ANALYTICAL

BIOCHEMISTRY

193,24-34

(1991)

Characterization of Glycosphingolipids by Supercritical Fluid Chromatography-Mass Spectrometry’ Margaret

V. Merritt,’

Department

of Nutrition,

Received

September

Douglas Harvard

M. Sheeley, School of Public

and Vernon Health,

N. Reinhold

665 Huntington

Auenue,

Boston, Massachusetts

02115

14,199O

Gangliosides have been characterized by supercritical fluid chromatography-chemical ionization mass spectrometry (SFC-CIMS) as permethyl and pertrimethylsilyl derivatives, using carbon dioxide as the SFC mobile phase and CI reagent gas. Ganglioside classes and ceramide heterogeneity within each class are well resolved by SFC. Direct SFC-interfacing allows the analytical manipulations of single-ion monitoring, total-ion plots, background subtraction, library searches, and spectral reconstruction algorithms. Addition of ammonia to the CI ion chamber (NH, as a CI reagent gas) yields abundant molecular-weight-related ions, (MH)+ and (MNH4)+ from analyte derivatives. Substitution of methanol for ammonia yields considerable parent-ion fragmentation, providing structural information on carbohydrate sequence, fatty acid, and sphingoid components. Under these latter conditions a unique c-cleavage fragment is observed which differentiates fatty acid from sphingosine heterogeneity. For ganglioside samples, the carboxyl group of neuraminyl residue(s) have been esterified with pentafluorobenzyl bromide and the products analyzed by negative ion chemical ionization MS. This modification improves chemical selectivity and greatly enhances detecting sensitivity. These “soft” ionization conditions provide abundant molecular-weight-related anions for collision-induced dissociation and subpicogram detection. 0 1991 Academic Press, Inc.

1 This project has been supported in part by USAMRIID, Fort Detrick, MD, DAMD17-88-C-8133, USPHS NIH Grant ROI A128215, and a Marie Curie Fellowship of the American Association of University Women to M.V.M. The views, opinions, and/or findings of the research do not necessarily reflect the position or decision of the U.S. Army and no official endorsement should be inferred. ’ Permanent address: Department of Chemistry, Wellesley College, Wellesley, MA 02181.

Glycosphingolipids (GSLS)~ are amphoteric conjugates of a long-chain base (sphingoid), a single fatty acid, and one or more carbohydrate residues. Gangliosides are a class of GSLs containing one or more neuraminic acid residues which terminate the hydrophilic carbohydrate chains. These structures are normal components of mammalian cell membranes and are particularly abundant in neuronal tissues. When the sphingoid moiety is conjugated with a fatty acid it is called a ceramide; it is this lipophilic portion that lies implanted within the cell membrane. The various classes of GSLs are defined by the sequence and number of carbohydrate residues, in combination with the number and position of neuraminic acid groups for gangliosides (1,2). The structural variation in the carbohydrate chains of GSLs between normal and tumor cells has been termed “aberrant glycosylation” to indicate an altered metabolic process that is tumor-associated (3). Within these defined classes, GSLs isolated from both normal and diseased tissue also exhibit considerable microheterogeneity in the ceramide portion of the molecule, (i.e., alkyl-chain modifications of unsaturation, hydroxylation, and chain length). Ceramide heterogeneity in relation to neoplastic degeneration was first reported in 1971 by Hakomori and co-workers (reviewed in Ref. (3)) and more recently aberrant fatty acid a-hydroxylation was suggested to be characteristic of tumor ganglioside metabolism (4). Understanding the structural differences between GSLs of tumorous and normal tissue is an indispensable prerequisite for unraveling their cellular function (5-8). Thus, a simple and sensitive method for

3 Abbreviations used: GSL, glycosphingolipid; SFC, supercritical Auid chromatography; MS, mass spectrometry; CI, chemical ionization; NICIMS, negative ion chemical ionization mass spectrometry; NANAL, N-acetylneuraminyllactose; TMS, trimethylsilyl; PFB, pentafluorobenzyl; CID, collision-induced dissociation; FAB, fast atom bombardment; DCI, direct chemical ionization.

24

0003-2697/91$3.00

Copyright All

rights

0

1991 of reproduction

by

Academic in any

form

Press, Inc. reserved.

