Anal. Chem. 1990, 62, 1731-1737
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Infrared Laser Desorption Mass Spectrometry of Oligosaccharides: Fragmentation Mechanisms and Isomer Analysis Bernhard Spengler, Joseph W. Dolce, and Robert J. Cotter* Department of Pharmacology and Molecular Sciences, Middle Atlantic Mass Spectrometry Facility, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
Infrared laser desorption/lonlzatlon of ollgosaccharldes and their naturally occurrlng derlvatlves leads to a dlstlnctive fragmentatlon pattern, which is useful both for the analysls of functlonal groups (e.g., the posltlons of fatty acyl groups In lipld A and other glycollpkls) and for distingulshbrg h e r s of underivatlred ollgosaccharldeswith dmerent glycosldlc linkages. The predominant mechanism of fragmentatlon can be understood as a pericyclic hydrogen rearrangement of the retro-aldol reaction type, leadlng to fragmentatkm of the sugar rlng. A prerequlslte for thls process appears to Involve opening of the hemlacetal saccharlde rlng to the llnear saccharide form by rapld heatlng from the Infrared laser lrradlatlon. Saccharkles derivatlzed at the anomerk hydroxyl group (Le., cycllc acetals) normally do not undergo thls rfng opening and thus do not exhlblt rlng fragmentatlon reactions. This fragmentatkin mechanism can be used to predlct and interpret fragmentation patterns of unknown oilgosaccharldes, glycosides, llpld A, and other giycoconJugates.
INTRODUCTION The direct mass spectrometric analysis of intact carbohydrates and glycoconjugates has been made possible by the introduction of several new ion desorption techniques. Because of the low vapor pressure and thermal instability of carbohydrates, standard mass spectrometric ionization techniques such as electron impact (EI) and chemical ionization (CI) fail to produce intact molecular ions. Thus, the most common strategies involve permethylation, reductive cleavage of the individual sugar units, acetylation of the resulting unmasked hydroxyl groups, and analysis of the products by combined gas chromatography/mass spectrometry (GC/MS) to reveal information on both the identity and linkage positions of the original carbohydrate (1-3). Desorption techniques such as fast atom bombardment (FAB), plasma desorption (PDMS), and field desorption (FD), on the other hand, can provide molecular mass information for many large and nonvolatile compounds but often reveal only minor structural information from specific fragmentation. In the case of carbohydrates, the most prominent fragmentation observed by using these techniques is the cleavage of the glycosidic bond, leading to information on the sequence (and branching) of the sugar units but not on the specific linkages involved or the positions of attachment of substituents such as fatty acyl groups. In addition, such fragmentation is generally weak or absent (4,5),so that both collision-induced dissociation (CID) using tandem mass spectrometers (6) and chemical derivatization (5) have been proposed as strategies for enhancing structural information. Recently Coates and Wilkins have observed that (in contrast to other desorption techniques) infrared laser desorption mass spectrometry (IR-LDMS) of carbohydratesproduces a unique fragmentation pattern that can be explained as two-bond ring
cleavages within the cyclic sugar units (7,8).The predominance of two-bond ring fragmentation over glycosidic bond cleavage following ionization by an infrared laser was observed as well as Cotter et al. (9, 10) for the phosphorylated diglucosamine known as lipid A, and its significance for the determination of substituent positions (9,lO) and linkages (I1,12) has been discussed. Lipid A, the anchor for the larger lipopolysaccharide (LPS) found on the outer wall of the outer membrane of Gram-negative bacteria, provided a unique opportunity to characterizethe locations of the two-bond ring cleavages. These compounds (Figure 1)contain from four to seven fatty acid groups attached (as acyl or acyloxyacyl species) by an ester linkage to the C i and C3 positions (R, and R3) and amide linkages to the C i and Cz positions (R, and R4) of the diglucosamine,respectively. Monophosphoryl lipid A (I) contains a single phosphate group, generally on the C4/ position of the distal sugar, while diphosphoryl lipid A (11) contains an additional phosphate group on the C1 position of the reducing glucosamine. For the monophosphoryl lipid A, losses of R,NHCH=CHOH, R30CH=CHNHR4and the observation of the distal portion + CH,CH=O + K+ from the MK+ ion were interpreted as simultaneous cleavages of the 0-C1 and C&3 bonds, the C1-C2 and C3-C4 bonds, and the 0-C1 and C4-C5 bonds, respectively. For the diphosphoryl lipid A, the phosphate group attached to the anomeric carbon was generally cleaved, and two-bond ring cleavages were generally diminished. This fragmentation pattern