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[38] Desorption mass spectrometry of glycosphingolipids
[38]
DESOP-,PTION M S OF GLYCOSPHINGOLIPIDS
713
[38] D e s o r p t i o n M a s s S p e c t r o m e t r y of Glycosphingolipids By JASNA PETER-KATALINICand HEINZ EGGE Introduction Glycosphingolipids, by definition glycosides of the ceramides, occur ubiquitously on the outer leaflet of cell surface membranes of vertebrates and invertebrates. Their carbohydrate moiety may be composed of one up to more than forty sugar residues. 1 The ceramide portion (Cer), composed of a sphingosine base and a fatty acid moiety linked to each other by an amide bond, can vary in type. More than sixty different sphingosine bases have been described so far. 2The alkyl chains of the fatty acid moiety vary from C~4to C26and may contain double bonds and/or hydroxy groups. The nomenclature of glycosphingolipids (GSLs) is based on the carbohydrate moiety. A glycosphingolipid thus defined may, however, vary in its lipid portion, depending on species, organ, and cell type 3 (Fig. 1). Glucose (Glc), galactose (Gal), N-acetylglucosamine (GIcNAc), N-acetylgalactosamine (GalNAc), fucose (Fuc), sialic acids, and glucuronic acid (GIcA) are the most common sugar components in the mammalian GSLs, whereas mannose (Man), xylose (Xyl), and arabinose (Ara) may occur in GSLs of invertebrates. It is the carbohydrate moiety of a GSL that determines its biological specificity as an antigen or receptor whereas the lipid portion may influence its membrane topology and the physicochemical properties of these membranes. The complete structural elucidation of a glycosphingolipid implies the determination of the following parameters: (1) the molecular weight; (2) the number and the type of sugar components; (3) their sequence and pattern of branching; (4) the sites of linkages; (5) anomeric configuration; (6) the presence of additional functional groups, e.g., phosphates and sulfates and their site of linkage; (7) the sphingosine base; (8) the fatty acid moiety; and (9) the conformation of the GSL molecule. The analysis of the structural parameters (1-3) and (6-8) of GSLs has been successfully probed by mass spectrometry for 20 years, generally using thermal evaporation and electron ionization of appropriate derivatives. 4 This methodology, still of general value for the determination of I B. A. M a c h e r a n d C. C. Sweeley, this series, Vol. 50, p. 236. 2 K.-A. K a r l s s o n , Lipids 5, 878 (1970). 3 K. N a k a m u r a , Y. H a s h i m o t o , I'. Y a m a k a w a , and A. Suzuki, J. Biochem. (Tokyo) 103, 201 (1988). 4 H. Egge, Chem. Phys. Lipids 21, 349 (1978).
METHODS IN ENZYMOLOGY.VOL. 193
Copyright © 1990by AcademicPress, Inc. All riots of reproduction in any form reserved.
714
GLYCOCONJUGATES
[38]
IO(X
N5
§99
t23
eO0
L~3
A.
~600-
(m-Hr... iN9
200
MASS P(R CHARGE
1000
1o 800-
II51
B. 669
600
( I~"H)"
J/,!,
ls3 j
599
LTO ~ qOG I
526 524.
I ~ - - ""~
"-~-"'"
I,
630
L. '%
r,ob..... "imb'"-'" ~'~'
.o .o
I?O$
li| i
i~ " "'"'~D5 ....... i ~J'liib ..... ]~i)""
MASS PI[R CHAImI[
F]6.1. Negative ion FAB mass spectrum of the native GM3fraction from (A) human liver, (B) human melanoma, and (C) bovine brain. Shown in (D) is the molecular ion region of the permethylated GM3 fraction from human melanoma depicted in (B) recorded in positive ion mode. The ganglioside GM3fractions show heterogeneity due to variations in the sphingosine moiety, documented in the molecular ion region by (M - H)- ions which appear separated by 28 mass units. The structurally unrelated impurity at m/z 599 [in (A) and (B)] is probably of biological origin and is destroyed during the permethylation.
the parameters (2-4) and (7-8), is, however, limited by thermal stability and the absolute necessity to derivatize under conditions that eliminate alkali-labile substituents. The soft ionization methods especially fast atom bombardment (FAB) 5 and liquid SIMS (secondary ion mass spectrometry),6 in combination with 5 M. Barber, R. Bardoli, R. D. Sedgwick, and A. N. Tyler, J. Chem. Soc. Chem. Commun. p. 325 (1981). 6 W. Aberth, K. M. Straub, and A. L. Burlingame, Anal. Chem. 54, 2029 (1982).
[38]
DESORPTION MS ov GLYCOSPHINGOLIPIDS
AJ.Ifd131N ! 3^I~V'13J
m...
A~ISN3Z41 3kI~V13~
715
716
GLYCOCONJUGATES
[3 8]
the development of high-field instruments, have brought substantial progress. It is now possible to analyze native or derivatized GSL structures within several minutes and to obtain intense molecular and sequence ions on molecules comprising more than 25 sugar residues. 7 Core Structures in Naturally Occurring Glycosphingolipids The following five major oligosaccharide structures have been observed in naturally occurring GSLs linked to Glc(fll--->l)Cer: Gal(fl I---~3)GalNAc(,81---~4)Gal(//l--~4)Gal(fl l--)3)GlcNAc(fl1--)3)Gal(fl 1---)4)Gal(/3 I---~4)GlcNAc~l--->3)Gal(fll-->4)GalNAc~ 1--->3)Gal(al--->4)Gal(fll--->4)GalNAc(/31--~4)GlcNAc(fl 1->3)Man(ill-->4)-
ganglio core lacto-N core neolacto-N core globo core arthro core
The sugar constituents and sequences of neutral oligosaccharides derived from these cores can be determined by desorption mass spectrometry from the fragment ions in terms of hexoses and N-acetylhexosamines in native or derivatized samples. Gangliosides (by definition GSLs, which contain sialic acids), can be derived from the ganglio, lacto-N, and/or neolacto-N series. The major representative of the sialic acids is 5-N-acetylneuraminic acid (Neu5NAc). It can be modified by additional hydroxy, deoxy, acetyl, methyl, lactyl, phosphate, and/or sulfate groups. Presently, thirty-two different sialic acids are known. 8 Beside sialic acids, other charged groups like carboxylate, sulfate, phosphate, phosphonate, and/or phosphoethanolamine may also be present. Such structural features play an important role in the proper choice of the most efficient method for analysis by mass spectrometry. Stereochemical factors may also be involved in the formation of parent as well as daughter ions in the gas phase, but no general rules have been established so far. Fragmentation Patterns
Molecular ions, (M - H ) - , and (M + H) ÷ or (M + cation) +, provide direct information on molecular weight, whereas information on type and number of sugar components can be deduced indirectly from these values. Primary or parent ions, formed after the cleavage of glycosidic bonds 7 H. Egge and J. Peter-Katalini~, Mass Spectrom. Reo. 6, 331 (1987). 8 R. Schauer, this series, Vol. 138, p. 132.
[38]
DESORPTIONMS oF GLYCOSPHINGOLIPIDS
717
° .2
I I I I SITE ~X~wa CLEAVAGE m
3
+2H~///// B "X~2H +~O~OR2 5
-0
o-
OR2 6
y
B~OR2 +
oH
O--
7
8_
SCHEME 1.
from both possible sites (pathway I and pathway II in Scheme 1), furnish information about sequence and branching pattern. The cations of types 1 and 4 should be observed in experiments performed in the positive ion mode and the anions of types 2 and 3 in the negative ion mode, if both pathways would randomly take place. In fact, the fragmentation pattern for native GSL in positive ion mode favors the pathway I, but beside the primary ion 1, the daughter ions 5 and 8, arising from the protonation of 2 and 3, respectively, can be observed. Therefore, an exact statement concerning the homogeneity of the molecular species with the same core structures and the branching patterns from these experiments is not possible. In addition, the parent ions are approximately of the same intensity as the daughter ions; consequently, a distribution of the total ion intensity over a number of peaks takes place. In the positive ion desorption mass spectra of derivatized (permethylated, peracetylated or perbenzoylated) GSL, the oxonium ion of type 1 is so much favored that the sugar sequence from the nonreducing terminus and the branching pattern, in addition to the molecular homogeneity,can easily be deduced directly from the spectrum. The cleavage of sugar C--C bonds, observed in direct chemical ionization (DCI) 9 or fast atom bombardment (FAB)-MS/MS, 1° does not take place.
9 V. N. Reinhold, this series, Vol. 138, p. 59. 10 B. Domon and C. Costeilo, Biochemistry 27, 1534 (1988).
718
GLYCOCONJUGATES
[38]
O
E
==
~.o
t~ Crl
~..-
~
.
