738

GLYCOCONJUGATES

[40]

from GC/MS of a seven-sugar oligosaccharide containing sialic acid obtained from a purified blood group B ganglioside. 15This eluted at 390° on a 10-m column. The endoglycoceramidase or ceramide-glycanase enzymes do not split monoglycosylceramides, but these small intact glycosphingolipids can be analyzed by GC/MS. 7 Oligosaccharides released from mucins and other glycoproteins by alkaline-NaBH4 treatment can be analyzed and the high resolution achieved is necessary for separating the complex mixtures found in nature. 6"s An example from neutral oligosaccharides of porcine small intestinal mucins is shown in Fig. 2, where three isomeric pentasaccharides are resolved and characterized. Permethylation using solid NaOH gives good yields for neutral oligosaccharides with a GalNAc-alditol, and neutral and sialic acid-containing oligosaccharides with a Glc-aldose. Oligosaccharides with a Glc-alditol give lower yields by permethylation, although these can be increased by modifying the permethylation procedure s (see above). Sialic acid-containing oligosaccharides with a GalNAc-alditol give poor yields. Acknowledgments This work was supported by grants from the Swedish Medical Research Council (No. 7461), the SwedishBoard for Technical Development,and Gabrielsson's research fund. The mass spectrometer was supported by the Swedish Medical Research Council (No. 3967). 15J.-F. Bouhours, D. Bouhours, and G. C. Hansson, J. Biol. Chem. 262, 163700987).

[40] T a n d e m

Mass Spectrometry of Glycolipids

By CATHERINE E. COSTELLO and JAMES E. VATH

Introduction The structural determination of glycolipids has been considerably advanced through the implementation of mass spectral methods, because of their sensitivity and the wealth of information they convey. There are, however, practical limitations to the utilization of the usual mass spectrometry-based approaches for the analysis of very small quantities of material, particularly when the sample consists of a mixture of closely related compounds. Tandem mass spectrometry (MS/MS) offers a means to adMETHODS IN ENZYMOLOGY, VOL. 193

Copyright © 1990by Academic Press, Inc. All rights of reproduction in any form reserved.

[40]

MS/MS OF GLYCOLIPIDS

739

dress these difficulties, because it reduces the contribution from background and allows the selective determinations of individual components within a complex mixture. In addition, MS/MS spectra, particularly those of derivatives designed to favor selected fragmentation pathways, may include structurally diagnostic ions not present in the normal mass spectrum. This chapter describes a systematic nomenclature for fragments observed in the mass spectra of glycolipids, the use of a high-performance tandem mass spectrometer (high mass range, with unit resolution or better available in both MS-1 and MS-2), and targeted derivatization procedures for glycolipid structural determinations. Investigation of the MS/MS spectra of underivatized glycolipids in the positive and negative ion modes has demonstrated the usefulness of the technique for structure elucidation at the microgram level. For smaller sample amounts and for compounds with poor fast atom bombardment (FAB) ionization or fragmentation characteristics, microscale derivatization procedures have been developed and the mass spectrometric properties of the derivatives investigated. The highenergy CID (5-10 keV) MS/MS properties of both the native and derivatized glycolipids are discussed and summaries are provided as guides for the analyst. Low-energy collisions have been demonstrated to be informative about carbohydrate structure during investigations of the behavior of mono- and disaccharides. 1-3 These conditions may furnish additional information about the types of compounds discussed here, but no systematic exploration of the low-energy collision spectra of glycolipids has yet been conducted. Some of the discussion is also relevant to interpretation of the two-sector FAB, field desorption (FD), and direct chemical ionization (DCI) spectra of these compounds, those obtained by normal magnetic scans, 4-7 by linked scans of the electric and magnetic

I R. Guevremont and J. L. C. Wright, Rapid Commun. Mass Spectrom. 1, 12 (1987). 2 R. A. Laine, K. M. Pamidimukkala, A. D. French, R. W. Hall, S. A. Abbas, R. K. Jain, and K. L. Matta, J. Am. Chem. Soc. 171, 6931 (1988). 3 D. R. Mueller, B. Domon, W. Blum, and W. Richter, Biomed. Environ. Mass Spectrom. 15, 441 (1988); see also [33] in this volume. 4 C. E. Costello, B. W. Wilson, K. Biemann, and V. N. Reinhold, in "Cell Surface Glycolipids" (C. C. Sweeley, ed.), p. 35. ACS Symp. Ser. No. 128, American Chemical Society, Washington, D.C., 1980. 5 T. H. Wang, T. F. Chen, and D. F. Barofsky, Biomed. Environ. Mass Spectrom. 16, 335 (1988). 6 M. E. Hemiing, R. K. Yu, R. D. Sedgwick, and K. L. Rinehart, Biochemistry 23, 5706 (1984). 7 j. Kuei, G. R. Her, and V. N. Reinhold, Anal. Biochem. 172, 228 (1988).

740

GLYCOCONJUGATES

[40]

fields of a double-focusing instrument, 8-~° by mass-analyzed ion kinetic energy (MIKES) analysis, 11-12 or by use of triple quadrupole and hybrid instruments. For the analysis of glycolipid mixtures, particularly when sample size is very limited, it is especially desirable to eliminate background interferences by using MS/MS. Unit mass resolution for the selection of the precursor ion (MS-l) in collision experiments is also important, because components that vary in the degree of unsaturation, the presence of amine or hydroxyl functions, or other modifications resulting in mass shifts of only a few daltons are often present as mixtures even in extensively purified extracts. The determination of base and fatty acid chain lengths of individual components, and localization of the site(s) of unsaturation or functional groups may be important for the understanding of biological activity. In addition, product ions may be present at adjacent m/z values even in the collision spectrum of a unit-resolved precursor, since multiple fragmentation pathways related to the various functional groups occur for these compounds. Therefore, while much of the information in this chapter is pertinent to the interpretation of spectra recorded under other instrumental conditions, limited mass resolution in either precursor selection or product ion spectra may result in some residual ambiguities and some of the fragmentations described may not be observed on low-energy collisions. Nomenclature Because the nomenclature systems previously employed for small carbohydrates were found to be unwieldy or inadequate when it became necessary to describe the range of fragments observed in the MS/MS spectra of large glycolipids, a nomenclature system for the designation of fragment ions in mass spectra and tandem mass spectra of glycolipids, oligosaccharides, and their derivatives has been developed 13-16and will be s H. Egge and J. Peter-Katalini~, Mass Spectrom. Rev. 6, 331 (1987); also [38] in this volume. 9 y . Ohashi, M. Iwamori, T. Ogawa, and Y. Nagai, Biochemistry 26, 3990 (1987). l0 S. Ladisch, C. C. Sweeley, H. Becker, and D. Gage, J. Biol. Che m. 264, 12097 (1989). ii R. B. Trimble, P. H. Atkinson, A. L. Tarentino, T. H. Hummer, Jr., F. Maley, and K. B. Tomer, J. Biol. Chem. 261, 12000 (1986). n G. Puzo, J.-J. Fournie, and J.-C. Prome, Anal. Chem. 57, 892 (1985). 13 B. Domon and C. E. Costello, Biochemistry 27, 1534 (1988). 14 B. Domon and C. E. Costello, Glycoconjugate J. 5, 397 (1988). 15 B. Domon, J. E. Vath, and C. E. Costello, Anal. Biochem. 184, 151 (1990). 16j. E. Vath, B. Domon, and C. E. Costello, Proc. 37th ASMS Conf. Mass Spectrom. Allied Topics, Miami Beach, FL p. 770 (1989).

