Analysis of gangliosides using fast atom bombardment mass spectrometry

Chemistry andPhysics of Lipids, 37 (1985) 127-141 Elsevier scientific Publishers Ireland Ltd.

127

ANALYSIS OF GANGLIOSIDES USING FAST ATOM BOMBARDMENT MASS SPECTROMETRY

H. EGGEa, J. PETER-KATALINICa, G. REUTERb, R. SCHAUERb, R. GHIDONIc, S. SONNINOe and G. TETTAMANTIc

alnstitut far Physiologische Chemic, Universitiit Bonn, Nussallee 11, D-5300 Bonn, bBio. chemisches Institut, Universitdt Kiel, Olshausenstrasse 40, 1)-2300 Kiel (F.R.G.) and eDepartment of Biological Chemistry, University of Milano (Italy) Received October 16th, 1984 accepted February 28th, 1985

revision received February 27th, 1985

The native gangliosides GM3, GMI, Fuc-GM1, GDla, GDIb, Fuc-GDlb, GTlb and GQlb were analysed by fast atom bombardment mass spectrometry (FAB-MS) in the negative ion mode in a matrix of thioglycerol. After permethylation the same gangliosides were analysed by electron impact (El) and FAB-MS in the positive ion mode. The negative ion mass spectra furnished information on the molecular weight, the ceramide moiety and the sequence of carbohydrate residues. The sites of attachment and the number of sialic acids present could be deduced directly from the pattern of sequence ions. After addition of sodium acetate positive ion FAB-spectra of the permethylated samples show intense pseudomolecular ions M+Na, that provide evidence on the homogeneity of the samples. In addition, the ceramide part, the oligosaccharide moiety obtained after cleavage of the glycosidic bond of the hexosamine residue, the whole carbohydrate chain and the sialic acids are represented by specific fragment ions. With EI-MS further information can be obtained on the sphingosine and fatty acid components of the ceramide residue. The data show, that the combination of soft ionization mass spectrometry with classical EI-MS gives valuable information on the structure and homogeneity of gangliosides. The method is also applicable to the structural elucidation or quantitation of more complex gangliosides or glycolipid mixtures using only micrograms of material.

Keywords: fast atom bombardment mass spectrometry; native gangliosides; negative ion mass spectrometry; permethylated gangliosides.

I. Introduction Gangliosides are glycosphingolipids which are characterized by the presence of one or more sialic acid residues. As components of the outer leaflet of the cell membrane they are involved in the control of the social behaviour of cells. As such they show species, tissue and developmental specificity [1,2] and can act as receptors or antigens. The growing perception of their biological importance has stimulated the development of more sensitive and specific methods of isolation, purification and analysis and in consequence has led to the identification of a large number of hitherto u n k n o w n gangliosides, as reviewed [2,3]. Especially the use of mass spectrometric methods [4,5] and nuclear magnetic resonance spectroscopy [ 6 - 9 ] has been shown to be useful for their structural elucidation. 0009-3084/85/$03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

128 With the introduction of FAB-MS [10,11] new horizons were opened in the applicability of mass spectrometry to the structural analysis of polar compounds [12]. Due to the 'cold desorption' of the molecular or pseudomolecular ions formed in a high boiling matrix under bombardment with rare gas ions, not only polar but also large molecules in excess of 6000 mol. wt. are amenable to the MS analysi: [13] A special problem encountered in FAB-MS analysis of gangliosides is their tendency to form high molecular weight micella in the presence of water. Hence a matrix has to be devised, favouring the presence of monomers that can be desorbed as pseudomolecular ions. Another problem encountered with native polysialosyl gangliosides is the presence of more than one negative charge in the molecule. In this case the pH of the solution in the matrix has to be modified in such a way that the formation of single charged molecules is favoured. A number of attempts has been made towards this aim using different solvents or mixtures of high boiling solvents as a matrix [ 14-20] for gangliosides carrying up tothree sialic acid residues. In this communication we show that with the use of thioglycerol as a matrix FAB spectra can be obtained in the positive and negative ion mode allowing the computerized subtraction of the signals produced by the matrix under standardized conditions of measurement. Thus also smaller ions in the range up to m/z 400 can be detected that are normally buried in the background produced by the matrix.