-~
~"---I -oo
°°~
OI
E~ m~
z~ ~o
'
='
'
~
IIISNI~NI lAl ~¥'111
'
"
'
X£1SN31NI 3^1~V~3~
[38]
DESORPTION MS OF GLYCOSPHINGOLIPIDS
719
In positive ion desorption mass spectra of permethylated acidic and neutral GSL, the preferred cleavage site in the sugar chain is that of Nmethyl-N-acetylamino sugar of type 9 where R is CH 3 or sugar residue. Oxonium ions of type 11 derived from mannose, are more abundant than 10, derived from glucose or galactose, as found in GSL of the arthro series, i i The daughter ions, diagnostically important for the discrimination between 1,3 and 1,4/6-substitution at 2-deoxy-2-acetamido sugars, are formed from the corresponding parent ions of type 9 by elimination of the substituent on C-3.12
Z~_~+R
CHzOR
H3C/N.~cOCH3
OR 10
Ch~OR
1"1
In negative ion desorption mass spectrometry, performed on the native GSL, generally the formation of alcoholate type of ion 2 (pathway I) is favored. The native GSLs carrying negatively charged substituents, like gangliosides and uronic acid-containing GSLs, however, also exhibit characteristic abundant sequence ions of type 3 (pathway II). By combining the information obtained from the fragmentation patterns via pathways I and II it is possible to obtain the complete sequence information from one single experiment. The sequence of two isomeric gangliosides, GMla and GM1b, with different attachment sites of the Neu5NAc, is depicted with their negative ion FAB mass spectra in Fig. 2. The structure of GSLs containing neutral sugars and alkali-labile substituents can be elucidated preferentially from native samples in the negative ion mode or peracetylated samples in positive or negative ion mode desorption mass spectra. The carbohydrate sequence can be deduced from appropriate abundant primary ions, as discussed previously. For the structure determination of the ceramide residue, four types of ions can be used: the ceramide cation of the type 12 and anion 13, in addition to a sphingosine-derived cation 14 and a cation of type 15 arising after the loss of the acyl group from the molecular ion and subsequent protonation:
11R. D. Dennis, R. Geyer, H. Egge,J. Peter-Katalini~,S.-C. Li, S. Stirm,and H. Wiegandt, J. Biol. Chem. 260, 5370 (1985). t2 N. K. Kochetkovand O. S. Chizhov,Ado. Carbohydr. Chem. 21, 39 (1966).
720
GLYCOCONJUGATES o
[38]
®
- CH:~CH- CH-CH-CH-(CH2)~ CH3 NR OR
~0 i
- 0- CH2-,CH-CH-C H=CH-(CH2)r~CH3 NR OR
co l {E,H2)n
(!H2}n CH3
CH3
12 ®
cH2- -cH
~ oR'
]
Sugar-O-CH2-CI-CH-CH=CH-ICH2)n~CH3 +H+ or Cation" NHR lS w h e r e R is H , C H 3 ; R' is H , C H 3 , C O C H 3 . I n the positive ion spectra f r o m
the permethylated GSLs, the ceramide moiety is generally represented by the cations 12, 14, and 15; in the peracetylated samples however, only 14 is of high abundance. The cation 14 is also abundant in the positive ion desorption mass spectra of the native GSLs. For the structure determination of the ceramide moiety in sulfatide, the positive ion FAB-MS of peracetylated sulfatide and the negative ion FAB-MS of lysosulfatide were used (Fig. 3).
Design of Appropriate Experimental Protocol for Analysis by Mass Spectrometry A rational protocol for the structural elucidation of a small amount (-I00/~g) of a G S L should include the following set of experiments: I. The negative ion mass spectrum of the native sample provides (M - H)-, (Ccr)-, and two series of ions, of type 2 and type 3 (Scheme I). With these data a structural proposal for the sugar moiety and the ccramidc, as a whole, can bc made. 2. The positive ion mass spectrum of the native sample furnishes (M + H) + or (M + cation)+, Ccr + 12, and sphingosinc-dcrivcd ion 14. 3. After peracetylation the positive ion mass spectrum providcs (M + H) + or (M + cation)+ beside oxonium sugar sequence ions of type I and a sphingosinc base-derived ion 14.
[38]
DESORPTION MS OF I001
GLYCOSPHINGOLIPIDS
o|
A. t
HO-S-O-Gat- 0 - C e r
3
6
I ~0
!.ooi
2N !?
~qO0"
+t, ,ootB"
332
tOOO 1060
~ S S PER CHARGE
0
5 H 0 - S - 0 - O a l - 0-Sph
,' BOO"4
721
8
/
~>
'
z soe
10?
(M-Hk- 540
ThgL
,ooi 20Q
~? ....
ThIL 215
El i
~
I
;
ThgL 321
!: 2+, 0 + T i !
' ~SS
l
PER CHARGE
FIG. 3. FAB mass spectra of the sulfatide: HSO3-GaI-Cer. (A) Positive ion spectrum of peracetylated sulfatide showing (M + H) ÷ at m/z 1060. The Cer ÷ fragment ion 12 at m/z 674 is not present, but the sphingosine ion 14 at m/z 264 indicates that the fatty acid is a lignoceric acid. (B) Negative ion FAB mass spectrum of lysosulfatide. The sugar moiety is unaffected by the change in the ceramide moiety, as documented by the fragment ions at m/z 97 (HSO4)-, rn/z 259/257 (HSO3-- Gal)-, and m/z 300 ( H S O 3 - - G a I - - O - - C H = C H - - N H 2 ) - .
4. After permethylation using NaOH/methyl iodide in dimethyl sulfoxide (DMSO) 13 the positive ion mass spectrum provides (M + H) + or (M + cation) +, oxonium sequence ions 1, their respective daughter ions, Cer + 12, sphingosine base-derived ion 14, and (MH - acyl + I) + ion IS. t3 I. Ciucanu and F. Kerek, Carbohydr. Res. 131, 209 (1984).
722
GLYCOCONJUGATES
[38]
Sample Preparation The isolation of GSLs from biological material is usually achieved by extraction, followed by several separation steps using adsorption and ionexchange chromatography adapted to the type of GSL under investigation. 14,15Frequently, as a final purification step, semipreparative chromatography on high-performance TLC plates is included. During the extraction from the plate, considerable amounts of salt, silica, and binder may be coeluted which may subsequently interfere with the ion desorption process. Here the use of high-performance TLC plates with gypsum as binder 16 or an additional clean-up step using reversed-phase C18 material is recommended. Alternatively, such contaminants can be cleaned out from the GSL sample by use of 5-cm Iatrobeads (silica gel beads of defined size, supplied by Macherey, Nagel and Co., D-5160 Dtiren, FRG) columns eluted by solvent mixtures (chloroform/methanol/water) of increasing polarity. Problems rising from minor salt contamination can be resolved by adding 1/xl of 1 N aqueous HC1 directly to the sample on the target. In some cases, the samples can be more efficiently purified if derivatized by permethylation, peracetylation, perbenzoylation, or by several derivatization steps, based on the differences in chemical reactivity between functional groups present (see [35] in this volume). The purified and thoroughly desalted native GSLs can be analyzed by positive or negative ion mode desorption.
Types of Ionization for Glycosphingolipids Field Desorption Field desorption (FD) ionization, developed by Beckey and Schulten, 17 was used for structural analysis of a few native neutral and acidic GSLs with 1 to 6 sugar residues. The FD mass spectra were dominated by molecular ions (M + H) ÷ or (M + cation)÷, but the fragments of type 1 and 4 were rather weak. In the permethylated samples, the fragments of type 1 became more abundant.18 14j. N. Kanfer and S. Hakomori, "Sphingolipid Biochemistry." Plenum, New York, 1983. 15R. Kannagi, K. Watanabe, and S. Hakomori, this series, Vol. 138, p. 3. 16 p. Hanttand, Fur. J. Biochem. 87, 161 (1978). t7 D. Beckey and H. R. Schulten, Angew. Chem. 87, 425 (1975). ts y. Kushi, S. Handa, H. Kambara, and K. Shizukuishi, J. Biochem. (Tokyo) 94, 1841 (1983).
[38]
DESORPTION M S OF GLYCOSPHINGOLIPIDS
723
Chemical Ionization Chemical ionization (CI) has been applied to the analysis of GSLs using ammonia as a reagent gas for native and for derivatized neutral 9 and acidic 19,2° GSLs. The oligosaccharides derived from gangliosides after oxidative cleavage of the lipid moiety by ozone and subsequent reduction were analyzed by CI using ammonia and isobutane as reagent gases. 2° The underivatized GSLs limited to three to five sugar units were analyzed by DCI. Theuseful results by CI for larger molecules up to the mass of 3000 u were obtained using different sets of derivatization procedures, like permethylation or, alternatively, permethylation with subsequent reduction by LiA1H4 , or permethylation, reduction, and trimethylsilylation. The fragmentation patterns are rather complicated because of potential alternate addition of water and/or reagent ga s to the parent ions.
Liquid Secondary Ion Mass Spectrometry In liquid secondary ion mass spectrometry (LSIMS) experiments with a primary beam of xenon ions, native and derivatized GSLs have been analyzed in the positive and in the negative ion mode. With positive ion LSIMS, the molecular ions (M + H) + and/or (M + cation) + appeared, in general, to be of lower intensity than the fragment ions. For acidic GSLs, like sulfoglycolipids, the negative ion LSIMS of underivatized samples appeared to give, similar to the FAB ionization, more structural information than with the positive ion mode. 2~ Abundant molecular ions (M - 1)- and/or (MNa + - 2)- were accompanied by significant fragment ions of type 2 and 3, useful for the determination of the sugar sequence and the site of sulfate substitution. Thus far, the desorption behavior and fragmentation patterns for LSIMS seem similar to those for FAB.
Fast Atom Bombardment The majority of fast atom bombardment (FAB) mass spectra of GSLs have been acquired on VG ZAB-IF or HF, VG 70-250, and VG 7070E (VG Analytical, UK), and JMS DX-300, JMS-DX 303, JMS HX-100, and JEOL HXll0/HXI10 (JEOL, Ltd., Japan) instruments. Ion guns operating with argon or xenon as bombarding gas produced an atom beam 19 S. A. Carr and V. N. Reinhold, Biomed. Mass Spectrom. 11, 633 (1984). 2o T. Ariga, R. K. Yu, M. Suzuki, S. Ando, and T. Miyatake, J. Lipid Res. 23, 437 (1982); Y. Tanaka, R. K. Yu, S. Ando, T. Ariga, and T. Itoh, Carbohydr. Res. 126, 1 (1984). 21 y. Kushi, S. Handa, and I. Ishizuka, J. Biochem. (Tokyo) 97, 419 (1985).