[40]

MS/MS OF GLYCOLIPIDS

741

used throughout this discussion. The core of this system will be presented first, and supplementary designations added in the subsequent discussion sections. This nomenclature system is conceptually similar to that in use for the description of FAB mass spectra and tandem mass spectra of peptides ~7,~8in that alphabetical symbols and numbers are used to designate bond cleavages along the backbone of the molecule. For glycoconjugates and oligosaccharides, A n , B n , Cn are used for fragments that retain the charge on the nonreducing end and X n , Yn, Zn are used for fragments that retain the charge on the reducing (lipid, peptide) end. Superscripts preceding the symbol are used to define cleavages within a carbohydrate ring, as discussed below. Lipid fragments are designated with other letters from the latter part of the alphabet. When lower case symbols are used for the peptide moiety, the system accommodates glycopeptides. The occurrences of these ion types are discussed in the individual sections that describe the spectral characteristics of underivatized and derivatized species. The same letters are used to designate positive and negative ions of similar origin. Hydrogen transfers may result in mass differences between ions of the same letter but opposite charge. When the number of hydrogen transfers that accompany bond-breaking has been found to be constant, the definition of the symbol assumes the transfer, e.g., Bi + = [Bi] ÷ but Bi- = [ n i - 2H]-. If the number of hydrogens transferred is a variable or differs from the definition, then the number needs to be specified when the symbol is utilized, e.g., 3'5.44 - 3H. Ceramide Portion

Scheme 1 indicates the bond fragmentations and hydrogen transfers included in the designations for product ions of ceramides and the ceramide portion of glycosphingolipids. Scheme 1A shows the product ions observed in the positive ion collision-induced desorption (CID) mass spectra of native and reduced compounds; Scheme 1B shows the product ions observed in negative ion CID mass spectra of native compounds, and includes representations for two important ions that arise via multiple bond cleavages. Carbohydrate Portion

Because structural variations and, therefore, fragmentation pathways (branching, ring cleavages) are more complex in carbohydrates than in peptides, a more elaborate nomenclature system is necessary. An addi17 p. Roepstorff and J. Fohlman, Biomed. Environ. Mass Spectrom. 11, 601 (1984). 18 Appendix 5, at the end of this volume.

742

A

GLYCOCONJOGATES

U0 (R=H)

~.U (R=car'bohydrate] v0

Yo

• p(R=car'bohydr'ate) . U

Zo.,,/

t

Zo~,l

Rfo'1 "01

[40]

(-

I,, HN', -H I

"~-

Me W

x a - 0. Ha, ~

NH

NH

O_

O_ S 10,'7

T ion

SCHEME 1. Designations for product ions that arise from fragmentations within the ceramide portion of glycolipids. (A) Positiveion fragmentations; (B) negativeion fragmentations. Designations of ion types include the hydrogen transfers indicated.

tional subscript (an, fin, etc.) has been added to indicate branching (an is the fragment originating from cleavage within the longest branch). Products of sequential cleavages are designated with a slash in the subscript, e.g., ~m~/n2. Left superscripts indicate the bonds broken during ring cleavages, as shown in Scheme 2.

Materials and Methods

Glycolipids Lactosylceramide [Gal(/31--~4)Glc(fll--*l)Cer, n16:0/18:0], galactosylceramide [Gal(fll--~l)Cer, nl6 : 0/18 : 1], N-palmitoyl-4E-sphingenine, N-oleoyl-4E-sphingenine, GMI {Gal(/31---~3)GalNAc(/31---~4)[NeuAc(o~2---~3)]Gal(/31---~4)Glc(fl 1---~1)Cer}, and Grlb {NeuAc(a2---~3)Gal(/31 ~ 3) GalNAc(/31 ~ 4) [NeuAc(ot2~ 8) NeuAc(a2 ~ 3)] Gal (/31 ~ 4)Glc/31---~ 1Cer} were obtained from Sigma Chemical Company, St. Louis, MO. The phospholipid samples were a gift from B. N. Singh, SUNY, Syracuse, NY. Sources of the reagents used for derivatizations are noted as the procedures are described.

[40]

MS/MS OF GLYCOLIPIDS

Y2 Z2

t~X1

743

Y~ Z~

CH20H

Yo Zo

CH20H O -

HO

O .R OH OH

i f

°'2A~

B, O,

CH2OH

B 2 Ce

Ba

zs^s

CH20H

4~O'0

HO~ ~ 2

z'A2

, O-R

1 OH

I 4/O~

5 0 --O.-R

. ~ L o -

/

3~P H 2

I OH

AcHN 4,/r--- o \ COOH

CHOH

I ~

I

OH

CH20H

R

SCHEME 2. Designations for product ions that arise from fragmentations within the carbohydrate portion of glycolipids. Because the number of hydrogen transfers during ring cleavages (A., X.) can vary, these are not included in the designations, but must be indicated in each case. Fragments that arise via glycosidic cleavages include predictable numbers of hydrogen transfers and the designations are thus defined: Bi+ = [Bi], Ci+ = [Ci + 2HI, Yj+ = [Yj + 2H], Zj + = [Zj]; Bi- = [ B i - 2H], Ci- = [Ci], Yj- = [Yj],Zj- = [Zj - 2]-. 14

Mass Spectrometry All s p e c t r a s h o w n w e r e r e c o r d e d with a J E O L H X 1 1 0 / H X 1 I0 (EIB~ E2B2) t a n d e m m a s s s p e c t r o m e t e r , o p e r a t e d at - 1 0 k V accelerating voltage, with --- 18 k V p o s t a c c e l e r a t i o n at the detector. H i g h - e n e r g y collisions (5 to 10 k e V ) with h e l i u m in a collision cell (that m a y be electrically floated at a selected potential) l o c a t e d b e t w e e n MS-1 and MS-2, at a p r e s s u r e sufficient to r e d u c e the p r e c u r s o r ion a b u n d a n c e to a b o u t 20% o f its initial value, w e r e e m p l o y e d . T h e p r i m a r y ion b e a m w a s either 6 k e V x e n o n neutrals o r 6 - 8 k e V Cs ÷ ions. (The c h o i c e o f p r i m a r y b e a m had little effect o n the s p e c t r a , a n d w a s m a d e on the basis o f compatibility with o t h e r o n g o i n g e x p e r i m e n t s . ) U n l e s s o t h e r w i s e n o t e d , the s p e c t r a w e r e r e c o r d e d with an e l e c t r o n multiplier during linked scans o f MS-2 at c o n s t a n t B/E. T h e J E O L D A 5 0 0 0 d a t a s y s t e m p r o v i d e d i n s t r u m e n t c o n t r o l

744

GLYCOCONJUGATES

[40]

and data acquisition and processing. MS-1 was operated at (or above) unit resolution. MS-2 was usually operated at 1 : 1000 resolution. Scan time was 1 min 30 sec to 2 min 30 sec, with filter (30-300 Hz) chosen so as to preserve mass resolution. Sample solutions (1-5/zg//zl) of underivatized compounds were mixed with matrix (1 : 1) and 0.3-0.5/~l of this solution was applied to a stainless steel FAB probe tip (3-mm diameter). For derivatized compounds, sample handling was modified and less sample was required, as noted in the discussions below. In order to allow for postmeasurement selection of parameters such as peak detection threshold and centroid vs. peak maxima mass assignments, and for examination of peak shapes and signal-to-noise (S/N) ratio, all spectra were recorded (and are presented) in the profile mode. Spectra shown are single scans, or the sum of a few scans added together after the measurement. Mass assignment accuracy is normally within 0.3 u of the calculated values. 19 In the figures, the calculated mass values for fragments are indicated on the structures and the experimentally observed values are indicated on the spectra and used in the text. Fractional mass values are included on spectra of compounds above Mr 1000, which have fragments whose fractional mass approaches or exceeds 1 d~lton (Da), in order to remove any ambiguity about the assignments.