11. Experimental Gangliosides were extracted from calf brain except GM3* which was prepared from human Gaucher spleen and Fuc-GM1 and Fuc-GDlb that were obtained from pig brain following the tetrahydrofurane/phosphate buffer procedure of Tettamanti et al. [22]. Each ganglioside was isolated from the total ganglioside extract, structurally identified and analysed as previously described [23-25]. Permethylation was performed essentially according to the method of Hakomori [26] as modified by Sanford and Conrad [27].

MS MS was performed on a ZAB HF instrument (VG Analytical, Manchester, U.K.) equipped with a fast atom gun. The samples were dissolved in methanol or methanol/ acetic acid (2 : 1) to give a concentration of 5 #g/#l. In the positive ion mode, the stainless steel target was first coated with 2 gl of 0.1% sodium acetate solution in methanol. After drying, 2 - 3 gl of the thioglycerol (1-mercapto-2,3-propanediol, Ega, Chemie, Steinheim, F.R.G.) were added to the target and 0.5-1.0 #1 of a methanolic solution of the sample. In the negative ion mode, the samples were added in methanol acetic acid solution to the thioglycerol matrix. During analysis,

*The paper follows the ganglioside nomenclature of Svennerholm [21 ].

129 the target was bombarded with xenon atoms having a kinetic energy equivalent to 8 - 9 keV. Spectra were routinely recorded in a mass controlled downfield scan of 2 - 5 min duration. The resolution of the instrument was set to 300 ppm giving unit resolution up to 3300 a.m.u. The spectra were evaluated by counting the spectral lines. The background produced by the matrix was subtracted between 20 and 950 a.m .u.

El-MS was performed on the same instrument. Approximately 5 - 1 0 gg of the sample were heated indirectly by the heater of the ion source to 250-300°C until relevant spectra were obtained. Spectra were recorded at 25 eV ionization energy and 8 kV acceleration voltage.

III. Results and Discussion The positive ion FAB spectra of permethylated gangliosides show a very clear fragmentation pattern. As an example the spectrum of GQlb is presented in Fig. 1. As shown in Scheme I four major groups of fragments can be discriminated. They are summarized in Table 1 for the gangliosides GM1 to GQlb. B~

I I GaI~GalNAc~I-Gal

A1

Fuc o r (NeuAc)o_ =

A=

D:

I I GIc I

I =.C I I Ceramide

c) t-=

S c h e m e I.

The cleavage of the glycosidic bond of the neuraminic acid residues gives rise to ions at m/z 376 and after loss of CH3OH m/z 344. The presence of the NeuAc-NeuAc residue is indicated by the ion pair at m/z 737 and 717. They are represented by A1 or A2 in Scheme I and Table I. The mass difference of 20 a.m.u, is observed in all FAB spectra of permethylated gangliosides having a disialosyl residue. This latter ion can be explained by the elimination of 43 a.m.u, from rn/z 737 and the addition of sodium. Like in other permethylated glycoconjugates containing Gal*GlcNAc- or Gal-GalNAc-residues [20] a highly preferred cleavage is observed at the glycosidic bond of the hexosamine residue. This gives rise to the ions of group B. These ions also indicate the type of substitution at the terminal galactose e.g. Neu5Ac or Neu5Gc. Due to the 1 - 3 linkage of this galactose to the N-acetyl-galactosamine, ion B in this series of compounds is always accompanied by m/z 228 which arises f r o m / ~ by elimination of the 3-1inked substituent without regard of its size. Depending on the ceramide constituents, fragment C can vary considerably. In the series presented here, the fatty acid is almost exclusively stearic acid with sphingosine and eicosasphingosine as long chain bases as established by EI-MS [4,5].

130 I00( NeuAc"

Nq

80O

qrlrj ( NeuA¢ ) i °

73/

Cer" /fJrJ ~

]7

'l~b"' ~bb....... 5bb'""" ~bb....... ~bb...... 6bb....... ~bh.....

a0b....... 9i~b"

4rJht ~qB I

(NeuAc)/Go t-Go LNA¢'

r''"

w5

n.No" "cO~m H'

M.Na"

! ~"'910

.

.

.

.

.

.

.

.