was observed for the well-characterized lipid A from E. coli (10) and was used subsequently for the structural elucidation of monophosphoryllipid A from Niesseria gonnorhoeae (9) and Rhodopseudomonas sphaeroides (13). The IR-LDMS fragmentation behavior for special cases like lipid A is thus well-known and predictable and can be extended to carbohydrates in general. In an earlier paper (14) we compared the IR laser desorption m a s spectra of a number of underivatized and derivatized (permethylated and peracetylated) neutral and amino sugars with spectra obtained by plasma desorption and fast atom bombardment. In general we found the ion signal produced by both PDMS and FAB to be of low intensity as well as an absence of fragmentation. Chemical derivatization increased both ion intensity and fragmentation, with fragmentation occurring primarily at the glycosidic bond and resulting in fragment ions with the charge retained on either the distal or reducing portion. In contrast, IR laser desorption of underivatized sugars resulted in abundant fragmentation of the sugar ring itself, with the charge retained almost exclusively on the distal portion. Sugars that did not have a reducing end (e.g., trehalose) did not undergo two-bond ring cleavages. In addition, chemical derivatization generally suppressed fragmentation. Thus, while IR laser desorption appears to offer a unique analytical approach for elucidating the structures of sugars and glycolipids, a general description of the underlying mechanism is stll lacking. The investigations described in this paper are intended to explain most of the observed fragmentations of
0003-2700/90/0362-1731$02.50/00 1990 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 17, SEPTEMBER 1, 1990
CH20H
0
ro\O-
CH 9
.
I00 L
z+
" I
I:
X = H
P
75
R4
11: X = POSH, General structures of the lipid anchor of the lipopolysaccharide found on the outer membrane of gram-negative bacteria known as lipid A: I = monophosphoryl lipid A I1 = dlphosphoryl Upid Flgure 1.
2
50
A. 25
saccharides and their derivatives by a single, simple mechanism. If this model will stand future proofs, IR-LDMS should become a very versatile and helpful tool for the structural analysis of carbohydrates found in biological systems, combining a high degree of certainty, high sensitivity, and short analysis times.
EXPERIMENTAL SECTION The IR laser desorption mass spectrometer has been described in detail previously (15, 16). A Tachisto Laser Systems Inc. (Needham, MA) Model 215G carbon dioxide laser generates pulses of infrared radiation (10.6 pm) of 50-ns width at half-maximum and a tail of about 2-ps duration, measured by means of a fast pyroelectric detector (Type 420, Eltec Instruments, Inc., Daytona Beach, FL). Desorbed ions are accelerated by the pulsed extraction system of the CVC Products (Rochester,NY)Model 2000 time-of-flight mass spectrometer and detected by a Galileo (Sturbridge, MA) dual channel plate detector. The analog signals from each laser shot are digitized and stored in a Lecroy (Chestnut Ridge, NY) Model 9400A digital oscilloscope/transient recorder as 32K X 8-bit data points of 10-ns resolution and signal averagedlintegrated by PC-based software. The spectra presented are averaged over various numbers of single-shot spectra to increase the reliability of relative intensity information. Carbohydratesamples were purchased from Sigma Chemical Co. (Kalamazoo, MI); 2 x M solutions of analyte in a 1:l water/methanol solvent mixture were prepared; 1pL of analyte solution was dried onto a vespel probe tip. RESULTS AND DISCUSSION To study the dependence of fragmentation on specific linkages, IR-LDMS spectra were obtained on the series of sugars summarized in Table I. These include different anomers (a-and @-linkedsaccharides) as well as isomers linked at different positions (1 4, 1 6, 1 3, and 1 1). Retro-Aldol-Type Reactions. The IR laser desorption mass spectrum of cellotriose is shown in Figure 2. In this and subsequent mass spectra the ions observed are generally formed as cationized species, Le., with the addition of Na+ or K+. The most prominent fragmentation of 1 4 and 1 6 linked sugars is the loss of 60 and 120 mass units (14). It can be observed in all saccharides of this type and has been observed by other groups as well (7,8,11,12). The mass losees of the fragment ions are listed in Table I. To use a unified nomenclature for the proposed fragmentations and the structures of the fragment ions observed, the nomenclature of Domon and Costello (17) has been used. It is described briefly in Figure 3. According to that system, for example, the fragment ion produced by cleavage of the O-C1and Cz-C3 bonds in the reducing end sugar of a disaccharide (with the charge retained on the distal portion) is called O,zAz. The proposed fragment ions observed for the various saccharides are listed in the right portion of Table 1.