724
GLYCOCONJUGATES
[38]
of an energy equivalent to 8-10 keV and a current of 5 x 10 -8 A. The acceleration voltage used varied from 10 to 3 kV, depending on the type of instrument and the extension of the mass range required. On the VG ZAB-HF instrument, an acceleration voltage of 4 kV was necessary to extend the mass range in order to obtain the molecular (M + Na) ÷ ion at m/z 6181 (nominal mass) of permethylated ceramide pentacosasaccharide from rabbit erythrocyte membranes. 22 As in the case of liquid SIMS, the solubility of the sample in the liquid matrix is the prerequisite. Matrices like glycerol, thioglycerol, a I : 1 mixture of glycerol/thioglycerol, triethanolamine, 1,1,3,3-tetramethylurea, a 5 : 1 mixture of triethanolamine/1, 1,3,3-tetramethylurea, and a 4 : 1 mixture of 1,4-dithioerythritol/1,4-dithiothreitol have been successfully applied to native and derivatized neutral and acidic GSLs. The final choice of the appropriate matrix must include considerations about polarity and intrinsic chemical properties of the molecule(s) to be analyzed. In our hands one of the most useful matrices is thioglycerol, having the single disadvantage of rapid evaporization in high vacuum. The sample of neutral GSL can be dissolved to a clear solution in methanol, chloroform/methanol, 1:1 (v/v), or chloroform/methanol/ water, 75 : 25 : 4 (v/v/v) containing 1-10 /xg//zl. Methanol/concentrated acetic acid (1 : 1, v/v) is considered to be the appropriate solvent for acidic GSLs. The sample solution is applied by a microsyringe to the stainless steel target, coated with about 2/zl of matrix. After the evaporation of the solvent by air or hair dryer, the probe with the sample is introduced to the vacuum and subjected to FAB. Due to the evaporation of the matrix during the acquisition of spectra, it is necessary to run in both up-scan and downscan mode in order to obtain intense fragments as well as molecular ions. The addition of 1/zl of I% solution of sodium acetate in methanol to the matrix is followed by an increase of the molecular ion and decrease in the intensity of fragment ions. The typical scan speed for instruments without a data system is approximately 1 sec/decade, depending on mass range and amplification. Use of a data system for multiple acquisitions or signal averaging, can increase the speed of a single scan to about 0.3 sec/decade, thus typically accumulating 10-20 scans. FAB mass spectrometry was applied in positive and negative mode to a large variety of native and derivatized GSLs in order to determine the primary structure of substances involved in recognition processes, pathological states or receptor phenomena. The potency of this method in obtaining several sets of clear-cut data
22 p. Hanfland, M. Kordowicz, J. Peter-Katalini~, H. Egge, J. Dabrowski, and U. Dabrowski, Carbohydr. Res. 171t, 1 (1988).
[38]
DESORPTIONMS OF GLYCOSPHINGOLIPIDS
725
and the simplicity of sample handling are the reasons for its wide and rapid acceptance .7,23-25 Combined Techniques TLC-LSIMS and TLC-FAB-MS Matrix-assisted SIMS and FAB-MS can be applied to the analysis of GSLs on TLC plates without prior elution. After TLC separation on an aluminum- or plastic-backed silica gel, the spots are visualized by iodine vapor or Coomassie blue staining. The area of interest on the plate is cut out (max. size 5 mm x 20 mm) and attached with double-face masking tape to a SIMS or FAB probe tip. After adding 1-2/~1 of solvent and 2-5 /zl matrix to the TLC strip, the probe tip is bombarded by Xe ÷ ions or Xe atoms under standard conditions. 26 Changes in ganglioside expression have been evaluated in rat fibroblasts after transfection with sarcoma virus DNA 27 using this method. Similarly, the glycolipid mapping of spleen and liver tissues from patients affected by metabolic glycolipid disorders caused by lysosomal enzyme deficiencies 28 and the structure of neoglycolipids after the reductive amination of human milk oligosaccharides with phosphoethanolamine-containing glycerides 29 have been established. Resuits from other experiments will be needed to evaluate the potency of this method on the higher GSLs. HPLC-C! and HPLC-FAB Positive ion CI mass spectra were obtained on HPLC separated neutral mono- to tetraglycosylceramides on-line using ammonia as the reagent gas. The observed fragmentation patterns were quite complex, even for ceramide mono- and disaccharides. As a general feature ofCI desorption, 3° the interpretation of these spectra is difficult, because primary ions tend to add or to eliminate water and/or ammonia. 23 A. Dell, Adv. Carbohydr. Chem. Biochem. 45, 20 (1987). 24 M. Arita, M. Iwamori, T. Higuchi, and Y. Nagai, J. Biochem. (Tokyo) 94, 249 (1983); Y. Ohashi, M. Iwamori, T. Ogawa, and Y. Nagai, Biochemistry 26, 3990 (1987). M. Suzuki, K. Nakamura, Y. Hashimoto, A. Suzuki, and T. Yamakawa, Carbohydr. Res. 151, 213 (1986). Y. Kushi and S. Handa, J. Biochem. (Tokyo) 98, 265 (1985). 27 H. Nakaishi, Y. Sanai, M. Shibuya, M. Iwamori, and Y. Nagai, Cancer. Res. 48, 1753 (1988). 2* y. Kushi, C. Rokukawa, and S. Handa, Anal. Biochem. 175, 167 (1988). 29 p. W. Tang and T. Feizi, Carbohydr. Res. 161, 133 (1987). 3o j. E. Evans and R. H. McCluer, Biomed. Environ. Mass Spectrom. 14, 149 (1987).
726
GLYCOCONJUGATES
[38]
S F C - C I and S F C - F A B
In supercritical fluid chromatography (SFC), a mobile phase consisting of a highly compressed gas near or above its critical temperature and pressure is used as a mobile phase. This separation method is more rapid than conventional HPLC and provides a higher number of effective plates at low temperature. The separation of GSLs by SFC requires permethylation in order to decrease their polarity and can be combined with CI or FAB mass spectrometry) 1 Analysis of Mixtures The complete separation of complex GSL mixtures into individual compounds can seldom be achieved even with the sophisticated methods available at the present time. 14,15The physical characteristics of individual molecular species of GSL arise from the structural variations of building blocks present in such a multifunctional molecule. The overlapping can occur due to the presence of (a) sugar chain homologs; (b) isomeric sugar sequences; (c) different sites of glycosidic linkages; (d) linearity vs. branching of the core structures; (e) the presence of other functi6nal groups like esters, lactones, sulfates, and phosphates; (f) the modifications of the basic structures by oxidation state (carboxy groups, deoxy sugars); (g) alkyl homologs arising from sphingosinc base and/or fatty acid; (h) modifications in thc lipid portion, such as in lysocompounds and in ncoglycolipids; (i) molecules structurally completely unrelated to GSLs; (j) stcrcochcmistry on the anomeric center of the glycosidic bond. A general discussion of analysis of mixtures is presented elsewhere in this volume [34] by Samuclsson et aL Only specific issues concerning desorption techniques will be discussed here. In Fig. 4, the methodological approach of handling a mixture by FAB mass spectrometry is shown. About 30 ~g of a ganglioside fraction was obtained after several chromatographic separation steps from a T-lymphoma cell line) 2 Whereas negative ion mode FAB did not give unequivocal results, the positive ion FAB spectrum obtained after permethylation revealed the presence of three molecular species. Their structures were definitely assigned by matching of fragment mass increments with molecular ions present in the mass spectrum of the mixture. If the analytc represents a mixture of analogs with the same core structure differing only in the absence of the terminal sugar unit, the 31 j. Kuei, G. R. Her, and V. N. Reinhold, Anal Biochem. 172, 228 (1988). 32 j. Mfithing, J. Peter-Katalinid, H. Egge, U. Neumann, B. Kniep, A. Loyter, and P. F. Mfihlradt, Proc. Xth Int. Syrup. Glycoconjugates, Jerusalem, September (1989).
[38]
DESORPTION M S OF GLYCOSPHINGOLIPIDS
727
Zo_.~ _o~.,E O
~
~,
oo
m
~
e-,
~=o.~.
~ .
•
0
z
X
,0
®o
~ o
~u m
o
m
•
.-,
¢~
,
?
m i
<.~
z
=
o-~
i
~"
~L[~3LN!
3^I~¥~3i
~
,...A ~
728
GLYCOCONJUGATES
[3 8]
°
uz
o
r-~-
r u
u
I N N
.
|
.-
O X % \
d
c~
d
:
*
l
A215N3~NI
*
\
•
3^]~¥73~
i
~
T
i
~
!
I
A£ISN]~NI
i
3A[LV'I]a
i
,
,
[38]
DESORPTIONMS OF GLYCOSPHINGOLIPIDS
729
negative ion spectra will not differentiate between ions arising from the cleavage of the glycosidic bond and the molecular ion of the lower homolog. This differentiation is, however, possible in positive ion desorption spectra of the derivatized samples due to pronounced expression of oxonium ions of type 1 and their specific mass increments. In Fig. 5 the positive ion FAB-MS of a permethylated ganglioside fraction, showing immunological activity toward VIM-2 antibody, is depicted. The only difference between the two components is the presence of an additional fucose .33 The presence of isomeric structures with the same molecular ion but different sugar sequences can be postulated if more than one set of sequence ions is detected both in negative ion desorption spectra of the native sample and in the positive ion mode of the derivatized sample. The presence of positional isomers can be detected to some extent if two daughter ions of different specificity occur in the same spectrum of the permethylated GSL. 34 This may be of particular importance for correlation of structural data of specific carbohydrate epitopes with immunological methods, e.g., immunostaining. 35 The aspect of linearity vs. branching of sugar cores in GSLs can successfully be analyzed in native and in derivatized s a m p l e s . 7,23,36 It is an important factor in studying the biosynthetic specificity of glycosyltransferases. Naturally occurring lactones have been found in GM~ and GDlb gangliosides as demonstrated in the negative ion desorption spectra of native 33 B. Kniep, J. Peter-Katalini~, H. Egge, W. Knapp, and P. F. Miihlradt, in preparation. H. Egge, J. Peter-Katalini6, M. Hergersberg, F.-G. Hanisch, R. Bruntz, and G. Uhlenbruck, Proc. Xlllth Int. Carbohydr. Symp. Ithaca, NY, August (1986). 35 j. Magnani, S. L. Spitalnik, and V. Ginsburg, this series, Vol. 138, p. 195. H. Egge, M. Kordowicz, J. Peter-Katalini~, and P. Hanfland, J. Biol. Chem. 260, 4927 (1985).