I000'....

w,,,llTO . . . . . . .

i,L ~''1139Q. . . .

RII55

w,,,~12O ........... 22b0

' .... ~'300'....

i .....

29101........

30010

PER CHRRGE

Fig. l. Positive ion FAB-MS of permethylated ganglioside GQlb in thioglycerol matrix and Scheme of fragmentation. Thus the ceramide residue is represented by the ions at m/z 576 and 604 with only minor signals at m/z 590 and 618. A fourth group ofionsD, that are of diagnostic value, is present only in low intensity. These ions are produced by a hitherto not completely elucidated elimination of the ceramide residue, oxygen and one methoxy group (M+Na-Cer-OCHa and M+Na-Cer-OCHa-O) from the pseudomolecular ion M+Na. They are valuable for the determination of the complete carbohydrate composition. The FAB mass spectrum of native GM3 prepared from human Gaucher spleen is shown in Fig. 2. Due to the presence of fatty acids, with a chain length of 16-24 carbon atoms, five groups of pseudomolecular ions M-1 can be observed between m/z l 151 and 1261. This latter species containing nervonic acid also forms an addition product with the thioglycerol matrix at m/z 1369. The ions representing the hexosylceramide and the dihexosylceramide fragments show a similar distribution of intensities as the M-1 ions between m/z 698 and 808, and m/z 860 and 970, respectively. The carbohydrate chain is also represented by three groups of ions at m/z 308 and 290 for the Neu5Ac residue at m/z 470/468 for the Neu5Ac-Hex residue and at rn/z 632/630 for the trisaccharide Neu5Ac-Hex2. It has to be noted, that the primary ions formed by fission of the glycosidic bonds with retention of the glycosidic oxygen are always accompanied by another ion two a.m.u, lower formed by elimination of H2. Thus the carbohydrate sequence is represented by

q~

Z

o ,,o

c)

z

0

0o

O

Z

z

Z

132 1900

I151 8OO

tlO

860 970

600

308-/ 290

?5~

.......

808

4.70J ~68

£32J 630

NeuA¢ -

2O0

630 q70

63~

GoL-GLc-Cer

Gtc-Cer" 75q

°

860 91B

970

11 1207

i ~

MRSS PER CHflf~GE

Fig. 2, MS of native GM3 preparedfrom humanGaucherspleen,recordedby negativeion FABMS. Signals derived from the thJoglycero|matrix are subtractedbetween 150 and 950 a.m.u.

two series of ions starting either from the ceramide residue or the 'nonreducing' terminal of the molecule. The same fragmentation pattern can be observed with the gangliosides GM1 [15], Fuc-GM1 an:d GDla as shown in Fig. 3a,b. The two series of fragment ions that are derived from both ends of the molecule allow an unequivocal determination of the sugar sequence in terms of deoxyhexose, hexose, N-acetyl hexosamine and N-acetyl neuraminic acid. The sugar residue of the tetrasaccharide backbone to which sialic acids are linked, can directly be identified from the spectra as indicated also in the schemes of fragmentation. The spectrum of Fuc-GMI (Fig. 3a) may serve as an example for the sensitivity of the method in evaluating the purity of a given sample. Thus the small signal representing the fragment Gal-GalNAc-Gal-NeuAc at m/z 835/833 points to a trace of GM1. The ion pairs at m/z 888/916 and m/z 1091/1119 on the other hand can be attributed to the fragments Gal-Glc-Cer and GalNAc-Gal-Glc-Cer, respectively. They indicate a minor component that does not carry a sialic acid at the second hexose. The presence of a second N-acetylneuraminic acid in GDla implies several changes in the region of the molecular ion. Here, only single-charged M-1 ions are recorded. Thus, one of the two Neu5Ac residues has to carry either a proton or another cation. Both is observed as documented by the M-1 ions at m/z 1855 and 1863 and the ions M-2+23 at m/z 1867 and 1885, that represent the mono-sodium salts. The occurrence of several additional pseudomolecular ions in this part of the spectrum can be attributed to the presence of other cations like potassium and calcium. The gangliosides GMI, Fuc-GM1 and GDla show the same ceramide-pentasac-

133

_

,...~

r--

O

Am

~"!- ~

__.

r.