-
- - -
-
-
250
300
450
350
500
W Z
Signal-averaged IR-LD mass spectrum of cellotriose (Glc[/31--4]),Glc, MW = 504 amu.
Flgure 2.
Structure of cellotriose, illustrating the fragment ion nomenclature, used according to ref 17. Figure 3.
The fact that simultaneous cleavage of two bonds is in general unlikely led to a model for the fragmentation of saccharides by IR-LDMS involving several consecutive steps. We assumed that the first step in the desorptionfionization of saccharides in IR-LDMS involves ring opening of the reducing sugar (generally drawn as the rightmost sugar mit and containing a free hydroxyl group at CJ. Saccharides that are not derivatized at the hydroxyl group of the anomeric carbon atom (cl) can exist in linear and cyclic forms. The cyclic hemiacetal form is in general strongly preferred in both aqueous solution and crystalline state, although changes of the stereochemistry at C1(known as mutarotation) that use the linear form as an intermediate are common. The equilibrium can as well be affected by changes in temperature. Therefore, pulsed IR laser irradiation is assumed (in our model) to result in thermal opening of the reducing sugar unit. Since there are minimal thermal effects in other desorption techniques (i.e., FAB or PDMS), this step can be expected to be more or less unique for IR laser desorption and would be the basis for explaining the different fragmentation behavior observed in that technique. Once the reducing sugar ring is opened, the molecule can undergo a series of thermal degradation processes. The complete proposed mechanism of the saccharide fragmentation is shown in Figure 4. The reaction following ring opening is known as a retro-aldol reaction (pathway a in Figure 4). Because IR laser desorption generally involves the gas-phase
ANALYTICAL CHEMISTRY, VOL. 62, NO. 17, SEPTEMBER 1, 1990 n, n"
1733
CHDH
F-\
Mo I11
b
J.
a
V (pulsed IR irradiation)
Figure 5. Proposed mechanism for the IR-laser induced fragmentation of palatinose (Glc [al+6]Fru).
VI1
J.
CIC.
Figure 4. Proposed mechanism of saccharide fragmentation in IRLDMS, using cellobiose as an example.