FIG. 5. Positive ion FAB mass spectrum of the permethylated ganglioside fraction isolated from human spleen, carrying the VIM-2 antigen epitope. (A) Lower mass region scanned in thioglycerol without addition of sodium acetate. (B) Molecular ion region scanned after addition of sodium acetate to the matrix. Two gangliosides, NeuAcHexsHexNAc3Cer (MNa + = 2718) and dHexNeuAcHexsHexNAc3Cer (MNa ÷ = 2892) show the same sugar core structure as documented by common fragment ions at m/z 376-344 (NeuAc ÷) and m/z 825-793 (NeuAcHexHexNAc +). The ions at m/z 1274-1242 (NeuAcHex2HexNAc 2+) versus m/z 1448 (dHexNeuAcHex2HexNAc2 ÷) and m/z 1723 (NeuAcHex3HexNAc3 ÷) versus m/z 1897 (dHexNeuAcHex3HexNAc 34) reflect the structural variation on the second lactosamine unit. Both gangliosides have their respective carbohydrate portion bound to the same type of ceramide, carrying palmitic acid.
730
GLYCOCONJUGATES
[38]
I
.m W e.L
N
c,O_
"I-
i ~J.ISN3J.NI 3 ^ I J.Y13~l
a'a'-
~
i
ALISNB£NI 3 ^ I £ V ~ 3 ~
I I I ,~P41/,.NI 1^ I ~V'l~141
[38]
DESORPTIONMS OF GLYCOSPHINGOLIPIDS
731
samples. 37The lactonization is a reversible reaction, which can be induced chemically by adding mineral acids to the samples; lactones will be easily hydrolyzed under basic conditions. Other base- and/or acid-labile esters are the O-acetates, s O-sulfates, 3s,39 and O-phosphoethanolamines. They can be localized in their desorption mass spectra by altered sugar mass increments and molecular ions. In Fig. 6 the negative ion FAB mass spectrum of a GSL isolated from Calliphora vicina is presented. To the sugar core of an arthro type, 11 a phosphoethanolamine substituent is attached. Its free amino group was acetylated by l : t mixture of (CH3CO)20/(CD3CO)20 and then peracetylated with (CH3CO)20. 4° The isotopic label in their respective molecular ions was visible in the negative mode FAB mass spectrum (Fig. 6B and C). Comparison with Other Analytical Methods The topo- and stereochemistry of the glycosidic linkages are the dominating factors that control the shape of the terminal epitopes of the carbohydrate antigens of GSLs. Information concerning glycosidic linkages can be obtained from experiments using nuclear magnetic resonance (NMR) and from combined GC/MS techniques; desorption mass spectrometric analysis has also been utilized. Immunostaining methods, based on antigen-antibody complex formation, have become available for searching for specific sugar-antigen epitopes in mixtures of GSLs, 35 particularly tumor-associated antigens. 41 If possible, such techniques should be used in concert with desorption mass spectrometry in order to characterize a yet unknown antigen or to define the specificity of a new antibody. The optimal approach is to permethylate the GSL antigen and to observe its specific parent and respective daughter ions in positive ion mode desorption mass spectra. The use of linked scan techniques a2 should also be considered. 37 S. Sonnino, G. Kirschner, G. Fronza, H. Egge, R. Ghidoni, D. Acquotti, and G. Tettamanti, Glycoconjugate J. 2, 343 (1985). 3g H. Leffler, G. C. Hansson, and N. Str/Smberg, J. Biol. Chem. 261, 1440 0986). 39 T. Ariga, T. Kohriyama, and L. Freddo, et al., J. Biol. Chem. 262, 848 (1987). 40 F. Helling, R. D. Dennis, M. Keller, J. Peter-Katalini6, H. Egge, and H. Wiegandt, in preparation. 41 S. Hakomori, Cancer Res. 45, 2405 (1985). 42 A. G. Brenton and J. H. Beynon, Eur. Spectrosc. News 29 (1980). FIG. 6. The negative ion FAB mass spectrum of the GSL from CaUiphora oicina pupae carrying a phosphoethanolamine substituent.40 (A) The native sample; (B) the molecular ion region of the same sample after N-acetylation with (CHaCO)20/(CD3CO)20; (C) the molecular ion region of the sample depicted in (B) after peracetylation.
732
GLYCOCON.IUGATES
[3 8]
Monitoring of Chemical and Enzymatic Reactions
The structural changes induced by chemical reactions of GSLs as polyfunctional molecules include derivatizations ([35] in this volume), oxidative and reductive cleavages of one or more building units ([30]-[32] in this volume), oxidations, and reductions. FAB mass spectrometry has been used to monitor the topospecifity of metaperiodate oxidation in GSLs from rabbit erythrocytes43 or for acid-catalyzed hydrolysis of neuraminic acid: 4 The synthesis of lyso compounds and their derivatives from GSLs by removal of the fatty acid moiety under basic conditions,45 leaves the carbohydrate moiety unaffected, as shown by negative ion mode FAB spectra (Fig. 3B). The removal of the lipid moiety from a GSL by the enzyme ceramidase (isolated from leach or from microorganisms)~ opens the possibility to study the liberated oligosaccharides by desorption mass spectrometry and by comparison with already known sugar structures. On the other side, reductive elimination of the terminal aldehyde group of oligosaccharides with nonpolar amines improves their desorption properties and increases the sensitivity of the measurement.29,47,48 Exoglycosidase degradation of the branched GSLs from rabbit erythrocytes by a-galactosidase,22,36or a ganglioside from mouse spleen showing choleragenoid-binding activity from mouse spleen by/3-galactosidase49 were monitored by FAB mass spectrometry. It was also shown by FAB methods, that the removal of the neuraminic acid by neuraminidase from Vibrio cholerae on the TLC plate is complete only in GDla,but not in GDI~ or GDIb .50
Summary Desorption mass spectrometry has become an important tool for sequencing and mapping of glycosphingolipids of natural, synthetic, or semisynthetic origin. The appropriate combination of different desorption mass 43 H. Egge and J. Peter-Katalini~, in "Mass Spectrometry in the Health and Life Sciences" (A. Burlingam¢ and N. Castagnoli, eds.). Elsevier, Amsterdam, 1985. 44 J.-E. Mansson, H. Mo, H. Egge, and L. Svennerholm, FEBS Lett. 196, 259 (1986). 45 G. Schwarzmann and K. Sandhoff, this series, Vol. 138, p. 319. 46 S.-C. Li, R. DeGasperi, J. E. Muldrey, and Y.-T. Li, Biochem. Biophys. Res. Commun. 141, 346 (1986); M. Ito and T. Yamagata, J. Biol. Chem. 261, 14278 (1986). 47 S. Dreyer, H. Egge, and J. Peter-Katalini~, in preparation. 4s L. Poulter and A. L. Burlingame, this volume [36]. 49 K. Nakamura, M. Suzuki, F. Inagaki, T. Yamagawa, and A. Suzuki, J. Biochem. (Tokyo) 101, 825 (1987). so j. Miithing, B. Schwinzer, J. Peter-Katalihi~, H. Egge, and P. F. Miihlradt, Biochemistry 28, 2923 (1989).
[39]
HIGH-MASS GC/IVIS OF PERMETHYLATED OLIGOSACCHARIDES
733
spectrometric techniques with other spectroscopic, enzymatic, chemical, and/or immunological methods represents the most direct and efficient way to establish frequent, yet unknown, molecular structure-function relationships. Acknowledgments We thank Dr. A. Vogel and Prof. K. Sandhoff, Bonn, for the sample of lysosulfatide. This work was generously supported by the Deutsche Forschungsgemeinschaft.
[39] H i g h - M a s s Gas C h r o m a t o g r a p h y - M a s s S p e c t r o m e t r y of P e r m e t h y l a t e d Oligosaccharides By
G U N N A R C . HANSSON a n d HASSE KARLSSON
Capillary gas chromatography-mass spectrometry (GC/MS) is a fast, sensitive, and high-resolving method for the characterization of biomolecules. It has been used in glycoconjugate research for analyzing the partially methylated alditol acetates after degradation of the glycoconjugates1 and, in a few cases, for the analysis of oligosaccharides after permethylation2-4 or trifluoroacetolysis. 5 The advent of high-temperature capillary GC using thin-film thermostable bonded stationary phases now allows the analysis of large permethylated oligosaccharides,6-8 an adaptation that should also be useful in other fields of biomedical research. Preparation of Oligosaccharides Glycosphingolipids
Glycan is cleaved7 from the ceramide portion of glycosphingolipids by endoglycoceramidase from Rhodococcus 9 (Genzyme Corp., Boston, MA) I This series, Vol. 50 [1]. 2 p. Hallgren and A. Lundblad, J. Biol. Chem. 252, 1014 (1977). 3 H. van Halbeek, L. Dorland, J. Haverkamp, G. A. Veldink, J. F. G. Vliegenthardt, B. Fournet, G. Ricart, J. Montreuil, W. D. Gathmann, and D. Aminoff, Eur. J. Biochem. 118, 487 (1981). 4 T. Tsuji and T. Osawa, Carbohydr. Res. 151, 391 (1986). 5 B. Nilsson and D. Zopf, Arch. Biochem. Biophys. 222, 628 (1983). 6 H. Karlsson, I. Carlstedt, and G. C. Hansson, FEBS Lett. 226, 23 (1987). 7 G. C. Hansson, Y.-T. Li, and H. Karlsson, Biochemistry 28, 6672 (1989). s H. Karlsson, I. Carlstedt, and G. C. Hansson, Anal. Biochem. 182, 438 (1989). 9 M. Ito and T. Yamagata, J. Biol. Chem. 261, 14278 (1986).