2 i-

®

c~ L~

o- " T o

o

o IIr~N31NI

oo

o

~^IIUlIU

~0

~io~ I

~

< ~E

' ~ u.

~

® o

: o

o

z~

o ~II~N31N[

3AII~I3U

~

134

charide structure. Therefore, the fragment ions that contain the ceramide residue are identical in all three compounds (Scheme II). 1544

I 1572

1382 1410

1179 1207

726 I'

564

754

! 592 R -

O -

Gal -- O -- GalNAc

-- O -

Gal -

O -

GIc -

O -

Cer

I O

I

Neu5Ac GM1 : Fuc-GM1

R = H :

GDla:

R = Fuc R = Neu5Ac

Scheme II.

A discrimination between these compounds can be made with the aid of the molecular ions and sequence ions that are formed starting from the non-reducing end of the molecules. In GDla the presence of a terminal Neu5Ac residue favours the formation of such ions as m/z 308/290 for Neu5Ac-, m/z 470/468 for Neu5Ac-Hexandm/z 673/671 for Neu5Ac-Hex-HexNAc-. The gangliosides G D I b and Fuc-GDlb are characterized by the occurrence of one Neu5Ac¢~2-8 Neu5Ac residue. This structural feature produces some very characteristic changes in the pattern of fragmentation as shown in Fig. 4a,b. The mass spectrum of G D l b (Fig. 4a) shows the same pseudomolecular ions M-1 as that of GDIa at m/z 1835 and 1863. Instead of salt formation like in GDla however, a pronounced tendency of lactone formation is observed as documented by the elimination of one molecule of water at m/z 1817 and 1845. This elimination of water also takes place after loss of one Neu5Ac residue m/z 1572 and 1544 as can be recognized by the fragment ions at m/z 1554 and 1526. The Neu5Aca2-8 Neu5Ac residue is represented by the very intense ion at m/z 599. This ion can form daughter ions at m/z 581 and 553 after the elimination of either 18 or 46 a.m.u. Linked scan measurements (B2/E) starting from m/z 553 showed, that this ion is derived directly from m/z 599 and that m/z 581 produced by elimination of H20 is not an intermediate (Scheme III). Thus the isomeric gangliosides GDla and G D l b can be clearly distinguished on the basis of their negative ion mass spectra. A very similar pattern of fragmentation is shown by Fuc-Gdlb (Fig. 4b). Three types of pseudomolecular ions are present: M-1 at m/z 1981 and 2009, M-2+Na at m/z 2003 and 2031 and M-l-18 at m/z 1963 and 1991.

135

IOCOI

®

O00 !

60C

581S99

......

....

""

. . . 60O ";.730

. . . . . . .

s o b "

. . .

~8~g

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IOOO 726

564-

754-

i592

BOO

600

599

,ooi

155~

0

1281 1

15~b. 1572

NIu, Ac

| __ I

I

I~Ei~

.... I

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llllT

1511115"/2

HRS5 PER CHRflGE 181~

eoO

1835 186~

~600

FUC

1655

1~52

Ik70

1673 1701 at

726 75~

1~98

aLNAc

at

I

~

0 =

599

169Q

56~ 592 tc

er

1399

1~27

NeUAC

125~~72 ...........

sab

sob

'

5 ~

'

'

hbb~'lb

~00~

'

"

lbbb'"tlhb

'l~bb''"j~b~ ~09

lalRS~ PER CHA~GE

Fig. 4. Negative ion FABoN$ of the native gangliosides GDlb (a) and Fuc43D1b (b).

136 OH H N ~ O-

m/z 599

Ac OH H ~ .jCH o

.~/~H~n~"~

OOH

/

CH20H

"~COoH HCOH H2COH OH H L/A~

m/z 553 OH

" ~ _ _~/-CO2

m/z581 OH H

,, ~

~cN~ ~ 7 0~/- H2

,,L---.L 0 / ~ CH c.2o.