attachment of alkali-metal ions to co-desorbed neutral molecules (18,19),such reactions of neutral molecules could occur in the condensed phase (prior to desorption) or in the gas phase (prior to alkali-metal ion attachment), although analogous reactions could also occur for ionized species. Related mechanisms are known in both synthetic organic chemistry and mass spectrometry, as the Norrish-type-I1reaction, ester pyrolysis, the Chugaev reaction, the retro-ene reaction, and the McLafferty rearrangement (if it occurs in the gas phase for the quasimolecular ion). All involve a six-membered planar ring structure as an intermediate for hydrogen transfer. While it is not clear whether these reactions take place in the condensed or gas phase or before or after ionization, if the first step (ring opening) is a thermally induced one, it is likely that subsequent fragmentation is as well a thermal degradation of neutral molecules prior to ionization. A reaction similar to a can occur by using a different proton of molecule 111, attached to carbon. This reaction (pathway b, Figure 4) might be favored in the fragmentation observed for lipid A, where the hydroxyl group at C3 is derivatized. Both reaction pathways would lead to the same neutral fragment (IV), resulting from the loss of 60 mass units. In the case of palatinose (VIII), which contains a furanose unit at the reducing end, the analogous fragmentation corresponds
to a loss of 90 mass units (IX), as can be seen in Figure 5. A further degradation of IV is possible, driven by the same mechanism and involving transfer of either a hydroxyl or a carbon proton to the aldehyde oxygen. It leads to structure V by loss of 120 mass units relative to the parent molecule. Structure V could then undergo a third retro-aldol reaction, transfering a y-proton to the aldehyde oxygen. However, this reaction, leading to structure VI1 and loss of 164 mass units relative to the parent molecular mass, is not observed. Instead a similar proton-transfer reaction may occur, leading to the opening of the adjacent saccharide ring (structure VI and loss of 162 mass units relative to the parent molecular mass). The fragment ion resulting from cation attachment to VI would, of course, have the same mass as an ion resulting from cleavage of the glycosidic bond ( O X 4 , accompanied by H transfer) of the MK+ molecular ion, analogous to that observed in FAB and PDMS (10,141. The resulting structure would then be the cyclic precursor to structure VI and might then undergo ring opening and additional cleavages of the remaining sugar ring (seebelow). However, the direct glycosidic bond cleavages observed in the latter techniques generally lead to ions in which the charge is carried by either the reducing or distal fragments. In addition, the fact that further (ring) cleavage is not observed in these techniques suggests that structure VI is formed (in IR-LDMS) by the same multistep thermal process that occurs prior to ionization. Structure VI has the same h i c structure as 111,so the same degradation mechanism can occur for the next sugar unit. Further fragmentation of the adjacent sugar units has been observed for all of the trisaccharides tested (indicated by etc. in Table I). Cellotriose, for example (Figure 2), shows Ov2A3, 2p4A3,C2, 294A2,and C1, which correspond to the mass peaks M - 60, M - 120, M - 162, M - 162 - 60, M - 162 - 120, and M - 162 - 162, respectively. 092A2,
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T a b l e I. D a t a f o r Sugars Investigated in This S t u d y OJA,
substance
mass losses
cellobiose = Glc[@1-+4]Glc cellotriose = Glc[fll-4]Glc[@l-4] Glc maltose = Glc[oll-+4]Glc maltotriose = Glc[otl+4]Glc[al-+4]Glc Gal[ (Y 1-41 Gal Gal[/31-+4]Gal[fl1-4]Glc gentiobiose = G k [P1--6]Glc isomaltose = Glc[ul-6]Glc isomaltotriose = Glc[~~1-61Glc[al-6]Glc nigerose = Glc[al-+S]Glc mannobiose = Man[cu 1-31 Man trehalose = Glc[al-l]Glc sucrose = Glc[ul-Z]Fru palatinose = Glc[nl+6]Fru raffinose = [Gal[a1-+6] Glc[al-Z]Fru y-cyclodextrin = c-(Glc[n1-4])6
-60, -120, (-1621," (-18), (-60-18) -60, -120, -162, etc. (-181, -60-18, -162-18
X X
-60, -120 -60, -120, -162, etc.
X X
-60, -120, (-162) -60, -120, -162, etc., -18, -60-18, -162-18
X X
-60, -120, (-162), (-go), -18
X
-60, -120, (-162), -(go) -60, -120, -90, -162, etc., -18
X X
a
(-901, -162, (-162-18) (-901, -162, (-60), (-120), (-162-18)
(-162) -162, -18 -90 -162, etc.
X
x
x
x
(X)
X
-102, -102-60, -102-120, -102-162, etc.
X
Parentheses indicate that ions of low intensity were observed.