MS/MS of Underivatized Compounds

Positive Ion MS~MS Spectra Information about the structure of the aglycon is most readily available from the positive ion MS/MS spectra of the (M + H) +, (M + cation) +, or aglycon-containing fragment ions. The behavior of (M + H) + ion is easiest to anticipate because prediction of its charge localization is more straightforward. Rules for the interpretation of the CID spectra have therefore been formulated primarily for (M + H) + ions. 13 Gangliosides and other negatively charged glycolipids are very prone to cationization, forming (M + Na) + and (M + K)+, and usually do not yield sufficient (M + H) + ion abundance for MS/MS analysis of the underivatized compounds. Ceramides and Neutral Glycosphingolipids. FAB sensitivity for underivatized ceramides, even in concentrated solutions, is poor and derivatization is therefore recommended (see below). For glycosphingolipids that contain one or two sugars, the situation is much improved and 0.5-5/zg of sample provides spectra of excellent quality. The CID spectrum of the 19 K. Sato, T. Asada, F. Kunihiro, Y. Kammei, E. Kubota, C. E. Costello, S. A. Martin, H. A. Scoble, and K. Biemann, Anal. Chem. 59, 1652 (1987).

[40]

MS/MS OF GLYCOLIPIDS

745

(M + H) + ion, m/z 864.6 of lactosyl-N-palmitoylsphinganine is shown in Fig. 1A. We have obtained the field desorption (FD)-CID MS/MS spectrum oflactosyl-N-palmitoylsphinganine(M + H) + ion and confirmed that the high-energy CID spectrum is quite independent of the ionization mode, i.e., the FAB-MS/MS and FD-MS/MS spectra are virtually identical. Glycosidic bond fragmentations within the carbohydrate give rise to low abundance ion(s) (m/z 702.6). The ion corresponding to the protonated aglycon (Y0, m/z 540.5) and its fragments generally have much higher abundances. In the CID spectra of compounds that contain sphinganine, (M + H - H20) + has only low abundance. Both the W and W' ions (m/z 302.3 and m/z 284.3) are present and indicate the length of the base. An abundant V ion (m/z 256.3) indicates the size of the fatty acyl group, and is accompanied by a V' ion if an hydroxyl group is present in the fatty acid. Remote site cleavages2° along the hydrocarbon chains lead to low abundance ions observed at masses below the aglycon fragment. When the base is (4E)-sphingenine (henceforth called sphingenine), water loss from the molecular ion or the aglycon is extremely facile, and results in the most abundant product ions. For these compounds, the W ion is not observed, but W' and W" are present and indicate the chain length of the base. The abundances of these ions are much greater than that of the V ion. The weight of the fatty acyl group can still easily be determined, however, by subtraction of the mass of the base from that of the aglycon. If modifications within the carbohydrate portion of the glycosphingolipid favor fragmentation pathways involving the carbohydrate moiety, thereby diminishing the relative abundance of lipid-related ions in the MS/ MS spectrum of (M + H) +, CID spectra of the Y0or Y0' ions can still yield the desired information about the lipid structure. These ions have CID spectra similar to those of the corresponding ceramides, with some variation in relative abundances, due to the preferential formation of one isomer when multiple structures of the fragment are possible. For example, in the case ofceramides and simple glycosphingolipids, the Y' ion may be formed by loss of either hydroxyl group, but loss of the 3-hydroxyl group seems to be preferred. Figure 2A shows the CID spectrum of the Y0' ion (m/z 520.5) from galactosyl-N-palmitoylsphingenine (n16:0/18: 1). This spectrum is dominated by the W' (m/z 282.3) and W" (m/z 264.3) ions originating from the Y0' ion which has undergone dehydration through loss of the 3-hydroxyl group. The ions at m/z 250.3 and 252.3 are derived by loss of methanol and formaldehyde, respectively, from the W' ion. The low abundance S ion at m/z 280.3 would arise by loss of HOCHnCH(CH2)I3CH3 from the Y0' = Z0 ion. When the carbohydrate moiety is 20 L. J. Deterding and M. L. Gross, Org. Mass Spectrom. 23, 169 (1988).

702

540

14ZX

/OH

,*/;iX

i I

HO

/OH

,

OH

i

2561"ZH

*0

"%, .

---~X 4

, [M+H] ÷

A

Yo

864.8

540

/ V 256

W' 284

1~s B, 163 JJ ........

.....

/

I'l'l'l'l'l'l'{'l'

m/z

100

522

302

J

~ li. I , I , i , i ~i • i', I , i Vi', i ,~i • i , i ,'i " "

I"

200

Yl 702

400

300

700

500

600

7[]0

800

538

°°f-%~ ,'v ~

..o, i ,

o,

, ,

i ' ~ O H ~

~ o .'I ~X

HN

I

: '--%. }341 "xO"~*'~''-'~"'

I h L ~ -a: ~ -H! ,-H / -2Hi ,19 1~1 1~9 221 / i 281

10

'

~

8

× 5

--->×1 Y1 700

YO 538

&2AI-H 119

[M-H]862 8

X

Ct 179

z

3 z

°'2A2 - H 281

2'4Az- H

Z1 682

(x

I

,-~ ,/[ m / z I00

253 /Tqn ]L 296

..rT-q~ r, r . r ~ [ q ~ c q - , 200

C2

/ /341

300

i ~ i , r, n, r, t, i-, ~ 400

500

. . . . . . . 600

700

800

FIG. 1. FAB-CID-MS/MS spectra of lactosyl-N-palmitoyisphinganine in DMSO. (A) (M + H) +, m/z 864.6, glycerol matrix. (B) (M - H ) - m/z 862.6, triethanolamine matrix. Collision energy in both spectra is l0 keV.

[40]

I.IJ ¢J Z

MS/MS OF GLYCOLIPIDS

747

A

[Y'o.]÷ 5205

w" 264

< t-~ Z

/

m
.. L.L A

T,',

~)1 I

v

P "'~f""~

E

.,'.