A~N~' oIeOOH

OH H /

Ac

H N ~ C OOg ~ C H H2OH

HCOH

HCOH

H2COH

H2COH

Scheme III. The disialo group is again represented by the three ions at m/z 599,581 and 553. Other ions that are of diagnostic importance for the determination of the sugar sequence are indicated in the scheme. The two ions at m/z 1544 and 1572 already discussed for G D l b indicate the presence of a contamination of a nonfucosylated mono- or disialosyl ganglioside. From the simultaneous presence of ions at m/z 1253/1281 and at m/z 1526/1554 it can be deduced that this nonfucosylated component is GDlb. The molecular ion region of the gangliosides with more than two sialic acid residues is characterized by a great complexity due to the formation of different salts. Together with the variation of the ceramide residue, a very complex pattern of pseudomolecular ions of relatively low signal to noise ratio is produced for GTlb between m/z 2120 and 2220 that is difficult to evaluate (Fig. 5a). Beside M-l: m/z 2126; 2154; M-2+Na: m/z 2148, 2176; M-3+2Na: 2170, 2198; M-3+Ca or M-2+K; 2164, 2192; M-3+Na+K: 2186, 2214 or M-3+2K: m/z 2202, 2230 may be present. This multiple salt formation also precludes the formation of clearcut sequence ions in the higher mass range. The major fragment ions are shown in Fig. 5a. The difficulties in observing pseudomolecular ions and higher sequence ions may be overcome by adding strong acid to the target. Under these conditions, elimination of water is observed leading to pseudomolecular ions 18 or 36 a.m.u, lower than M-l, e.g. m/z 2136, 2118, 2108, 2090 (Fig. 5b). This elimination of water can be explained by formation of a six membered lactone ring, possibly between the hydroxyl group of C: of the inner galactose and the adjacent Neu5Ac residue as shown in the Scheme IV. Concomitant with the elimination of water, the three ions representing the

137

00 '°° I J,,,®

SS]

~BO0 Ce~"

400-

NeuAc-G~ -

NeuAC*

l

I '

240

q40

300

t-GolNAc"

58%

SO0

/ G~3

GO0

Otc -Cer"

700

800

900

lO00

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® /,5

z~

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(

~

/

/

/

~20

/

-

i

~ 6oo-

i i i i i

200 -

.oo I

3OO

600

14~S~ P £ R

CHanGE

Fig. 5. Negative ion FAB-MS of native GTlb added to the matrix in methanol/acetic acid solution (a) and after addition of 1/~1 0.1 N HC1 to the matrix (b). Matrix derived signals up to 950 a.m.u, were subtracted.

138 1452 1655

r~ ' 1 4

f"~ 1683 l I I

H~COH

~u

I

H~,OH

~

~

~_l\

726 I/'W 7 5 4 i

80

* 673¢ i--'~

671

OH

J

q

--.

,N,*~

y-

,

/

/ ~

O ~

.

1817

,&~O~/coo; ----",8,s HCOH

~

,~,~,o~/~c.

\ O /,O

OH

\

564

I

H2COH

~w592

H2COH

~

NH OH

oH

~-o

C17H33

," C17H35

-

' 2108

H~COH

.~OH H2COH

Scheme IV. disialosyl-group at m/z 599, 581 and 553 are absent or of very low intensity, The terminal Neu5Ac-Gal-GalNAc residue on the other hand is represented as expected by m/z 6 7 3 , 6 7 1 without any sign of lactone formation (Fig. 5a,b). Elimination of one or two sialic acid residues is observed in both spectra irrespective of the addition

]ooo

i600

~5

371

311

~oo

6oot ~oo

M-1

~(NeuA¢ J2 GoLNAC-(NeuAC)2-GoL-GLc-Cer" .. ~

-HzO ISl?

Fig. 6. Negative ion FAB-MS of native GQlb recorded after addition of HCl to the matrix. Major fragment ions are indicated in the accompanying Scheme V. Background subtraction was applied up to 700 a.m.u.

139 _ 946 "1 * 1452 P ~71480 I 944

1655 ~e1683 H2C'OH

:

.0/t--o.

H2COH '1

: H2COH

.o~r--o

0

t

726 +~'754

O

!

",t--o

'+

,

564 I->592

H2COH

"J---o

"

,

O--Cer

,,,/ o.

o

,3

o."