J
c>--qi>c C y OAc
AcO
OAc
1
M-60
c>-c-&
AcO NHRd
UHR4
X
XI
hl- 108
M-94
OAc
+
XI1
Figure 6. Left: two fragmentation pathways involving the loss of the methylated phosphate group from the reducing glucosamine of diphosphotyl lipid A. Right: mechanism of ester pyrolysis in a peracetylated saccharide.
The assumption that ring opening of the reducing sugar is a prerequisite for the observation of ring cleavages is strongly
supported by the investigation of three saccharides: trehalose (Glc[al+l]Glc), sucrose (Glc[al-S]Fru), and raffinose
ANALYTICAL CHEMISTRY, VOL. 62, NO. 17, SEPTEMBER 1, 1990
(Gal[a1-6]Glc[al-+B]Fru), which do not have a reducing terminus. The anomeric oxygens of the rightmost sugar units are linked to the distal ends of these oligosaccharides. Thus there is no equilibrium between the linear and the cyclic form of the saccharide and the retrc-aldol mechanism will not be induced. Consistent with this model, no ring cleavages have been observed in the rightmost sugar unit of trehalose, raffinose, and sucrose (contrary to the very similar 1 6 linked palatinose, Figure 5 ) . Trehalose instead shows a weak fragmentation at the glycosidic bond, which is stronger in the case of sucrose and raffinose. Raffinose shows weak retro-aldol type fragmentation in the adjacent sugar unit. Similar arguments hold for the observed lack of fragmentation starting at the distal end of oligosaccharides. In the absence of a free hydroxyl group on the anomeric carbon of the distal sugar, no ring opening and subsequent fragmentation occurs to the distal sugar unit, leaving the reducing end of the saccharide intact. Ring opening and fragmentation can as well be hindered by derivatizing the anomeric hydroxyl group. Peracetylated oligosaccharides (14), for example, do not show any fragmentation except for a weak loss of 60 mass units in some cases. This scheme would thus explain the unique behavior of IR-LDMS toward derivatization. In contrast to the improvement in fragmentation in the FAB and PDMS spectra of derivatized sugars, the reverse is observed for IR laser desorption. This is not necessarily true for all derivatives. As noted above, the diphosphoryl lipid A from Gram-negativebacteria generally contains a phosphate group attached to the C1 position as well as the C i position of the diglucosamine backbone. In our laboratory (10) these compounds have generally been analyzed as their fully methylated phosphoesters. Thus, the IR-LDMS spectra of tetramethyldiphosphoryl lipid A generally show slightly less but not vanishing ring fragmentation, compared to dimethylmonophosphoryl lipid A, which contains a free hydroxyl group at the anomeric carbon. This small but observable fragmentation for the diphosphoryl structure can be explained by the facile loss of the reducing end P03Me2moiety, observed as losses of 94 or 108 mass units. Mechanisms that illustrate the loss of the reducing phosphate accompanied by ring opening are shown in Figure 6, left, and can be used to explain subsequent ring fragmentation. Methyl transfer from the phosphate to the ring oxygen as well as hydrogen transfer from other SOUTCW leads to structures (X and XI) similar to 111in Figure 4,which will undergo the described ring fragmentations. In contrast, peracetylated saccharides undergo weak fragmentation of the acetate residue, leading to structures that will not undergo further fragmentation. To permit the pericyclic proton transfer to occur, the transition state must have a planar structure, which is possible only if the y-proton is in a cis position with respect to the anomeric acetyl group, as e.g., in peracetylated saccharides ending in a P-glucose unit. The mechanism is shown in Figure 6, right. While the reaction is of the same type as the described retro-aldol reactions, it is more appropriately known as ester pyrolysis. The reaction seems to occur only once (XII) and not to the other acetyl groups. Regioselective elimination of the acetyl group at C1 has also been observed in preparative organic chemistry by thermal ester pyrolysis in acetone at 350 "C (20). Lipid A. The strongest support for the described model can be obtained from the fragmentationof lipid A, investigated earlier (9, IO),where the natural derivatization of the glucosamine units by various fatty acids had provided a means for determining the cleavage sites. The fragmentation observed for a lipid A with known structure (IO)was described by three separate two-bond ring cleavages and was subsequently used
1735
CI~OII
-
v
I
R* (pulsed IR irradiation)
hbo0
XI11
NH
1
02
RA'= A2
MCO I
xv
R2
eic. Figure 7. Proposed mechanism for the fragmentation of lipid A. R,-R,
are fatty acids.