METHODS IN ENZYMOLOGY,VOL. 193
Copyright© 1990by AcademicPress. Inc. All fightsof reproductionin any formreserved.
DESOP-,PTION M S OF GLYCOSPHINGOLIPIDS
713
[38] D e s o r p t i o n M a s s S p e c t r o m e t r y of Glycosphingolipids By JASNA PETER-KATALINICand HEINZ EGGE Introduction Glycosphingolipids, by definition glycosides of the ceramides, occur ubiquitously on the outer leaflet of cell surface membranes of vertebrates and invertebrates. Their carbohydrate moiety may be composed of one up to more than forty sugar residues. 1 The ceramide portion (Cer), composed of a sphingosine base and a fatty acid moiety linked to each other by an amide bond, can vary in type. More than sixty different sphingosine bases have been described so far. 2The alkyl chains of the fatty acid moiety vary from C~4to C26and may contain double bonds and/or hydroxy groups. The nomenclature of glycosphingolipids (GSLs) is based on the carbohydrate moiety. A glycosphingolipid thus defined may, however, vary in its lipid portion, depending on species, organ, and cell type 3 (Fig. 1). Glucose (Glc), galactose (Gal), N-acetylglucosamine (GIcNAc), N-acetylgalactosamine (GalNAc), fucose (Fuc), sialic acids, and glucuronic acid (GIcA) are the most common sugar components in the mammalian GSLs, whereas mannose (Man), xylose (Xyl), and arabinose (Ara) may occur in GSLs of invertebrates. It is the carbohydrate moiety of a GSL that determines its biological specificity as an antigen or receptor whereas the lipid portion may influence its membrane topology and the physicochemical properties of these membranes. The complete structural elucidation of a glycosphingolipid implies the determination of the following parameters: (1) the molecular weight; (2) the number and the type of sugar components; (3) their sequence and pattern of branching; (4) the sites of linkages; (5) anomeric configuration; (6) the presence of additional functional groups, e.g., phosphates and sulfates and their site of linkage; (7) the sphingosine base; (8) the fatty acid moiety; and (9) the conformation of the GSL molecule. The analysis of the structural parameters (1-3) and (6-8) of GSLs has been successfully probed by mass spectrometry for 20 years, generally using thermal evaporation and electron ionization of appropriate derivatives. 4 This methodology, still of general value for the determination of I B. A. M a c h e r a n d C. C. Sweeley, this series, Vol. 50, p. 236. 2 K.-A. K a r l s s o n , Lipids 5, 878 (1970). 3 K. N a k a m u r a , Y. H a s h i m o t o , I'. Y a m a k a w a , and A. Suzuki, J. Biochem. (Tokyo) 103, 201 (1988). 4 H. Egge, Chem. Phys. Lipids 21, 349 (1978).
METHODS IN ENZYMOLOGY.VOL. 193
Copyright © 1990by AcademicPress, Inc. All riots of reproduction in any form reserved.
714
GLYCOCONJUGATES
[38]
IO(X
N5
§99
t23
eO0
L~3
A.
~600-
(m-Hr... iN9
200
MASS P(R CHARGE
1000
1o 800-
II51
B. 669
600
( I~"H)"
J/,!,
ls3 j
599
LTO ~ qOG I
526 524.
I ~ - - ""~
"-~-"'"
I,
630
L. '%
r,ob..... "imb'"-'" ~'~'
.o .o
I?O$
li| i
i~ " "'"'~D5 ....... i ~J'liib ..... ]~i)""
MASS PI[R CHAImI[
F]6.1. Negative ion FAB mass spectrum of the native GM3fraction from (A) human liver, (B) human melanoma, and (C) bovine brain. Shown in (D) is the molecular ion region of the permethylated GM3 fraction from human melanoma depicted in (B) recorded in positive ion mode. The ganglioside GM3fractions show heterogeneity due to variations in the sphingosine moiety, documented in the molecular ion region by (M - H)- ions which appear separated by 28 mass units. The structurally unrelated impurity at m/z 599 [in (A) and (B)] is probably of biological origin and is destroyed during the permethylation.
the parameters (2-4) and (7-8), is, however, limited by thermal stability and the absolute necessity to derivatize under conditions that eliminate alkali-labile substituents. The soft ionization methods especially fast atom bombardment (FAB) 5 and liquid SIMS (secondary ion mass spectrometry),6 in combination with 5 M. Barber, R. Bardoli, R. D. Sedgwick, and A. N. Tyler, J. Chem. Soc. Chem. Commun. p. 325 (1981). 6 W. Aberth, K. M. Straub, and A. L. Burlingame, Anal. Chem. 54, 2029 (1982).
[38]
DESORPTION MS ov GLYCOSPHINGOLIPIDS
AJ.Ifd131N ! 3^I~V'13J
m...
A~ISN3Z41 3kI~V13~
715
716
GLYCOCONJUGATES
[3 8]
the development of high-field instruments, have brought substantial progress. It is now possible to analyze native or derivatized GSL structures within several minutes and to obtain intense molecular and sequence ions on molecules comprising more than 25 sugar residues. 7 Core Structures in Naturally Occurring Glycosphingolipids The following five major oligosaccharide structures have been observed in naturally occurring GSLs linked to Glc(fll--->l)Cer: Gal(fl I---~3)GalNAc(,81---~4)Gal(//l--~4)Gal(fl l--)3)GlcNAc(fl1--)3)Gal(fl 1---)4)Gal(/3 I---~4)GlcNAc~l--->3)Gal(fll-->4)GalNAc~ 1--->3)Gal(al--->4)Gal(fll--->4)GalNAc(/31--~4)GlcNAc(fl 1->3)Man(ill-->4)-
ganglio core lacto-N core neolacto-N core globo core arthro core
The sugar constituents and sequences of neutral oligosaccharides derived from these cores can be determined by desorption mass spectrometry from the fragment ions in terms of hexoses and N-acetylhexosamines in native or derivatized samples. Gangliosides (by definition GSLs, which contain sialic acids), can be derived from the ganglio, lacto-N, and/or neolacto-N series. The major representative of the sialic acids is 5-N-acetylneuraminic acid (Neu5NAc). It can be modified by additional hydroxy, deoxy, acetyl, methyl, lactyl, phosphate, and/or sulfate groups. Presently, thirty-two different sialic acids are known. 8 Beside sialic acids, other charged groups like carboxylate, sulfate, phosphate, phosphonate, and/or phosphoethanolamine may also be present. Such structural features play an important role in the proper choice of the most efficient method for analysis by mass spectrometry. Stereochemical factors may also be involved in the formation of parent as well as daughter ions in the gas phase, but no general rules have been established so far. Fragmentation Patterns
Molecular ions, (M - H ) - , and (M + H) ÷ or (M + cation) +, provide direct information on molecular weight, whereas information on type and number of sugar components can be deduced indirectly from these values. Primary or parent ions, formed after the cleavage of glycosidic bonds 7 H. Egge and J. Peter-Katalini~, Mass Spectrom. Reo. 6, 331 (1987). 8 R. Schauer, this series, Vol. 138, p. 132.
[38]
DESORPTIONMS oF GLYCOSPHINGOLIPIDS
717
° .2
I I I I SITE ~X~wa CLEAVAGE m
3
+2H~///// B "X~2H +~O~OR2 5
-0
o-
OR2 6
y
B~OR2 +
oH
O--
7
8_
SCHEME 1.
from both possible sites (pathway I and pathway II in Scheme 1), furnish information about sequence and branching pattern. The cations of types 1 and 4 should be observed in experiments performed in the positive ion mode and the anions of types 2 and 3 in the negative ion mode, if both pathways would randomly take place. In fact, the fragmentation pattern for native GSL in positive ion mode favors the pathway I, but beside the primary ion 1, the daughter ions 5 and 8, arising from the protonation of 2 and 3, respectively, can be observed. Therefore, an exact statement concerning the homogeneity of the molecular species with the same core structures and the branching patterns from these experiments is not possible. In addition, the parent ions are approximately of the same intensity as the daughter ions; consequently, a distribution of the total ion intensity over a number of peaks takes place. In the positive ion desorption mass spectra of derivatized (permethylated, peracetylated or perbenzoylated) GSL, the oxonium ion of type 1 is so much favored that the sugar sequence from the nonreducing terminus and the branching pattern, in addition to the molecular homogeneity,can easily be deduced directly from the spectrum. The cleavage of sugar C--C bonds, observed in direct chemical ionization (DCI) 9 or fast atom bombardment (FAB)-MS/MS, 1° does not take place.
9 V. N. Reinhold, this series, Vol. 138, p. 59. 10 B. Domon and C. Costeilo, Biochemistry 27, 1534 (1988).
718
GLYCOCONJUGATES
[38]
O
E
==
~.o
t~ Crl
~..-
~
.