~l.e'~u •

'

H2COH

eoo.', HCOH \ H2COH

v/~ 308 290

HCkOH H2COH

I~ 308 290

Scheme V.

of HC1. In the acid milieu however, the 'M-Neu5Ac' ions are shifted by 18 a.m.u. due to the elimination of water thus giving rise to the intense m/z 1845 and 1817. When spectra of GTlb are recorded at acidic pH, the ions at m/z 1544 and 1572 (GMl-ions) are accompanied by a group of fragments around m/z 1500 that are formed by elimination of H20, CO2 and H2 (64 a.m.u.). The ion m/z 1480 coincides with that formed after cleavage of the GalNAc-Gal bond. These data are in agreement with the structure proposed in the Scheme IV. The negative ion FAB spectrum of GQlb is characterized by an even higher complexity. Without the addition of acid during measurement, multiple salt formation is observed in the molecular ion region shifting the major ions to m/z 2483, 2511 (spectrum not shown) above the calculated M-1 values at m/z 2417 and 2445. The spectrum is changed drastically after the additon of HCI to the matrix. Now, the major pseudomolecular ions are M-1-2H20 at m/z 2381 and 2409 (Fig. 6). The elimination of three molecules of water is also observed (m/z 2363). After the loss of one disialosyl group the ions m/z 1817 and 1845 still indicate the presence of one lactone ring as shown in the Scheme V. Hence it can be concluded, that both disialosyl groups are involved in a lactone ring formation. In agreement with this finding, the terminal trisaccharide ion gives rise to m/z 743 and the terminal tetrasaccharide ion Neu5Ac2-Gal-GalNAc appears at m/z 946, 944. Other structurally important fragment ions are indicated in the Scheme V. Discussion

A comparison of CI or EI mass spectra of permethylated gangliosides with those obtained by FAB-MS clearly shows the advantages of this latter method. The high

140 intensity of pseudomolecular and structurally important fragment ions together with a very clearcut fragmentation greatly facilitates the establishing of structurespectra relationship. This is especially true for gangliosides with molecular weights in excess of 2000 a.m.u. In contrast, only very few CI or El spectra of permethylated, reduced and trimethylsilylated gangliosides were reported, that showed sufficiently intense fragment ions in the high mass range that could be used for structural analysis [4,29,30]. The highest molecular ion of a ganglioside recorded so far using e.i. ionization is that of GDla as reported by K.-A. Karlsson et al. [31]. Recently a number of minor or alkali labile gangliosides has been characterized that possibly play a role in cellular adhesion, signal recognition, differentiation and neoplastic degeneration [32-35]. In order to obtain however more information about the long chain base and fatty acid composition, CI or E1 mass spectra of permethylated gangliosides have to be recorded. As reviewed earlier [5,28] fatty acid residues and sphingosine bases give rise to characteristic rearrangement ions under E1 that can be used in combination with the data from FAB-MS for a quantitative calculation of the different molecular species present. Positive ion MS using CI, El or FAB ionization is however not adequate enough for establishing unambiguously the complete saccharide structure of gangliosides mainly because no significant ions are formed that carry the sialic acid residues linked to the inner galactose of the gangliotetraose. Hence, the site of attachment of these sialic acids is not directly discernible by these methods. The presented examples of FAB spectra of native gangliosides do show however, that such characteristic ions are formed preferrentially when recording negative ion spectra. Therefore the occurrence of overlapping sequence ions in the negative ion FAB spectra which are derived from both ends of the molecule is invaluable for the determination of the carbohydrate structure. We have shown further, that with the use of thioglycerol as a matrix, FAB spectra can be obtained from 2-5/ag of native gangliosides with a high signal to noise ratio for the pseudomolecular and most of the fragment ions. Due to the presence of only one type of matrix molecule, a highly reproducible matrix spectrum is obtained especially in the negative ion mode that allows computerized subtraction of these signals. Thus, less intense fragment ions of the sample under investigation can easily be identified also in the low mass range up to rn/z 600. It can be anticipated, that FAB-MS data of native and derivatized gangliosides combined with those obtained by high resolution proton nuclear magnetic resonance spectroscopy [7-9] will play a crucial role in the structural analysis of such biologically important compounds.

Acknowledgements The authors are indebted to the expert technical assistance of B. Barnhusen and M. Pfliiger. This investigation was supported by the Deutsche Forschungsgemeinschaft.

141

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