on the nitrogen drives the dissociation. Carbons 3 and 4 are lost as a diol unit, followed by C5 and C6. (b) Loss of C5H9NO3 from Precursor Ion m/z 426 (Figure 8). The origin of this neutral loss, as determined from isotopic labeling studies, is carbons 1, 2, and 6 (Table 3B). Because carbon 6 is not directly adjoined to carbons 1 or 2, major rearrangement of the monosaccharide would be necessary before these carbons could leave in a concerted fashion. However, if the dissociation were the result of two consecutive losses, no major rearrangement is required. Figure 8 depicts one such consecutive mechanism where C6 is eliminated followed by the loss of C1 and C2. In order for this elimination to occur as depicted in Figure 8, the C1 oxygen and the N-acetyl nitrogen must be protonated and the C6 oxygen and the C3 oxygen become deprotonated. While this complex is certainly higher in energy than the proposed starting complex, previous computational work involving similar monosaccharides in the gas phase suggests that the existence of this higher-energy species is possible in small quantities.24 Thus, once this species is generated, carbons 1, 2, and 6 can easily dissociate, leaving the negative charge on the C4 oxygen. Analytical Chemistry, Vol. 71, No. 10, May 15, 1999

2001

Figure 10. Mechanism of dissociation for loss of 60 Da (C2H4O2).

Figure 9. Mechanism of dissociation for loss of (A) 30 Da (CH2O); (B) 90 Da (C3H6O3). R ) Co(DAP).

(c) Loss of CH2O and C3H6O3 from Precursor Ion m/z 352 (Figure 9). In these cases, the mechanism begins with a series of proton transfers that ultimately result in the deprotonation of C6. This deprotonation is supported by the isotopic labeling studies. The 2H-labeling studies confirm that the CH2O loss consists of C6 and the C6 oxygen, but the acidic proton on the C6 hydroxyl group is not part of the 30 Da (CH2O) loss (Table 4). Thus, this proton must be transferred before dissociation can occur, or a portion of the m/z 426 ion population with C6 deprotonated, a priori, must exist. Once the C6 oxygen is deprotonated (as shown in Figure 9, top), two different dissociation pathways are possible. The dissociation of CH2O occurs by a concerted elimination of formaldehyde and subsequent extraction of the proton on the C4 oxygen (Figure 9A). If the C4 proton is not easily extractable, the loss of CH2O could be followed by a loss of carbons 4 and 5 (Figure 9B.) In this case, two consecutive losses of CH2O and C2H4O2 (making a combined loss of C3H6O3) are followed by a proton extraction at C3. This consecutive loss, involving carbons 4, 5, and 6 is consistent with the isotopic labeling studies that confirm the loss of C3H6O3 contains exactly these carbons. The two mechanisms described above illustrate that two dissociation pathways are possible once C6 is deprotonated. The formaldehyde elimination occurs when the proton on the C4 oxygen is transferred, and if this proton is not transferred, the concerted elimination of CH2O followed by C2H4O2 occurs. Thus, the accessibility of the proton on the C4 hydroxyl group must play a role in determining which reaction is favorable. Therefore, it is not surprising that GalNAc (with the C4 hydroxyl group axial) undergoes the CH2O elimination readily while GlcNAc (with the less accessible, equatorial C4 hydroxyl group) preferentially dissociates through the concerted loss of CH2O followed by C2H4O2. (d) Loss of C2H4O2 from Precursor Ion m/z 352 (Figure 10). Labeling studies confirm that the 60 Da loss (C2H4O2) includes carbons 5 and 6. Again, the dissociation mechanism must start with a site of deprotonation; accordingly, the negative charge on the C1 oxygen can initiate the elimination of C5 and C6 by rehybridizing to sp2. This shift of electrons drives the dissociation of C2H4O2 as a leaving group (Figure 10). The elimination likely 2002 Analytical Chemistry, Vol. 71, No. 10, May 15, 1999

occurs via the concerted abstraction of the proton from the C3 oxygen. While other mechanistic pathways are feasible for this dissociation,16,24 the one depicted in Figure 10 avoids the presence of a high-energy carbanion intermediate. While preliminary mechanisms were proposed for all of the dissociations observed, (Figures 7-10) we acknowledge that without further mechanistic studies (including the use of deoxysaccharides,16 kinetic energy release data, or performing double resonance studies) we cannot assert with certainty how each dissociation is occurring. For example, the possibility exists in which different ion populations with different deprotonation sites make up the total precursor ion population. Some ion populations may be more thermodynamically favorable than others, thus accounting for the observed differences. It is very likely that the mechanisms for these dissociations are similar to those reported for the hexose monomers.16,22-24 However, the hexose monomers displayed stereoselectivity different from the N-acetylhexosamines for each of the reported dissociations. To reconcile these differences, further mechanistic studies are currently being pursued. CONCLUSIONS Coordination of N-acetylhexosamines to a metal-ligand system allows for stereochemical differentiation, when the complexes are analyzed by tandem mass spectrometry in a quadrupole ion trap. Multiple MSn studies showed unique spectra that unambiguously differentiated the diastereomers, and this technique can be used to identify the stereochemistry of N-acetylhexosamines in an oligosaccharide. Furthermore, isotopic labeling studies were used to determine the origins of each of the neutral losses observed, and mechanisms that explained the observed stereoselectivity were postulated. This work has provided a sound demonstration of the crucial role that mass spectrometry can play in distinguishing biologically relevant diastereomers. Furthermore, we have recently completed studies that indicate this method can be used quantitatively in predicting the precise amount of each diastereomer present in an unknown mixture. (Data to be published separately.) In combining these methods along with our previous work in determining branch points, linkage position, anomericity, and our current efforts involving on-line derivatization of subnanomole quantities of saccharide, we are one step closer to complete structural elucidation of oligosaccharides using tandem mass spectrometry. ACKNOWLEDGMENT The authors gratefully acknowledge NIH Grant GM47356 for financial support. Received for review September 22, 1998. Accepted February 24, 1999. AC981052Y