GLYCOSPHINGOLIPID

STRUCTURE

BY

CHROMATOGRAPHY-MASS

routine analysis of GSL mixtures would be most helpful in defining functional relationships. Current MS analysis of GSL fractions result in a family of molecular-weight-related ions in which their parent-ion distribution is a measure of composition. For an excellent review on this topic, see Egge and Peter-Katalinic (9). Attributing this heterogeneity to specific components within the molecule is most difficult, although impressive progress has been made with collision-induced dissociation (CID) using high-performance tandem mass spectrometry (10). These approaches are less effective, however, with compositional isomers, where mass selection is not a possibility. Recently, three classes of permethylated GSLs, including gangliosides, were shown to be well resolved by supercritical fluid chromatography (SFC) (11). The present paper extends that work with mass spectrometry interfacing (SFC-MS) and applies this combined instrumental approach to the structural analysis of neuraminyl-containing GSLs. To achieve greater chemical ionization (CI) fragmentation, a CO,-methanol mixture has been utilized as a reagent gas (5,ll). In addition, we report the application of pentafluorobenzyl (PFB) ester derivatization to improve NICIMS detection, analogous to procedures utilized for prostaglandin detection by GC-MS (12-16). MATERIALS

AND

METHODS

Materials Monosialoganglioside, GMi4; disialoganglioside, GDl,; and trisialoganglioside, GTlb (containing lo-30% GDlb) were obtained from Supelco, Inc. (Bellefonte, PA) or Matreya, Inc. (Pleasant Gap, PA). Purified mixed bovine brain gangliosides, N-acetylneuraminyllactose (NANAL), and N-acetylneuraminic acid were purchased from Sigma Chemical Co. (St. Louis, MO). The silylating reagent, Sylon BFT, bis(trimethylsilyl)trifluoroacetamide containing 1% trimethylchlorosilane, was obtained from Supelco, Inc. PFB bromide and reagent grade pyridine were obtained from Aldrich Chemical Co. (Milwaukee, WI); the pyridine was stored over barium oxide to remove water. Solvents and other reagents were of the highest purity available and used as received. Chromatography Supercritical fluid chromatographic separations were performed on Model 501B or Series 600 instruments (Lee Scientific, Inc., Salt Lake City, UT), each equipped with a flame ionization detector and a pneumatically activated submicroliter internal sample loop injection valve (Valco Instruments Co., Inc., Houston, TX). For 4 The nomenclature for the gangliosides recommendations (1,Z).

follows

IUB-IUPAC

CBN

SPECTROMETRY

25

sample concentrations of 1 pglpl, this injector was programmed to deliver 20 to 40 ng of sample on column for each chromatogram. The SFC pump cylinder, injection valve, oven, and detector temperatures were held constant at 15, 15, 120, and 39O”C, respectively. The 10-m capillary columns (50 pm i.d. X 375 pm o.d.) used in this work had one of two different 0.25-pm stationary-phase coatings: SB-cyanopropyl-50 and SBphenyl-5 (Lee Scientific, Inc., Salt Lake City, UT). The mobile phase was SFC grade carbon dioxide (Scott Gases, Plumbsteadville, PA) and the pressure and flow were maintained with an integral flow restrictor fabricated in-house (17). Component resolution and elution were carried out by a programmed increase in the mobile-phase pressure. Unless otherwise specified, the mobile-phase pressure was held at 115 atm for 10 min following injection and then increased at a rate of 10 atm/min to 415 atm. Mass Spectrometry SFC-MS was performed using the above chromatograph coupled to a high-performance mass spectrometer (ZAB-SE, VG Analytical, Manchester, UK) operating at an ion source potential of 8 kV. The instrument interfacing has been previously described (18,19). Under NICI operation, the carbon dioxide mobile phase served as the reagent gas. For positive-ion CI operation, ammonia or a CO,-methanol mixture served as the reagent gas. The latter mixture provided enhanced CI fragmentation; the reagent gas was made up with 12% methanol in carbon dioxide (Scott Gases, Plumbsteadville, PA). All mass spectra have been corrected to nominal values; C, 12.0 amu. Preparation of GSL Derivatives For each derivative type, 2- to 200~pg portions of glycolipid were subjected to the appropriate reaction conditions. The products were dissolved in methylene chloride to bring the final concentration to 1 pglpl for subsequent SFC and SFC-MS analysis. Permethylation. The method of Ciucanu and Kerek (20) was used for permethylation as adapted for glycosphingolipids by Larson et al. (21). Acetylation. Samples were placed in a Teflon-lined screw-cap vial with 100 ~1 each of acetic anhydride and pyridine. Acetylation was carried out at 65°C for 1 h, or overnight at room temperature. The reagents were dried to a white residue with a stream of dry nitrogen or by vacuum centrifugation. Residues were dissolved in methylene chloride. Pentafluorobenzylation. Ganglioside PFB ester derivatives were prepared by dissolving the gangliosides in 200 ~1 of an acetonitrile solution containing, by volume, 18% PFB bromide and 5% pyridine. The sealed Teflonlined screw cap vials were heated for 1 h or allowed to sit