• . [ . rl'l'l'['lrl'l%

100

(C,H~I,5-C H3

"2s9 300

3O0

'r"

"-(c.,j,o-C.,

i I-IN' I I

-., : +./', / '6 ~1

m/z

[M-H]780 5

,vn

,, q

H2PO 4 97

[40]

200

,

300

U

Y°PO3H 618

, , iI .,.,,'].-,'."r','"t.'l.',,~,, ,l L ~ l l l , J J l l l J J l | '

400

500

' l ' f ' l ' l ' l ' l ' l ' l ' l

600

i

700

Fro. 3. CID-MS/MS spectrum of the (M - H ) - , m/z 780.5, ofphosphoinositol N-stearoylhexadecasphinganine (hi8 : 0/16 : 0) isolated from Leishmania, 21 in chloroform/methanol/ triethanolamine, 3 : l : 4 (v/v/v). Collision energy, 7 keV.

the carbohydrate rings as well as by glycosidic cleavages. Assignments of these ions are indicated on the spectrum. In the spectra of ceramides and cerebrosides that have small carbohydrate moieties, low abundance ions (designated S and T) characterize the lipid portion of the molecule. These ions do not have useful abundances in the spectra of glycosphingolipids that contain four or more sugars, but CID-MS/MS spectra of the abundant II, series fragments (n = 0 - 2) of the normal FAB mass spectrum may be used to determine the ceramide structures of such compounds. In the negative ion CID spectra, the series of remote site cleavage ions begins at the molecular ion, rather than from the aglycon, as was described above for positive ion spectra. Ions related to phosphoric acid (m/z 97.0, H2PO4- and m/z 79.0, POaand the (B, + 80)- and (C, + 80)-, where n equals the number of sugar rings, dominate the negative ion MS/MS spectra of phosphoglycolipids. Figure 3 shows the CID-MS/MS spectrum of the (M - H)- ion, m/z 780.5, of phosphoinositol N-stearoylsphinganine (n18 : 0/16 : 0), one of several closely related glycolipids from Leishmania. 21 Cleavage adjacent to the phosphate at the glycosidic bond results in an abundant YoPO3H ion, m/z 618.5, and cleavages within the ceramide yield Uo- (m/z 566.3) and E-type (m/z 300.0) products, all retaining the phosphate group. Gangliosides. Localization of the negative charge on the carboxyl group of sialic acid results in a drastic change in the spectra of gangliosides compared to those of cerebrosides, so that the MS/MS spectrum of the

[40]

MS/MS OF GLYCOLIPIDS 1382 ~, HOCHI, ~..~.n

i('-2H t

*tIOC,H~

1179 I ~-|H I HOCH,

~ -~2Hi tHOCH,

751

995 .~,3 )

OH

2hat 2 .

At

891.2

I.SXI-3H EII 2901

14106 CI

C4-2H

\,308.1

995.3

154

/,2 ....................... l ..... .., .VVl,V),V~,l,VVvvl,vv).Vl,Vrvvrv~,Vrl,VVl,Vl,VVrVl,l,~,VVl,ln 1192

100

200

300

Y~

400

500

600

700

800 m/z

900

'~= 138~,6 I

Ill

11794

~L \ l . . . . . a.[l,l, r'q r[,v), v v l , v i . v v i, v v v ~,l,V H , V l , V w,l'rr,'~,l,t 1000

1100

1200

1300

1400

1500

FIG. 4. FAB-CID-MS/MS spectrum of the (M - H)- ion of the lower molecular weight GM1 homolog, m/z 1544.9. Collision energy, 7 keV.

(M - H ) - ion is dominated by ions related to the sialic acid moiety. 13 Figure 4 shows the CID-MS/MS spectrum of the (M - H ) - ion, m/z 1544.9, recorded for the lower homolog of GMI. The most abundant product ions are those that arise by (C - 2H)-type glycosidic cleavage beyond the carbohydrate ring that carries the sialie acid substituent (m/z 833.2, m/z 995.3) and by 3'sX-type cleavage of the carbohydrate ring adjacent to the one substituted (m/z 891.2). Y- and Z-type fragments are also observed only beyond the sialic acid attachment site (m/z 1382.6 and 1179.4, m/z 1161.4). Loss of sialic acid results in a fragment at m/z 1253.3. Abundant ions at m/z 290.1 and m/z 308.1 represent (M - H ) - and (M - H H20)- for sialic acid, and lower mass product ions are mostly due to fragmentation within the sialic acid moiety. When more than one sialic acid group is present, the CID-MS/MS spectra of the (M - H ) - or (M - 2H + Na)- ions allow differentiation of the isomeric possibilities.13 Information about details of the cerarrfide structure (other than its total weight) is lacking in the normal FAB mass spectra and the (M - H ) - collision spectra of gangliosides but, once again, may be obtained from CID spectra of ions in the Y, series (n = 0 - 2) in the FAB mass spectrum. Figure 5 shows the CID-MS/MS spectrum of the Y0 ion, m/z 564.5, of the lower homolog of GMI. This feature has recently

752

[40]

GLYCOCONJUGATES

23~ 534 /.I~I

[

H--O , ~ ( C H ~ } a 2 C H 3 2ea ~ HN _m131~ " ~l~x, (CH~115CH3 0

:~65

[~ ]-

[yo] 564.5

.%

S

"~/"~ (CH2}15CH s O_

Uz

508

/

S m/z 308

z

,•/0

< F-

5 w

m/z 564

237 265

T

V

O_

•324

,i,~,l,l,l,t,l,l,[,i,l,l,i, m/z 200 250

I 300

534 {CH2)15CH3

5J1 6 J

T m/z 32,1

i,i,i. .350

.i,i.i,r,r.l,l,l,r,l,l. 400 450

[ ........ 500 550

FIG. 5. FAB-CID-MS/MSspectrum of the Y0fragment ion, m/z 564.5, due to ceramide portion of the lower homolog, in the FAB mass spectrum of Gin. Collisionenergy, 7 keV. been exploited by Sweeley and co-workers for the analysis of gangliosides in tumor cells, m The sensitivity for underivatized gangliosides is much lower than that observed for glycolipids which contain only a few sugars and the possibility for adverse effects from the presence of endogenous salts makes CID-MS/MS analysis more problematic for gangliosides. Derivatization overcomes many of these difficulties, improves sensitivity, and introduces control over fragmentation pathways, as described below. Other Glycolipids. Fragmentation within the carbohydrate portion of other types of glycolipids follows the general patterns described for glycosphingolipids. Unless the lipid has a very favorable site for charge localization, the product ion abundance will generally be distributed primarily over fragments that arise as a result of bond cleavages within the carbohydrate moiety. Some minor fragmentation may also occur within the lipid portion, and may provide unique information about its structure. 22The most abundant ions in the CID spectra of glycerophosphoinositol compounds are

[40]

MS/MS OF GLYCOLIPIDS

753

those related to the phospho sugar, and, therefore, resemble the spectrum shown in Fig. 3, but also having abundant ions that correspond to the carboxylate anions of fatty acid substituents. 21 MS/MS of Reduced and Permethylated Compounds