to determine the positions of fatty acyl groups in the lipid A obtained from other bacteria (9,131. Two of these two-bond ring cleavages (0-C1 and C2-C3, and 0-C1 and C4-C5) are consistent with the described first and second retro-aldol reactions (ov2A,, and OPA,, respectively). The third, described as the simultaneous cleavage of C1-C2 and C3-C4, appears (from our current model) to be less likely. Rather, a fragment ion of the observed mass could evolve as well from the first retro-aldol reaction, followed by an a-cleavage of the linked fatty acid, leading to structure XV in Figure 7. Cyclodextrin. The fragmentation pattern of y-cyclodextrin (XVI in Figure 8), a cyclic structure containing eight glucose rings, seems to be somewhat different from that of the linear saccharides. The IR-LDMS spectrum for this
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ANALYTICAL CHEMISTRY, VOL.
62, NO. 17, SEPTEMBER 1, 1990
OH
1
'"An
t.1 Y
6H
m1 ,'\
I
1
M.102 M=1194
/
p 3 *
b-'",
r;
01 I
-
Figure 9. Possible mechanism for the OS3Anfragmentation of 1 3-linked and 1 6-linked saccharides.
I
I
300
400
'
I
600
'
I
800
'
I
"
"
loo0
Mlr
Figure 8. Proposed mechanism for the first fragmentation step and IR laser desorption mass spectrum of y-cyclodextrin. compound is shown in Figure 8. The expected series of losses of 60,120, and 162 mass units relative to the parent molecule is not observed instead, one observes an initial loss of 102 mass units, followed by further losses of the above series. The cyclic structure of cyclodextrin is a special case which, nevertheless, can be explained by the same basic mechanism of retro-aldol reactions. As observed for trehalose, sucrose, and raffinose, linkage at the anomeric hydroxyl group of the reducing sugar normally prevents opening of the ring and, therefore, fragmentation by the retro-aldol mechanism. These sugars, however, undergo fragmentation by cleavage of the glycosidic bond, leading to a loss of 162 mass units. Thus the first step in the fragmentation of the cyclodextrin can be assumed to be cleavage of one of the glycosidic bonds. The proposed mechanism is shown in Figure 8. I t leads to the opening of the superring-structure of cyclodextrin, as well as the opening of one sugar unit, initiating the known retro-aldol fragmentation of the new molecule (XVII) of mass M - 102. The complete series of fragmentation has been observed, starting with Ov2A7, 2,4A7,C6 and continuing down to C1, 0*2A,. Differences in Fragmentation between Isomeric Saccharides. Retro-aldol type fragmentations for 1 4 and 1 6 linked sugars can lead to the same mass losses (-60, -120, -162), regardless of the site of linkage and anomeric configuration (aor p) and thus do not appear to be distinguishable. In contrast, 1 3 linked saccharides exhibit quite different
-
-
-
-+
-
behavior (-90, -162), while 1 1 linked sugars are distinguishable by their lack of fragmentation. There are, however, other fragment ions that can provide valuable information on the type of the sugar isomer under investigation. As can be seen from Table I, all of the P-linked 1 4 and 1 6 saccharides tested showed one or more additional peaks due to the loss of water from the parent molecule or from a fragment. None of the a-linked 1 4 and 1 6 saccharides (except a minor peak in the case of isomaltotriose) showed any peaks due to losses of water. The observed fragmentation pattern is also sensitive to the site of the glycosidic linkage. In general, 1 6 linked pyranoses showed additional fragmentation of type Ov3An,leading to a loss of 90 mass units, while 1 4 linked saccharides did not. Distinguishing these two most common types of saccharides therefore should be possible by IR-LDMS. As noted above, 1 1 linked pyranoses and 1 2 linked furanoses did not show any ring fragmentations, so they can be easily distinguished. The 1 3 linked saccharides showed only weak but clearly detectable ring fragmentation, allowing one to distinguish them from the other linkage types as well. The ring fragmentation of type Ov3An,leading to losses of 90 mass units, cannot be explained by the retro-aldol type reaction. Its mechanism is not clear yet. The fact that the intensity of these fragments is always very low could indicate that this fragmentation type is due to some second-order mechanisms. Assuming a similar mechanism as in the case of retro-aldol reactions, one could suggest a retro-ene reaction, described in Figure 9 for the case of 1 3 and 1 6 linked saccharides, respectively.