-~
~"---I -oo
°°~
OI
E~ m~
z~ ~o
'
='
'
~
IIISNI~NI lAl ~¥'111
'
"
'
X£1SN31NI 3^1~V~3~
[38]
DESORPTION MS OF GLYCOSPHINGOLIPIDS
719
In positive ion desorption mass spectra of permethylated acidic and neutral GSL, the preferred cleavage site in the sugar chain is that of Nmethyl-N-acetylamino sugar of type 9 where R is CH 3 or sugar residue. Oxonium ions of type 11 derived from mannose, are more abundant than 10, derived from glucose or galactose, as found in GSL of the arthro series, i i The daughter ions, diagnostically important for the discrimination between 1,3 and 1,4/6-substitution at 2-deoxy-2-acetamido sugars, are formed from the corresponding parent ions of type 9 by elimination of the substituent on C-3.12
Z~_~+R
CHzOR
H3C/N.~cOCH3
OR 10
Ch~OR
1"1
In negative ion desorption mass spectrometry, performed on the native GSL, generally the formation of alcoholate type of ion 2 (pathway I) is favored. The native GSLs carrying negatively charged substituents, like gangliosides and uronic acid-containing GSLs, however, also exhibit characteristic abundant sequence ions of type 3 (pathway II). By combining the information obtained from the fragmentation patterns via pathways I and II it is possible to obtain the complete sequence information from one single experiment. The sequence of two isomeric gangliosides, GMla and GM1b, with different attachment sites of the Neu5NAc, is depicted with their negative ion FAB mass spectra in Fig. 2. The structure of GSLs containing neutral sugars and alkali-labile substituents can be elucidated preferentially from native samples in the negative ion mode or peracetylated samples in positive or negative ion mode desorption mass spectra. The carbohydrate sequence can be deduced from appropriate abundant primary ions, as discussed previously. For the structure determination of the ceramide residue, four types of ions can be used: the ceramide cation of the type 12 and anion 13, in addition to a sphingosine-derived cation 14 and a cation of type 15 arising after the loss of the acyl group from the molecular ion and subsequent protonation:
11R. D. Dennis, R. Geyer, H. Egge,J. Peter-Katalini~,S.-C. Li, S. Stirm,and H. Wiegandt, J. Biol. Chem. 260, 5370 (1985). t2 N. K. Kochetkovand O. S. Chizhov,Ado. Carbohydr. Chem. 21, 39 (1966).
720
GLYCOCONJUGATES o
[38]
®
- CH:~CH- CH-CH-CH-(CH2)~ CH3 NR OR
~0 i
- 0- CH2-,CH-CH-C H=CH-(CH2)r~CH3 NR OR
co l {E,H2)n
(!H2}n CH3
CH3
12 ®
cH2- -cH
~ oR'
]
Sugar-O-CH2-CI-CH-CH=CH-ICH2)n~CH3 +H+ or Cation" NHR lS w h e r e R is H , C H 3 ; R' is H , C H 3 , C O C H 3 . I n the positive ion spectra f r o m
the permethylated GSLs, the ceramide moiety is generally represented by the cations 12, 14, and 15; in the peracetylated samples however, only 14 is of high abundance. The cation 14 is also abundant in the positive ion desorption mass spectra of the native GSLs. For the structure determination of the ceramide moiety in sulfatide, the positive ion FAB-MS of peracetylated sulfatide and the negative ion FAB-MS of lysosulfatide were used (Fig. 3).
Design of Appropriate Experimental Protocol for Analysis by Mass Spectrometry A rational protocol for the structural elucidation of a small amount (-I00/~g) of a G S L should include the following set of experiments: I. The negative ion mass spectrum of the native sample provides (M - H)-, (Ccr)-, and two series of ions, of type 2 and type 3 (Scheme I). With these data a structural proposal for the sugar moiety and the ccramidc, as a whole, can bc made. 2. The positive ion mass spectrum of the native sample furnishes (M + H) + or (M + cation)+, Ccr + 12, and sphingosinc-dcrivcd ion 14. 3. After peracetylation the positive ion mass spectrum providcs (M + H) + or (M + cation)+ beside oxonium sugar sequence ions of type I and a sphingosinc base-derived ion 14.
[38]
DESORPTION MS OF I001
GLYCOSPHINGOLIPIDS
o|
A. t
HO-S-O-Gat- 0 - C e r
3
6
I ~0
!.ooi
2N !?
~qO0"
+t, ,ootB"
332
tOOO 1060
~ S S PER CHARGE
0
5 H 0 - S - 0 - O a l - 0-Sph
,' BOO"4
721
8
/
~>
'
z soe
10?
(M-Hk- 540
ThgL
,ooi 20Q
~? ....
ThIL 215
El i
~
I
;
ThgL 321
!: 2+, 0 + T i !
' ~SS
l
PER CHARGE
FIG. 3. FAB mass spectra of the sulfatide: HSO3-GaI-Cer. (A) Positive ion spectrum of peracetylated sulfatide showing (M + H) ÷ at m/z 1060. The Cer ÷ fragment ion 12 at m/z 674 is not present, but the sphingosine ion 14 at m/z 264 indicates that the fatty acid is a lignoceric acid. (B) Negative ion FAB mass spectrum of lysosulfatide. The sugar moiety is unaffected by the change in the ceramide moiety, as documented by the fragment ions at m/z 97 (HSO4)-, rn/z 259/257 (HSO3-- Gal)-, and m/z 300 ( H S O 3 - - G a I - - O - - C H = C H - - N H 2 ) - .
4. After permethylation using NaOH/methyl iodide in dimethyl sulfoxide (DMSO) 13 the positive ion mass spectrum provides (M + H) + or (M + cation) +, oxonium sequence ions 1, their respective daughter ions, Cer + 12, sphingosine base-derived ion 14, and (MH - acyl + I) + ion IS. t3 I. Ciucanu and F. Kerek, Carbohydr. Res. 131, 209 (1984).
722
GLYCOCONJUGATES
[38]
Sample Preparation The isolation of GSLs from biological material is usually achieved by extraction, followed by several separation steps using adsorption and ionexchange chromatography adapted to the type of GSL under investigation. 14,15Frequently, as a final purification step, semipreparative chromatography on high-performance TLC plates is included. During the extraction from the plate, considerable amounts of salt, silica, and binder may be coeluted which may subsequently interfere with the ion desorption process. Here the use of high-performance TLC plates with gypsum as binder 16 or an additional clean-up step using reversed-phase C18 material is recommended. Alternatively, such contaminants can be cleaned out from the GSL sample by use of 5-cm Iatrobeads (silica gel beads of defined size, supplied by Macherey, Nagel and Co., D-5160 Dtiren, FRG) columns eluted by solvent mixtures (chloroform/methanol/water) of increasing polarity. Problems rising from minor salt contamination can be resolved by adding 1/xl of 1 N aqueous HC1 directly to the sample on the target. In some cases, the samples can be more efficiently purified if derivatized by permethylation, peracetylation, perbenzoylation, or by several derivatization steps, based on the differences in chemical reactivity between functional groups present (see [35] in this volume). The purified and thoroughly desalted native GSLs can be analyzed by positive or negative ion mode desorption.
Types of Ionization for Glycosphingolipids Field Desorption Field desorption (FD) ionization, developed by Beckey and Schulten, 17 was used for structural analysis of a few native neutral and acidic GSLs with 1 to 6 sugar residues. The FD mass spectra were dominated by molecular ions (M + H) ÷ or (M + cation)÷, but the fragments of type 1 and 4 were rather weak. In the permethylated samples, the fragments of type 1 became more abundant.18 14j. N. Kanfer and S. Hakomori, "Sphingolipid Biochemistry." Plenum, New York, 1983. 15R. Kannagi, K. Watanabe, and S. Hakomori, this series, Vol. 138, p. 3. 16 p. Hanttand, Fur. J. Biochem. 87, 161 (1978). t7 D. Beckey and H. R. Schulten, Angew. Chem. 87, 425 (1975). ts y. Kushi, S. Handa, H. Kambara, and K. Shizukuishi, J. Biochem. (Tokyo) 94, 1841 (1983).
[38]
DESORPTION M S OF GLYCOSPHINGOLIPIDS
723
Chemical Ionization Chemical ionization (CI) has been applied to the analysis of GSLs using ammonia as a reagent gas for native and for derivatized neutral 9 and acidic 19,2° GSLs. The oligosaccharides derived from gangliosides after oxidative cleavage of the lipid moiety by ozone and subsequent reduction were analyzed by CI using ammonia and isobutane as reagent gases. 2° The underivatized GSLs limited to three to five sugar units were analyzed by DCI. Theuseful results by CI for larger molecules up to the mass of 3000 u were obtained using different sets of derivatization procedures, like permethylation or, alternatively, permethylation with subsequent reduction by LiA1H4 , or permethylation, reduction, and trimethylsilylation. The fragmentation patterns are rather complicated because of potential alternate addition of water and/or reagent ga s to the parent ions.
Liquid Secondary Ion Mass Spectrometry In liquid secondary ion mass spectrometry (LSIMS) experiments with a primary beam of xenon ions, native and derivatized GSLs have been analyzed in the positive and in the negative ion mode. With positive ion LSIMS, the molecular ions (M + H) + and/or (M + cation) + appeared, in general, to be of lower intensity than the fragment ions. For acidic GSLs, like sulfoglycolipids, the negative ion LSIMS of underivatized samples appeared to give, similar to the FAB ionization, more structural information than with the positive ion mode. 2~ Abundant molecular ions (M - 1)- and/or (MNa + - 2)- were accompanied by significant fragment ions of type 2 and 3, useful for the determination of the sugar sequence and the site of sulfate substitution. Thus far, the desorption behavior and fragmentation patterns for LSIMS seem similar to those for FAB.
Fast Atom Bombardment The majority of fast atom bombardment (FAB) mass spectra of GSLs have been acquired on VG ZAB-IF or HF, VG 70-250, and VG 7070E (VG Analytical, UK), and JMS DX-300, JMS-DX 303, JMS HX-100, and JEOL HXll0/HXI10 (JEOL, Ltd., Japan) instruments. Ion guns operating with argon or xenon as bombarding gas produced an atom beam 19 S. A. Carr and V. N. Reinhold, Biomed. Mass Spectrom. 11, 633 (1984). 2o T. Ariga, R. K. Yu, M. Suzuki, S. Ando, and T. Miyatake, J. Lipid Res. 23, 437 (1982); Y. Tanaka, R. K. Yu, S. Ando, T. Ariga, and T. Itoh, Carbohydr. Res. 126, 1 (1984). 21 y. Kushi, S. Handa, and I. Ishizuka, J. Biochem. (Tokyo) 97, 419 (1985).