Derivatization Methods In many instances it is desirable to derivatize glycolipids prior to MS/ MS analysis. Derivatization can be used to improve secondary ion yield and to direct fragmentation. The types of derivatization that will be described here are permethylation of amide and hydroxyl groups (to improve sensitivity for gangliosides in the positive ion mode), borane reduction of amides to amines (to increase sensitivity and improve MS/MS fragmentation characteristics of both ceramides and glycolipids), hydroboration of olefins (to locate their positions in the lipid chains), and the combined use of permethylation and borane reduction. Borane reduction of N-acyl groups is performed with condensed reagent vapor and the sample requirement for this reaction is thus decreased to the picomole level. Reactions performed in this manner are able to efficiently convert very small sample amounts into a derivative in a form that permits complete transfer of the sample to the mass spectrometer for GC, EI, and FAB. Contamination is minimized since the sample comes in contact only with minute quantities of the volatile components of the reagent(s). Losses due to surface adsorption and sample transfers are minimized because the sample remains exposed to only a small area on the inside of a capillary tube support during derivatization. The sample is transferred only once, to the instrument for measurement. The additional reactivity of borane with carbon-carbon double bonds in lipids to yield an alkylborane permits the location of unsaturation in the acid and/or sphingoid base of sphingolipids following its conversion to an alcohol using a reagent vapor oxidation step. The reaction conditions employed in the following derivatization reactions and their effect on all of the common functional groups found in glycosphingolipids are summarized in Table I. Permethylation. The formation of permethylated derivatives for the analysis of glycolipids by positive ion FAB-MS is a well-established strategy. 8'23 The increased sensitivity upon permethylation (about 20- to 50fold) allows the detection of submicrogram quantities of materials. For MS/MS, increased secondary ion yield is important because the quality of the product ion spectrum obtained upon CID is directly linked to the 23 A. Dell, Adv. Carbohydr. Chem. Biochem. 45, 19 (1987); see also [35] in this volume.

754

GLYCOCONJUGATES

[40]

I I z

[--,o

II I ~F~: X

©

°~ u

II

I

e

I

Z Q.

°~

× ~o

I Z

I

© m

I

OX

O

???

?~ -6 ¢D

.1

O

©

I ,.~

I

e~

II

O

~;I Z

r

e.

±

o r.

e~

I~

I

< m

~

I

0

I O

I e~

i. i , o.~

I

[40]

MS/MS OF GLYCOLIPIDS

755

amount of precursor ion available, among other variables. The goal of many of the derivatization strategies described here was to develop methods where sample waste is minimized. Generally, the techniques developed start with one to two times the sample amount required for a single MS/MS experiment and consume all of the derivatized sample in the measurements (FAB-MS and MS/MS). The permethylation technique best suited for this approach is essentially that of Ciucanu and Kerek24 as modified by Gunnarsson. 25The sample (1-10/zg) is dried in the bottom of a screw-top culture tube and subsequently dissolved in 200-300/xl of anhydrous dimethyl sulfoxide (DMSO) (Pierce Chemical, Rockford, IL) which contains about 10 mg of finely powdered NaOH. The DMSO/NaOH solution is kept as a stock solution prepared by finely pulverizing NaOH pellets with a mortar and pestle and combining with dry DMSO in a dry culture tube under a nitrogen atmosphere to form a saturated solution. The stock solution is vortexed and the 200-300-/zl aliquot for the reaction is drawn from the area of cloudy suspension above the solid. Such a stock solution can be used reliably for several weeks, if the solution is discarded when discoloration or odor develop. The sample in the DMSO/NaOH solution is then sonicated for 5 min at room temperature. Methyl iodide (Aldrich Chemicals, Milwaukee, WI) (10-20/zl) is added and the solution sonicated at room temperature for an additional hour. Water (1 ml) containing 1-5% acetic acid (enough to neutralize the NaOH) is added to the reaction mixture. The neutralization of the NaOH is important to avoid methyl ester hydrolysis in the NeuAc residues of gangliosides. The samples are purified on Sep-Pak cartridge (Millipore Corp., Milford, MA) which has been successively conditioned with 20-50 ml chloroform, 20 ml methanol, 5 ml acetonitrile, and 5 ml water. Adequate washing of the cartridge with chloroform is important for small sample amounts in order to minimize silica contamination from the cartridge which can be detrimental to FAB-MS analysis. The entire sample is applied to the conditioned cartridge which is then washed with 5 ml water, 5 ml acetonitrile, and 1 ml methanol. The sample is eluted with 1 ml of chloroform and this fraction is concentrated to about 50/~l by evaporation with a nitrogen stream. The solution is either transferred to a melting point capillary tube for further reagent vapor derivatization (described below) or 1-2/~1 of 3-nitrobenzyl alcohol is added and the sample concentrated in vacuo for FAB-MS and MS/MS analysis. Borane Reduction. Permethylated, reduced derivatives of glycosphingolipids have made an important contribution to extending the upper limit 24 1. Ciucanu and F. Kerek, Carbohydr. Res. 131, 209 (1984). 25 A. Gunnarsson, Glycoconjugate J. 4, 239 (1987).

756

GLYCOCONJUGATES

[40]

of the mass range and improving the information contained in the EI mass spectra of these compounds. 26-27 Similar reduced and permethylated-reduced derivatives of sphingolipids have also proved useful for FAB-MS/ MS. For this application, the borane reduction and associated reactions (solvolysis and oxidation) are conducted with condensed reagent vapor derivatization. 28 With this technique the sample is deposited as a solid on a small area of the inner wall of a capillary tube. A 1- to 20-/zl aliquot of sphingolipid solution [either native or permethylated in 2 : 1 (v/v) chloroform/methanol solution] is placed into the bottom of a melting point capillary (100 mm long, 1.5 mm id) using a 10-/zl fused-silica capillary syringe (J&W Scientific, Folsom, CA) as shown in Fig. 6A, left-hand side. The tube is placed in a vacuum centrifuge and the solvent evaporated in o a c u o leaving a thin film of solid on the walls at the bottom of the capillary tube (Fig. 6A, right-hand side). The sample tube is placed into an apparatus for conducting reagent vapor reactions (shown in Fig. 6B). The apparatus has a side arm well which contains about 200/zl of the appropriate reagent or reagent mixture. The reagent well is cooled with either dry ice or liquid nitrogen and the apparatus is evacuated (X8

) - - - - - ~ / - . +~..

A 228.2

(,

re+m+

,~.'

~__:-~,,,.0

,,2,.2

A OOR~I '~Tm Ac C ~ C OOR 376.2"x t.o, c~

Z Z

82(,

I" - ) X 40

ri++l _ • .,,.l J., ......

,o~+g.,

200

300

1+1++

,

1.,,,,...,.,L,,,,+.,.,,.,l..,. 4J,,+,,,.,.,1,,.,J,J.

•r l ' V l V V l ' l . l . l + r l . V l , r V l . l , v V l . l . V r ) . l , l , r i . l . l . l . l . l . l . l . l . l . l . l . l . l . l . l . l . r l . l . r l . r

100

Ira++,

400

500

600

700

800

900

I . l - i . r l - i , i . r i . r l . r rl.l,l.rl.).m-l-lrrl.W.l-i,i.t'~.l,i.~.i.i

1000 1100 1200 1300 1400 1500 1600 1700 1800

m/z

1125.4 ROCH2 !

----

-

tt~u~;e*

~i

I

li04.1 +

ROCHt

(

~Hz

I

/2. ~- ~,, ~ ~_,js~..orCOO.