-
-
-
-
-
-
-
-
-
-
-
CONCLUSION The investigation of isomeric oligosaccharideshas been used to determine the mechanism of fragmentation in IR laser desorption mass spectrometry. It has been suggested that the most prominant fragmentation pathway involves a series of
Anal. Chem. 1QQO. 62. 1737-1746
retro-aldol reactions, assumed to take place prior to ionization, in this case by attachment of an alkali-metal ion to desorbed neutral species. This mechanism can account for most of the observed fragmentation of sugars and their natural derivatives. The model can also be used to explain the observed reduction of fragmentation for permethylated and peracetylated sugars and for 1 1 linked sugars, since both involve the absence of a free hydroxyl group on the anomeric carbon and prevent the type of ring opening what would lead to additional ring fragmentation via a retro-aldol mechanism. Differences in fragmentation have been observed for different isomers of oligosaccharides. 1 -.6 linked pyranoses can be distinguished from their 1-.4-linked isomers by an additional fragment ion. fl-homers, on the other hand, show a distinctive loss of water from the parent molecule and specific fragments, while the a-anomers in general do not. IR laser desorption mass spectrometry provides a distinctively different and complementaryapproach to the structural analysis of carbohydrates and glycoconjugates, since fragmentation occurs primarily in the sugar rings rather than at the glycosidic bond. It has been an invaluable tool for the analysis of glycolipids such as lipid A, where ring fragmentation can be used to reveal the specific locations of fatty acyl groups. While such fragmentation has been reliable and predictable, the current study helps to establish with some confidence the mechanisms that give rise to the cleavages observed. An understanding of these mechanisms can, in turn, be applied to unsubstituted oligosaccharides and will be invaluable for the elucidation of the structures and linkages of complex carbohydrates containing several branches.
mann. C. J., McGinnis. G. D., Eds.; CRC Press; Boca Raton, FL, 1989; pp 27-41. Carpita, N. C.; Shea, E. M. I n Analysis of Carbohydrates by GLC and MS; Biermann, C. J., McGinnis, G. D., Eds.; CRC Press: Boca Raton, FL. 1989;pp 157-216. Reinhold, V. N.; Carr, S. A. Mass Spectrom. Rev. 1983, 2, 153-221. Dell, A.; Tiller, P. R. Biochem. Biophys. Res. Commun. 1988, 135, 1 126-1 134. Cai: S. A.;Reinhold, V. N.; Green. E. N.; Haas, J. R. B/omed. Mass Spectrom. 1985, 12,288-295. Coates, M. L.; Wilkins, C. L. Biomsd. Mass Spectrom. 1985, 12,
-
LITERATURE CITED (1) Hakamori, S.-I. J. Biochem. (Tokyo) 1984, 55,205-208. (2) Biermann, C. J. I n Analysls of Carbohydrates by OLC and Ms; Bier-
1737
424-428.