724
GLYCOCONJUGATES
[38]
of an energy equivalent to 8-10 keV and a current of 5 x 10 -8 A. The acceleration voltage used varied from 10 to 3 kV, depending on the type of instrument and the extension of the mass range required. On the VG ZAB-HF instrument, an acceleration voltage of 4 kV was necessary to extend the mass range in order to obtain the molecular (M + Na) ÷ ion at m/z 6181 (nominal mass) of permethylated ceramide pentacosasaccharide from rabbit erythrocyte membranes. 22 As in the case of liquid SIMS, the solubility of the sample in the liquid matrix is the prerequisite. Matrices like glycerol, thioglycerol, a I : 1 mixture of glycerol/thioglycerol, triethanolamine, 1,1,3,3-tetramethylurea, a 5 : 1 mixture of triethanolamine/1, 1,3,3-tetramethylurea, and a 4 : 1 mixture of 1,4-dithioerythritol/1,4-dithiothreitol have been successfully applied to native and derivatized neutral and acidic GSLs. The final choice of the appropriate matrix must include considerations about polarity and intrinsic chemical properties of the molecule(s) to be analyzed. In our hands one of the most useful matrices is thioglycerol, having the single disadvantage of rapid evaporization in high vacuum. The sample of neutral GSL can be dissolved to a clear solution in methanol, chloroform/methanol, 1:1 (v/v), or chloroform/methanol/ water, 75 : 25 : 4 (v/v/v) containing 1-10 /xg//zl. Methanol/concentrated acetic acid (1 : 1, v/v) is considered to be the appropriate solvent for acidic GSLs. The sample solution is applied by a microsyringe to the stainless steel target, coated with about 2/zl of matrix. After the evaporation of the solvent by air or hair dryer, the probe with the sample is introduced to the vacuum and subjected to FAB. Due to the evaporation of the matrix during the acquisition of spectra, it is necessary to run in both up-scan and downscan mode in order to obtain intense fragments as well as molecular ions. The addition of 1/zl of I% solution of sodium acetate in methanol to the matrix is followed by an increase of the molecular ion and decrease in the intensity of fragment ions. The typical scan speed for instruments without a data system is approximately 1 sec/decade, depending on mass range and amplification. Use of a data system for multiple acquisitions or signal averaging, can increase the speed of a single scan to about 0.3 sec/decade, thus typically accumulating 10-20 scans. FAB mass spectrometry was applied in positive and negative mode to a large variety of native and derivatized GSLs in order to determine the primary structure of substances involved in recognition processes, pathological states or receptor phenomena. The potency of this method in obtaining several sets of clear-cut data
22 p. Hanfland, M. Kordowicz, J. Peter-Katalini~, H. Egge, J. Dabrowski, and U. Dabrowski, Carbohydr. Res. 171t, 1 (1988).
[38]
DESORPTIONMS OF GLYCOSPHINGOLIPIDS
725
and the simplicity of sample handling are the reasons for its wide and rapid acceptance .7,23-25 Combined Techniques TLC-LSIMS and TLC-FAB-MS Matrix-assisted SIMS and FAB-MS can be applied to the analysis of GSLs on TLC plates without prior elution. After TLC separation on an aluminum- or plastic-backed silica gel, the spots are visualized by iodine vapor or Coomassie blue staining. The area of interest on the plate is cut out (max. size 5 mm x 20 mm) and attached with double-face masking tape to a SIMS or FAB probe tip. After adding 1-2/~1 of solvent and 2-5 /zl matrix to the TLC strip, the probe tip is bombarded by Xe ÷ ions or Xe atoms under standard conditions. 26 Changes in ganglioside expression have been evaluated in rat fibroblasts after transfection with sarcoma virus DNA 27 using this method. Similarly, the glycolipid mapping of spleen and liver tissues from patients affected by metabolic glycolipid disorders caused by lysosomal enzyme deficiencies 28 and the structure of neoglycolipids after the reductive amination of human milk oligosaccharides with phosphoethanolamine-containing glycerides 29 have been established. Resuits from other experiments will be needed to evaluate the potency of this method on the higher GSLs. HPLC-C! and HPLC-FAB Positive ion CI mass spectra were obtained on HPLC separated neutral mono- to tetraglycosylceramides on-line using ammonia as the reagent gas. The observed fragmentation patterns were quite complex, even for ceramide mono- and disaccharides. As a general feature ofCI desorption, 3° the interpretation of these spectra is difficult, because primary ions tend to add or to eliminate water and/or ammonia. 23 A. Dell, Adv. Carbohydr. Chem. Biochem. 45, 20 (1987). 24 M. Arita, M. Iwamori, T. Higuchi, and Y. Nagai, J. Biochem. (Tokyo) 94, 249 (1983); Y. Ohashi, M. Iwamori, T. Ogawa, and Y. Nagai, Biochemistry 26, 3990 (1987). M. Suzuki, K. Nakamura, Y. Hashimoto, A. Suzuki, and T. Yamakawa, Carbohydr. Res. 151, 213 (1986). Y. Kushi and S. Handa, J. Biochem. (Tokyo) 98, 265 (1985). 27 H. Nakaishi, Y. Sanai, M. Shibuya, M. Iwamori, and Y. Nagai, Cancer. Res. 48, 1753 (1988). 2* y. Kushi, C. Rokukawa, and S. Handa, Anal. Biochem. 175, 167 (1988). 29 p. W. Tang and T. Feizi, Carbohydr. Res. 161, 133 (1987). 3o j. E. Evans and R. H. McCluer, Biomed. Environ. Mass Spectrom. 14, 149 (1987).
726
GLYCOCONJUGATES
[38]
S F C - C I and S F C - F A B
In supercritical fluid chromatography (SFC), a mobile phase consisting of a highly compressed gas near or above its critical temperature and pressure is used as a mobile phase. This separation method is more rapid than conventional HPLC and provides a higher number of effective plates at low temperature. The separation of GSLs by SFC requires permethylation in order to decrease their polarity and can be combined with CI or FAB mass spectrometry) 1 Analysis of Mixtures The complete separation of complex GSL mixtures into individual compounds can seldom be achieved even with the sophisticated methods available at the present time. 14,15The physical characteristics of individual molecular species of GSL arise from the structural variations of building blocks present in such a multifunctional molecule. The overlapping can occur due to the presence of (a) sugar chain homologs; (b) isomeric sugar sequences; (c) different sites of glycosidic linkages; (d) linearity vs. branching of the core structures; (e) the presence of other functi6nal groups like esters, lactones, sulfates, and phosphates; (f) the modifications of the basic structures by oxidation state (carboxy groups, deoxy sugars); (g) alkyl homologs arising from sphingosinc base and/or fatty acid; (h) modifications in thc lipid portion, such as in lysocompounds and in ncoglycolipids; (i) molecules structurally completely unrelated to GSLs; (j) stcrcochcmistry on the anomeric center of the glycosidic bond. A general discussion of analysis of mixtures is presented elsewhere in this volume [34] by Samuclsson et aL Only specific issues concerning desorption techniques will be discussed here. In Fig. 4, the methodological approach of handling a mixture by FAB mass spectrometry is shown. About 30 ~g of a ganglioside fraction was obtained after several chromatographic separation steps from a T-lymphoma cell line) 2 Whereas negative ion mode FAB did not give unequivocal results, the positive ion FAB spectrum obtained after permethylation revealed the presence of three molecular species. Their structures were definitely assigned by matching of fragment mass increments with molecular ions present in the mass spectrum of the mixture. If the analytc represents a mixture of analogs with the same core structure differing only in the absence of the terminal sugar unit, the 31 j. Kuei, G. R. Her, and V. N. Reinhold, Anal Biochem. 172, 228 (1988). 32 j. Mfithing, J. Peter-Katalinid, H. Egge, U. Neumann, B. Kniep, A. Loyter, and P. F. Mfihlradt, Proc. Xth Int. Syrup. Glycoconjugates, Jerusalem, September (1989).
[38]
DESORPTION M S OF GLYCOSPHINGOLIPIDS
727
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[38]
DESORPTIONMS OF GLYCOSPHINGOLIPIDS
729
negative ion spectra will not differentiate between ions arising from the cleavage of the glycosidic bond and the molecular ion of the lower homolog. This differentiation is, however, possible in positive ion desorption spectra of the derivatized samples due to pronounced expression of oxonium ions of type 1 and their specific mass increments. In Fig. 5 the positive ion FAB-MS of a permethylated ganglioside fraction, showing immunological activity toward VIM-2 antibody, is depicted. The only difference between the two components is the presence of an additional fucose .33 The presence of isomeric structures with the same molecular ion but different sugar sequences can be postulated if more than one set of sequence ions is detected both in negative ion desorption spectra of the native sample and in the positive ion mode of the derivatized sample. The presence of positional isomers can be detected to some extent if two daughter ions of different specificity occur in the same spectrum of the permethylated GSL. 34 This may be of particular importance for correlation of structural data of specific carbohydrate epitopes with immunological methods, e.g., immunostaining. 35 The aspect of linearity vs. branching of sugar cores in GSLs can successfully be analyzed in native and in derivatized s a m p l e s . 7,23,36 It is an important factor in studying the biosynthetic specificity of glycosyltransferases. Naturally occurring lactones have been found in GM~ and GDlb gangliosides as demonstrated in the negative ion desorption spectra of native 33 B. Kniep, J. Peter-Katalini~, H. Egge, W. Knapp, and P. F. Miihlradt, in preparation. H. Egge, J. Peter-Katalini6, M. Hergersberg, F.-G. Hanisch, R. Bruntz, and G. Uhlenbruck, Proc. Xlllth Int. Carbohydr. Symp. Ithaca, NY, August (1986). 35 j. Magnani, S. L. Spitalnik, and V. Ginsburg, this series, Vol. 138, p. 195. H. Egge, M. Kordowicz, J. Peter-Katalini~, and P. Hanfland, J. Biol. Chem. 260, 4927 (1985).