"U.+ o +tab

376,2

1 I ....

100

£.J.L.J, 300

Zp.

I

+,,

[p

B~.

. . . . . . .

700

(M+H) 2577.6

82s6

°ui.°ll " I I I I. . . . . L...I / LI. .

SO0

.... ,,,..,

~ j l +,,,., ~l~:M~ , . v + . R ~

73~5 2282

"~

.o.N--~oo.

, , 344.1

OR

.+,"~.,s. ..... .

+ ~j

|

900

,,

~.,-.°o. 1810.2 . . . . . . . . 1100

1300 m/z

1500

L.I ..., . . . . . 1700

?900

~,+-.°o. 2;7+6

Lm. 2100

/I . J,l

. . . . . . .

2300

2500

[40]

MS/MS OF GLYCOLIPIDS

759

the capillary tube with the fused-silica syringe and placed onto the FAB probe which has been previously coated with a very thin layer of glycerol (0.1 /xl) in order to increase the longevity of the sample signal. In this manner the reagent vapor derivatized sample can either be used entirely or in portions for FAB-MS and MS/MS, which are often performed at different times.

MS~MS Spectra of Derivatized Glycosphingolipids Permethylated Gangliosides. As previously stated, the MS/MS analysis of low microgram or submicrogram quantities of gangliosides in the positive ion mode usually requires permethylation. An example of a FABMS/MS spectrum of such a derivative is given for the lower homolog of GMI, (M + H) + m/z 1827.2, in Fig. 7A. The product ion spectrum is primarily composed of fragment ions resulting from cleavages of the interglycosidic linkages (B and Y ions) with minimal fragmentation in the ceramide portion of the molecule. The B ions occurring adjacent to the amino sugars (Bza at m/z 464.3 next to GalNAc and Bit~ at m/z 376.2 next to NeuAc) are particularly abundant fragment ions that indicate to which residue sialic acid is attached. Even though the spectrum shown in Fig. 7A is informative, it is not ideal for low-level structural characterization by MS/MS. The center and high mass portion of the spectrum, containing much of the sugar sequence information, is very weak in this derivative and with slightly less material, or with larger molecules, this region would disappear. This effect can be observed in the FAB-MS/MS spectrum of p e r m e t h y l a t e d GTIb, (M + H) + m/z 2577.6, in Fig. 7B. For this spectrum, the B ions are once again very informative for the sialic acid attachment. In addition to the B~ ion at m/z 376.2, the serial attachment of two sialic acids beyond the GalNAc residue is indicated by the B2~ ion at m/z 737.5 and the fact that only one of the sialic acids present is attached at the nonreducing end of the carbohydrate is apparent from the B3a ion at m/z 825.6. The middle and high mass regions of this spectrum are extremely weak considering the amount of material used to obtain this spectrum is the same as that in Fig. 7A for permethylated GMl (5 /~g). The only structurally informative ions that appear above the noise in this region are Y2,-methanol and Y4,-methanol at m/z 1810.2 and 2171.6, respectively. Despite the increased signal size over the native compound, the fragmenta-

FIG. 7. FAB-CID-MS/MS spectra of permethylated gangliosides at 7 keV collision energy. (A) (M + H) ÷ m/z 1827.2 of the lower molecular weight homolog of permethylated GMI. (B) (M + H) ÷ m/z 2577.6 of the lower molecular weight homolog of permethylated GTIb-

760

GLYCOCONJUGATES

[40]

tion characteristics of the permethylated gangliosides in MS/MS limits the amount of sequence information for small sample amounts and those with extensive carbohydrate moieties. The combined use of amide reduction with permethylation, described below, is able to overcome some of these problems. Amide-Reduced Glycolipids. For ceramides and small neutral glycosphingolipids there is a considerable sensitivity enhancement upon reduction of the amide group in the sphingolipid to an amine. Borane reduction, as opposed to reduction with lithium aluminum hydride, offers additional reactivity with alkenes as well as the ability to conduct the derivatization at much lower levels, Figures 8A and 8B compare the FAB-MS spectrum of 1 /zg of N-palmitoylsphingenine [(M + H) ÷ m/z 538.5] with that of 10 ng of the BHa-derivatized sample [(M + H) + m/z 542.5], respectively. The most striking difference between the spectra of these two samples is the enhancement in ion yield upon derivatization. For ceramides, the increase in sensitivity upon reduction is about l03 while for monoglycosyl glycosphingolipids it is about l 0 2. The ion at m/z 520.5 in the spectrum of the underivatized compound is the result of dehydration of the (M + H) ÷ species while the ion at m/z 526.5 in the spectrum of the derivatized compound is due to an elimination-rehydroboration by-product of derivatization that is typical for the hydroboration of allylic a l c o h o l s 29 such as that present in most sphingolipids. This by-product is most noticeable in derivatized ceramides where it represents about 20% of the desired product (its yield is much lower during derivatization of cerebrosides and gangliosides). The sensitivity enhancement of the major product over that observed for the underivatized compound is such that the formation of the by-product is only a minor inconvenience. The major product of hydroboration and oxidation of the allylic alcohol is a vicinal diol by virtue of the directing influence of the hydroxyl group in the 3-position of the sphingoid base. This product can be differentiated from the naturally occurring 4-hydroxysphinganine (phytosphingosine) when the reduction is performed with BD3 because a deuterium will be placed at the nonhydroxylated carbon atom (C-5). As opposed to allylic carbon-carbon double bonds, hydroboration and oxidation of an isolated olefinic bond results in a mixture of two isomeric alcohols with the hydroxyl group attached to either carbon atom of the original double bond. A deuterium atom is introduced at the nonhydroxylated carbon atom upon BD 3 reduction. This reactivity permits the location of double bonds present in either the acyl or sphingoid base chains. Figure

29 G. M. L. Cragg, "Organoboranes in Organic Synthesis." Dekker, New York, 1973.

*

A

(,~ Z r", Z :C) t'~

_>

5

~.J

.~ '

l

'

,

:

:

i

,

t

,

I

~

,

500

45O

i

538 ,

i

~

~

,

i

~

550

i

,

i

,

i

,

j

:

t

,

600

i

~

i

t"'t

,

L

,'

653

B 54-2

(,,D Z r~

Z rn

:>

5 tY

' t " i

450

' l ' J

I ' l ' l ' t ' J ' l

500

I ' l ' ~ ' J ' l ' l ' i ' P ' l ' ~ '

,550

600

650

m/z FIG. 8. FAB mass spectra of N-palmitoylsphingenine (A) before and (B) after reduction/ hydroboration. Asterisks (*) indicate matrix-related cluster ions.

762

GLYCOCONJUGATES .HiOH

H

2

'~ o ~

H

(

t

\

[40]

14r42C1"13

..,,

(c"2>r0.3

CH2(C~)7 '~ ~"T": ~, O-tH 44~.,16a

EM+H] +

U 328

586.5.~.. 568

V 286

z c~ z o3

172

V'

5 t.d

I'l' I'l'l 1O0

I'l'l'l'l' 200

'1' 300

I'J'

J'l'~'l'l'l'l'l'l' 400

'1 500

m/z FIG. 9. FAB-CID-MS/MS spectrum of the (M + H) ÷ ion, m/z 586.5, obtained for Noleoylsphingenine, after reduction/hydroboration, showing product ions that indicate the location of the double bond(s) in the starting material. Collision energy, 7 keV.