Coates. M. L.; Wilkins. C. L. Anal. Chem. 1987, 59. 197-200. Takayama, K.; Qureshi, N.; Hyver, K.; Honovich, J.; Cotter, R. J.; M a 5 cagni, P.; Schneider, H. J. Bid. Chem. 1988, 261, 10624-10631. Cotter. R. J.; Honovich. J.; Qureshi, N.; Takayama, K. Blomed. Envlron. ass Spectrom. 1987, 14,591-598. Lam, 2.; Comisarow, M. E.; Dutton, G. 0. S.; Weil, D. A.; Bjarnason, A. Rapid Commun. Mass Spectrom. 1987, I , 83-87. Lam, 2.; Comisarow, M. B.; Dutton, G. S. Anal. Chem. 1988, 6 0 ,
2304-2306. Qureshi, N.; Honovich, J. P.; Hara, H.; Cotter, R. J.; Takayama, K. J. Biol. Chem. 1988, 262,5502-5504. Martin, W. B.; Silty, L.; Murphy, C. M.; Raley, T. J., Jr.; Cotter, R. J.; Bean, M. F. Int. J. Mass Spectrom. l o n Processes 1989, 92,
243-265. VanBreemen, R. B.; Snow, M.; Cotter, R. J. I n t . J . Mass Spectrom. Ion Phys. 1983, 49,35-50. Otthoff, J. K.; Lys, I.; Demirev, P.; Cotter, R. J. Anal. Instrum. 1987,
16,93-115. Domon, B.; Costello, C. E. GlycoconjugateJ. 1988, 5 . 397-409. Van der Peyi, G. J. Q.; Isa, K.; Haverkamp, J.; Kistemaker, P. 0. Org. Mass Spectrom. 1981, 16, 416-420. Tabet. J.-C.; Cotter, R. J. Anal. Chem. 1984, 56, 1662-1667. Koell, P.; Steinweg, E.; Meyer, B.; Metzger, J. Llebigs Ann. Chem. 1982, 6 , 1039-1051.
RECEIVED for Review January 4, 1990. Accepted April 16, 1990. This work was supported by a grant (BBS 86-10589) from the National Science Foundation. B.S. was supported by a Deutsche Forschungsgemeinschaft Fellowship. Research was conducted a t the Middle Atlantic Mass Spectrometry Laboratory, an NSF-supported Regional Instrumentation Facility.
Determination of Complexing Ability of Natural Ligands in Seawater for Various Metal Ions Using Ion Selective Electrodes Takashi Midorikawa,*Eiichiro Tanoue, and Y ukio Sugimura Geochemical Laboratory, Meteorological Research Institute, Nagamine 1 - 2 , Tsukuba, Ibaraki 305, Japan
A newly developed method Is presented for the measurement of the aMllty of organic ligands In seawater to form complexes wlth metal ions. Organlc ligands (nominal molecular weight 2 1000) are concentrated from seawater and desalted by lyophlllzatlon and dialysis. The concentrated solution of natural ligands Is electrodialyzed wlth ethylenediaminetetraacetic acid to remove metal Ions. The demetallzed ligands obtained In thts way are tltrated wlth a metal ion to determine the complexhg abliky of the natural ligands. The advantages of this method are as follows: the formation of a complex between organic ligands and a speclflc metal Ion can be studied wlthout consideratlon of simultaneous side-reactlons; samples from dlfferent sources (sallne and freshwater, blologlcal, sedimentary, etc.) can be compared on the same basis; repeated measurements can be made wlth the same sample afler removal of the exogenously added metal ions. The abiilty of natural ligands In seawater to form complexes with copper( 11) and cadmium( 11) is discussed.
INTRODUCTION During the past 3 decades, increased attention has been paid to metal-organic associations in natural aquatic systems. The ability of dissolved organic matter to form complexes with metal ions in natural water is of interest because of the associated biological implications, such as the bioavailabdity and toxicity of metals to living organisms (1,2), and because of its relevance to efforts at understanding geochemical cycles of metals in the environment (3). In marine and, especially, in oceanic environments, the concentrations of metals and of organic complexing ligands are extremely low. Moreover, seawater contains high levels of inorganic salts with a complex composition. Therefore, analytical difficulties are inherent in efforts at measuring the ability of dissolved organic matter to form complexes with metals in natural seawater. Two general techniques have been used in attempts to determine the complexing ability of natural ligands in sea-
0003-2700/90/0362-1737$02.50/0@ 1990 American Chemical Society