FIG. 5. Positive ion FAB mass spectrum of the permethylated ganglioside fraction isolated from human spleen, carrying the VIM-2 antigen epitope. (A) Lower mass region scanned in thioglycerol without addition of sodium acetate. (B) Molecular ion region scanned after addition of sodium acetate to the matrix. Two gangliosides, NeuAcHexsHexNAc3Cer (MNa + = 2718) and dHexNeuAcHexsHexNAc3Cer (MNa ÷ = 2892) show the same sugar core structure as documented by common fragment ions at m/z 376-344 (NeuAc ÷) and m/z 825-793 (NeuAcHexHexNAc +). The ions at m/z 1274-1242 (NeuAcHex2HexNAc 2+) versus m/z 1448 (dHexNeuAcHex2HexNAc2 ÷) and m/z 1723 (NeuAcHex3HexNAc3 ÷) versus m/z 1897 (dHexNeuAcHex3HexNAc 34) reflect the structural variation on the second lactosamine unit. Both gangliosides have their respective carbohydrate portion bound to the same type of ceramide, carrying palmitic acid.
730
GLYCOCONJUGATES
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[38]
DESORPTIONMS OF GLYCOSPHINGOLIPIDS
731
samples. 37The lactonization is a reversible reaction, which can be induced chemically by adding mineral acids to the samples; lactones will be easily hydrolyzed under basic conditions. Other base- and/or acid-labile esters are the O-acetates, s O-sulfates, 3s,39 and O-phosphoethanolamines. They can be localized in their desorption mass spectra by altered sugar mass increments and molecular ions. In Fig. 6 the negative ion FAB mass spectrum of a GSL isolated from Calliphora vicina is presented. To the sugar core of an arthro type, 11 a phosphoethanolamine substituent is attached. Its free amino group was acetylated by l : t mixture of (CH3CO)20/(CD3CO)20 and then peracetylated with (CH3CO)20. 4° The isotopic label in their respective molecular ions was visible in the negative mode FAB mass spectrum (Fig. 6B and C). Comparison with Other Analytical Methods The topo- and stereochemistry of the glycosidic linkages are the dominating factors that control the shape of the terminal epitopes of the carbohydrate antigens of GSLs. Information concerning glycosidic linkages can be obtained from experiments using nuclear magnetic resonance (NMR) and from combined GC/MS techniques; desorption mass spectrometric analysis has also been utilized. Immunostaining methods, based on antigen-antibody complex formation, have become available for searching for specific sugar-antigen epitopes in mixtures of GSLs, 35 particularly tumor-associated antigens. 41 If possible, such techniques should be used in concert with desorption mass spectrometry in order to characterize a yet unknown antigen or to define the specificity of a new antibody. The optimal approach is to permethylate the GSL antigen and to observe its specific parent and respective daughter ions in positive ion mode desorption mass spectra. The use of linked scan techniques a2 should also be considered. 37 S. Sonnino, G. Kirschner, G. Fronza, H. Egge, R. Ghidoni, D. Acquotti, and G. Tettamanti, Glycoconjugate J. 2, 343 (1985). 3g H. Leffler, G. C. Hansson, and N. Str/Smberg, J. Biol. Chem. 261, 1440 0986). 39 T. Ariga, T. Kohriyama, and L. Freddo, et al., J. Biol. Chem. 262, 848 (1987). 40 F. Helling, R. D. Dennis, M. Keller, J. Peter-Katalini6, H. Egge, and H. Wiegandt, in preparation. 41 S. Hakomori, Cancer Res. 45, 2405 (1985). 42 A. G. Brenton and J. H. Beynon, Eur. Spectrosc. News 29 (1980). FIG. 6. The negative ion FAB mass spectrum of the GSL from CaUiphora oicina pupae carrying a phosphoethanolamine substituent.40 (A) The native sample; (B) the molecular ion region of the same sample after N-acetylation with (CHaCO)20/(CD3CO)20; (C) the molecular ion region of the sample depicted in (B) after peracetylation.
732
GLYCOCON.IUGATES
[3 8]
Monitoring of Chemical and Enzymatic Reactions
The structural changes induced by chemical reactions of GSLs as polyfunctional molecules include derivatizations ([35] in this volume), oxidative and reductive cleavages of one or more building units ([30]-[32] in this volume), oxidations, and reductions. FAB mass spectrometry has been used to monitor the topospecifity of metaperiodate oxidation in GSLs from rabbit erythrocytes43 or for acid-catalyzed hydrolysis of neuraminic acid: 4 The synthesis of lyso compounds and their derivatives from GSLs by removal of the fatty acid moiety under basic conditions,45 leaves the carbohydrate moiety unaffected, as shown by negative ion mode FAB spectra (Fig. 3B). The removal of the lipid moiety from a GSL by the enzyme ceramidase (isolated from leach or from microorganisms)~ opens the possibility to study the liberated oligosaccharides by desorption mass spectrometry and by comparison with already known sugar structures. On the other side, reductive elimination of the terminal aldehyde group of oligosaccharides with nonpolar amines improves their desorption properties and increases the sensitivity of the measurement.29,47,48 Exoglycosidase degradation of the branched GSLs from rabbit erythrocytes by a-galactosidase,22,36or a ganglioside from mouse spleen showing choleragenoid-binding activity from mouse spleen by/3-galactosidase49 were monitored by FAB mass spectrometry. It was also shown by FAB methods, that the removal of the neuraminic acid by neuraminidase from Vibrio cholerae on the TLC plate is complete only in GDla,but not in GDI~ or GDIb .50
Summary Desorption mass spectrometry has become an important tool for sequencing and mapping of glycosphingolipids of natural, synthetic, or semisynthetic origin. The appropriate combination of different desorption mass 43 H. Egge and J. Peter-Katalini~, in "Mass Spectrometry in the Health and Life Sciences" (A. Burlingam¢ and N. Castagnoli, eds.). Elsevier, Amsterdam, 1985. 44 J.-E. Mansson, H. Mo, H. Egge, and L. Svennerholm, FEBS Lett. 196, 259 (1986). 45 G. Schwarzmann and K. Sandhoff, this series, Vol. 138, p. 319. 46 S.-C. Li, R. DeGasperi, J. E. Muldrey, and Y.-T. Li, Biochem. Biophys. Res. Commun. 141, 346 (1986); M. Ito and T. Yamagata, J. Biol. Chem. 261, 14278 (1986). 47 S. Dreyer, H. Egge, and J. Peter-Katalini~, in preparation. 4s L. Poulter and A. L. Burlingame, this volume [36]. 49 K. Nakamura, M. Suzuki, F. Inagaki, T. Yamagawa, and A. Suzuki, J. Biochem. (Tokyo) 101, 825 (1987). so j. Miithing, B. Schwinzer, J. Peter-Katalihi~, H. Egge, and P. F. Miihlradt, Biochemistry 28, 2923 (1989).
[39]
HIGH-MASS GC/IVIS OF PERMETHYLATED OLIGOSACCHARIDES
733
spectrometric techniques with other spectroscopic, enzymatic, chemical, and/or immunological methods represents the most direct and efficient way to establish frequent, yet unknown, molecular structure-function relationships. Acknowledgments We thank Dr. A. Vogel and Prof. K. Sandhoff, Bonn, for the sample of lysosulfatide. This work was generously supported by the Deutsche Forschungsgemeinschaft.
[39] H i g h - M a s s Gas C h r o m a t o g r a p h y - M a s s S p e c t r o m e t r y of P e r m e t h y l a t e d Oligosaccharides By
G U N N A R C . HANSSON a n d HASSE KARLSSON
Capillary gas chromatography-mass spectrometry (GC/MS) is a fast, sensitive, and high-resolving method for the characterization of biomolecules. It has been used in glycoconjugate research for analyzing the partially methylated alditol acetates after degradation of the glycoconjugates1 and, in a few cases, for the analysis of oligosaccharides after permethylation2-4 or trifluoroacetolysis. 5 The advent of high-temperature capillary GC using thin-film thermostable bonded stationary phases now allows the analysis of large permethylated oligosaccharides,6-8 an adaptation that should also be useful in other fields of biomedical research. Preparation of Oligosaccharides Glycosphingolipids
Glycan is cleaved7 from the ceramide portion of glycosphingolipids by endoglycoceramidase from Rhodococcus 9 (Genzyme Corp., Boston, MA) I This series, Vol. 50 [1]. 2 p. Hallgren and A. Lundblad, J. Biol. Chem. 252, 1014 (1977). 3 H. van Halbeek, L. Dorland, J. Haverkamp, G. A. Veldink, J. F. G. Vliegenthardt, B. Fournet, G. Ricart, J. Montreuil, W. D. Gathmann, and D. Aminoff, Eur. J. Biochem. 118, 487 (1981). 4 T. Tsuji and T. Osawa, Carbohydr. Res. 151, 391 (1986). 5 B. Nilsson and D. Zopf, Arch. Biochem. Biophys. 222, 628 (1983). 6 H. Karlsson, I. Carlstedt, and G. C. Hansson, FEBS Lett. 226, 23 (1987). 7 G. C. Hansson, Y.-T. Li, and H. Karlsson, Biochemistry 28, 6672 (1989). s H. Karlsson, I. Carlstedt, and G. C. Hansson, Anal. Biochem. 182, 438 (1989). 9 M. Ito and T. Yamagata, J. Biol. Chem. 261, 14278 (1986).
METHODS IN ENZYMOLOGY,VOL. 193
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