9 shows the CID mass spectrum of the (M + H) ÷ m/z 586.5 ion produced by reagent vapor hydroboration and oxidation of N-oleoylsphingenine. The major fragment ions for this derivative, the U and V ions at m/z 328.3 and 286.3, respectively, are typical for amide-reduced ceramides. These ions confirm the location of the alkene in the acyl chain of the native molecule. Also, additional ions due to loss of water from the U and V ions are present (labeled U' and V' at m/z 310.3 and 268.3, respectively). These ions are indicative of the presence of hydroxyl groups in the acyl chain and were used to provide extended information with regard to the characterization of hydroxyacyl and hydroxysphinganine-containing cerebrosides from cestodes. 15,30The new hydroxyl group introduced in the derivatization shifts the mass at the carbon atoms of the former double bond and

6OO

[40]

MS/MS OF GLYCOLIPIDS

763

608 ~OH

-H I I OH

~OH

~OH

~,

OHN~IOm~'\

', +NH2

{+X 242

U° 284

Yo 526

2~2 145

127

Zo Xl (MH-H20) 716 832

163 II

u"

Ill & III

lli

m/z I O0

"~'i~'"I

200

''

'I''"I"~'I''"I''"I

300

400

' ~'I"'

500

iO0

700

800

FIG. 10. FAB-CID-MS/MS spectrum obtained for the (M + H) + m/z 850.7 of the product obtained by reduction of 1 ng (1.2 pmol) lactosyl-N-palmitoylsphingenine. Collision energy, 5 keV. The spectrum was recorded with a photodiode array detector on MS-2, 10 x 0.3 sec integrations per segmentf1,32 Asterisks (*) indicate matrix-related fragments.

generally stabilizes elimination of alkene and H 2 to the new C-OH bond in charge remote site fragmentation. The increased intensity of the charge remote site fragment ions at m/z 472.4 and 458.4 allow almost instant visual identification of the former double bond location between C-9 and C-10 of the acyl chain. This technique was used to locate the position of the carbon-carbon double bond in the sphingolipid base of a ceramide isolated from soft coral. ~5 As an example of amide-reduced neutral glycosphingolipids, the FABMS/MS spectrum of reduced lactosylsphinganine (M + H) ÷ m/z 850.7 is shown in Fig. 10. A 1.0 ng sample was used for the borane reduction and acquisition of the CID spectrum. In addition, the spectrum was recorded 3o B. N. Singh, C. E. Costello, S. B. Levery, R. W. Walenga, D. H. Beach, J. F. Mueller, and G. G. Holz, Mol. Biochem. Parasitol. 26, 99 (1987).

764

GLYCOCONJUGATES

[40]

with a multichannel array detector 31,32which lends an additional factor of 50 to 100 increase in sensitivity above that of derivatization. As was the case for reduced ceramides, the U (m/z 608.3) and V (m/z 242.3) ions are present in this spectrum. Now there is a U 0 ion at m/z 284.3 which represents the same bond cleavage in the sphingoid base as the U ion, but without the carbohydrate unit attached. The Y0 ion at m/z 526.5, an ion encompassing the entire aglycon, is also prominent in the normal FAB mass spectrum of the derivative and can be used to thoroughly characterize the ceramide (i.e., locate double bonds) by obtaining its MS/MS spectrum analogous to the case shown in Fig. 5. The fragment ions for the carbohydrate moiety of the derivative are quite different than those of the native compound. 13,15 Instead of the familiar cleavages of the interglycosidic linkage seen in the spectra of the native and permethylated compounds, the major fragmentations in the sugars occur within the ring yielding LsX0 (m/z 554.1) and t'sX1(m/z 716.5) ions. It shall be shown in a later series of examples for GM~ (Figs. 12A and 13) that this type of fragmentation is due to specific charge location in the ceramide portion of the derivative at the site of high proton affinity (the amino group resulting from reduction). This results in charge remote site-induced fragmentation in the carbohydrate rather than the site-induced protonation and cleavage which is responsible for the B and Y ions in compounds which lack such a directing influence. A very similar spectrum is observed for the amide-reduced derivative of the ganglioside GM3, (M + H) ÷ m/z 1171.8 shown in Fig. 11. The Y0, U, X 0, and Xj ions are found at m/z 570.6, 913.5, 598.6, and 760.5, respectively, although, in this case, the U ion abundance is decreased significantly. The difference that now exists in the derivative of this compound compared to Lac-Cer is the presence of the amine in the carbohydrate from the reduced amide in the NeuAc residue. This residue provides a strong B1 ion at m/z 278.2 and an additional Y2 ion at m/z 894.5. Overall the amine present in the lipid still seems to dictate the fragmentation of this derivative. Permethylated and Amide-Reduced Gangliosides. Reduction of amides to amines in gangliosides can create one or more sites of enhanced basicity in the molecule, which can protonate preferentially in the formation of the (M + H) ÷ ion and provide discrete charge locations which are able to channel the fragmentation toward specific pathways. Combined with an additional 2- to 5-fold increase in sensitivity over that of the 3z j. A. Hill, S. A. Martin, J. E. Biller, and K. Biemann, Biomed. Environ. Mass Spectrom. 17, 147 (1988). 32 j. A. Hill, S. A. Martin, J. E. Biller, and K. Biemann, Int. J. Mass Spectrom. Ion Proc. 92, 211 (1989).

[40]

MS/MS OF GLYCOLIPIDS 732.6

HO]~-O,_

570.6

765

9t3.5

', )------0 I0 ~ ~ ~)'~.OH " "~r~l "~ + ~ ~ H

"(CH')'CH'

'

i-l, _

_

t,~;%~olCOO"

(M+H) ÷

x ~HOH CH~DH

Yo 570.5

1171 8

B. 278.2 Yz 8945 5986 p

'-

X1 760 5

' i' I ' l ' l ' l ' I' I'~'l'~'~' z'~'l'J' ~' ~'~ '1' I'l'r' I' 111' I ' l ' i ' l ' l ' l ' I ' l ' I' I ' l ' l ' l ' l ' l ' t ' 100 200 300 400 500 600 700 800

I ' l ' l ' l ' I' ~' I' I ' ~' I'l'l'l'~'~'l' I' I 900 1000 11 O0 1200

m/z

FIG. 11. FAB-CID-MS/MSspectrum of the lower molecularweighthomologof amidereduced Gm (M + H)+ 1171.9. Collisionenergy, 7 keV.

permethylated alone, the improved fragmentation of permethylated and reduced gangliosides significantly lowers the limit for obtaining CID mass spectra of these compounds. Figure 12A is the FAB-CID mass spectra of the permethylated and reduced C~4-sphingoid homolog of GM1 at (M + H) ÷ m/z 1803.1. In contrast to that of the permethylated-only compound in Fig. 7A, the spectrum contains many more significant ions, even though it was acquired with less than one-half the material (2/~.g). Cleavages still occur adjacent to the reduced amino sugars, B2~ m/z 450.4 and BIa m/z 362.3, but now more fragmentation is observed near the reducing end of the carbohydrate portion giving rise to C and A ions. The C4-methanol ion at m/z 1191.8 is very abundant and, in fact, the C,-methanol ion (where n = the number of sugars in the longest chain) is the base peak in the spectra of most permethylated, amide-reduced gangliosides. This ion defines the size of the carbohydrate unit. Loss of the substituent in the 3position of the terminal residue of C ions is common and has been observed in the FAB mass spectra of permethylated glycoconjugates.8 Thus, the C3 ion also loses sialic acid (SA) to yield an abundant ion at m/z 640.6. Instead of the normal }1, ion, such as in the spectrum of permethyl derivatives,

~2z3.0

766 450.3 T -~

-

ROCH~

A

~

1001.6 ROCHI

.2H~l

"~

)~V-I,,,., tlNl~lJCOOil I

ROCHI

I

OR

Is"

....

H

%;~

640.6

E}2a 450 4 C3-2 6406 I /

B1B 36255

J

10178

83

I

1001.8

Y2-2 1441 1 - -

•

/

J

./h

rl,l.lrl,l.l,l,rl,l,r.l,rl.l,l,l,r/,i I'r'l'l'l'l'lrl'l'l'l'l'r'l,l'l 'I'I'I'I'UI'I ,i,i,f,l,r Vl.l.r,Vl,l,i, v rl,l,l,i,l,l,l.l.l,r ui,i,l.rl.l.l,l.rl,lrl.l.l,l,l,l,i,i ,i, 1oo 200 ,300 400 ,500 600 700 800 900 1000 1100 1200 15500 1400 1500 1600 1700 1800 m/z

797.4

I~lH'

-

1918.0

ROCH.- "'l ROCHe,',

ROCH, I

I

/~V-I-~ I~

1695 9 - - ,,

" "R

~

~HOR

OR

H

_

~

_-

B

I

7 ~ < 1 - I , o .., I-""

" ~I,IOR

C~

"Z~'\

RO

/

EM+H]*

25256--

Cs-MeOH

/~'~u ~ Cl41~R

.... •

18,86.7

I

.=H' 218'~'q

, ,1oo 988.0

JII i, t00

300

,,,lJ,l," .500

700

I.SA4zI 1686.7

_~.J 900

1100

1500

21655 1 /

,11 I,,,I 1, ,~,~

~, 1300 m/z

II'

1700

1900

2100

25500

2fi00

FIG. 12. FAB-CID-MS/MS spectra of the lower molecular weight homologs of gangliosides obtained after permethylation and reduction with BH3. (A) Permethylated GM] fully amide-reduced, (M + H) + 1803.1. (B)Permethylated GT]b fully amide-reduced, (M + H) + 2525.6. Collision energy, 7 keV.

[40]

MS/MS OF GLYCOLIPIDS 464.2

,o.,,_

RO

^

2282

I

~

15.58.9

"~

-'\

'.

:....

~

B~8 B~ _AdOrneDICOOR " 5795 46-~.~ "

I

767

I I

Xe Zo 626.7 5807 754.8 XI

[M+H3÷

X.~ 15U116413 I'Sx2 1"4X2

J'v I'm u'v l'l'l'm'l'm'H'iq u'L'~'luu'm'l'm'~'~,l'l'~'F. I,l,l,l,~ihu,~, 6I' m'~'F'l'm'Vi'l'Vm'Vl'V m,~,F~,~'rm]qm~qm'rmTTL,m,~,l,m, 100 200 500 400 500 600 700 800 900 1000 1100 1200 1500 '1400 1500 1600 1700 1800 m/z FIG. 13. FAB-CID-MS/MS spectrum of the lower molecular weight hornolog of permethylated, partially reduced Gut (M + H) ÷ 1831.2. Collision energy, 7 keV.

which include an hydroxyl group at the site of cleavage as well as a proton due to ionization at the same site or elsewhere in the ion, a Y2-2 ion is observed at m/z 1441.1. This ion would appear to result from a charge remote site process. Virtually all of the analogous ions are present in the spectrum of permethylated, amide-reduced GT]b (M + H) + m/z 2525.6 shown in Fig. 12B. Note that the C,-methanol ion again reveals the size of the carbohydrate (Cs-methanol m/z 1886.7). The attachment of a single sialic acid on the Gal residue of the nonreducing end of GT]b is diagnosed from the B3~ ion at m/z 797.9 as was the case in the permethylated-only derivative. The fact that the other two sialic acids are consecutively linked is again indicated by the B2a ion at m/z 709.7. The reactivity difference between amides in ceramides versus those in N-acetyl sugars in gangliosides with respect to hydroboration allows for the generation of a ceramide-only reduced ganglioside derivative, having the strong directing influence of a single amino group in the aglycon. The same Gu] homolog reacted with a shorter reduction time (10 rain) is shown in Fig. 13. A complete series of X, ions (m/z 626.7, 830.8, 1396.2, and 1641.3) and a U ion at m/z 1559.1 are present. The C4-methanol ion, which

l

l /

768

GLYCOCONJUGATES

[40]

was the most abundant in the permethylated, totally amide-reduced case, is now absent as well as most other ions with charge retention on the nonreducing end due to the absence of amine in the carbohydrate portion. The B ion adjacent to the GalNAc residue (B2~m/z 464.3) remains, which is useful for confirming the position of sialic acid residues. The spectrum also contains some ions resulting from the elimination of two neighboring substituents on a particular sugar residue as noted on the structure in Fig. 13, This occurs mostly with the substituents in positions 3 and 4 of Glc and Gal residues (m/z 754.8 and 959.0, respectively). The MS/MS spectrum of this ceramide-only reduced species is clearly an improvement over that of the permethylated or permethylated, amide-reduced derivatives. The sequence of the sugars is more efficiently determined since there is a complete series of a single ion type. The problem with this derivative lies in the fact that the reduction time required for a suitable yield of ceramidereduced derivative varies depending on the ganglioside (i.e., shorter reaction times are required for GM1 than Go1). Also with these short reaction times, variables such as temperature regulation and strength of the borane reagent (which changes with age) can make a substantial difference in yield of the derivative. Consequently, the reaction has to be optimized individually for each compound type. This is best done by running a range of reactions from 5 to 30 min, and determining by FAB-MS the reaction time most advantageous for MS/MS analysis. Conclusion Tandem mass spectrometry of glycolipids represents a powerful new approach to the determination of structural details, especially when only small quantities of the compound of interest are available and may be present as components of complex mixtures of closely related species. Derivatization can improve sensitivity and can direct the fragmentation along pathways that maximize spectral information content. This approach is still in its early stages of development, and should increase in importance as more experimental data becomes available for a broader range of native compounds, for sets of rationally designed derivatives, and for a variety of collision regimes. Acknowledgments The authors wish to acknowledgeextensive contributions of B. Domonto development of the methodologyand the nomenclature system described in this chapter, helpful discussions with K. Biemann, and the recording of the array detector spectrum by S. A. Martin, J. A. Hill, and J. E. BlUer.The MIT Mass SpectrometryFacility is supported by Grant No. RR00317from the NIH Center for Research Resources.