26

MERRITT,

SHEELEY,

overnight at room temperature. The solution was brought to a dry, white residue with a stream of nitrogen, or by vacuum centrifugation. Reagents for subsequent derivatization (acetylation or trimethylsilylation) were added directly to this residue. Trimethylsilylation. PFB-derivatized gangliosides, or other GSL samples, were prepared as trimethylsilyl ether derivatives by the addition of 300 ~1 of a SylonBFT/pyridine/acetonitrile solution (0.7/1.3/1.0) followed by vigorous mixing to facilitate solubilization. The vials were heated for 1 h at 65°C and allowed to sit an additional 24 h at room temperature. Reagents were removed by vacuum centrifugation to yield an orangecolored residue. To this residue were added 500 ~1 each of methylene chloride and water with vigorous mixing. The aqueous layer was discarded and the methylene chloride, containing the desired product, was dried by vacuum centrifugation. The residue was dissolved in dry methylene chloride and immediately analyzed by SFC or SFC-MS. Samples were stored in 200 ~1 of a silylation reagent mixture (Sylon-BT/pyridine/methylene chloride; l/2/4). RESULTS

AND

DISCUSSION

Supercritical fluids, with physical properties intermediate between those of gases and liquids, are able to solubilize and transport high-molecular-weight biopolymers through chromatographic columns. In comparison to HPLC, analytes in supercritical fluids exhibit larger diffusion coefficients, and, thus, improved chromatographic etliciency. The lower viscosity of these fluids results in a smaller pressure drop across the column, allowing the use of capillary tubing for enhanced resolution. The resultant decrease in volumetric flow contributes to the ease of MS interfacing. Carbon dioxide has been widely used as a mobile phase and is likely to continue in popularity because of its low toxicity, cost, and minimal detector response. Moreover, the gas is easy to purify and maintain above its critical point. In the separation of polar biopolymers, however, the low polarity of CO, introduces problems of analyte solubility which can be offset by sample derivatization. This approach has been very successful in extending the applications of SFC to many amphoteric and highly polar materials and, as seen below, these techniques are consistent with the goals for improved MS sensitivity and structural specificity. An earlier application of capillary SFC to permethylated samples had demonstrated the separation of GSLs based on the degree of glycosylation, number of neuraminyl groups, and sphingoid chain variations within each class (11). In separate studies of purified GSL samples, MS has provided extensive detail for understanding unknown structures (4,5,9,10). The importance of these applications, and the recent reports describing SFC-MS interfacing (l&19,22) suggest that the com-

AND

REINHOLD

bined technology could be most effective for understanding complex GSL mixtures. The major goal of this study was to extend the earlier study of GSL mixture separation by SFC to structural analysis using on-line SFC-MS. The primary focus was to develop derivatization strategies that would maintain chromatographic efficiency and resolution, and be compatible with mass spectral analysis. A second goal was to introduce greater selectivity and enhance detecting sensitivity by PFB-labeling of ganglioside samples. Three general derivatization procedures were utilized during this study: methylation, acetylation, and trimethylsilylation. SFC-MS

of Permethylated Gangliosides

Consistent with the earlier work (ll), SFC-CIMS analysis of GM1exhibited two peaks which are presented as ion plots eluting at scans 222 and 226 (Fig. la, insert). Direct MS analysis of the same sample had also indicated two components differing by 28 Da which are resolved by SFC and provide the spectra in Figs. lb and lc. Each spectrum shows one major and one minor parent ion, (MH)+ and (MNH,)+, respectively, with several smaller fragments. Ammonia as a reagent gas provides a rich source of protons and rapidly forms adducts with minimal excess energy. This “soft” ionization, coupled with cold sample injection, due to mobile-phase vaporization, results in little fragmentation. The minor fragments observed are most likely the result of CO,-induced charge-exchange CI. The parent-ion mass, m/z 1826 (scan 222), is consistent with the replacement of 20 hydrogens with methyl groups in GM, containing sphingosine as the ceramide base. The second spectrum is also consistent with this composition, but shows a 28Da increment for the eicosasphingosine moiety, m/z 1854 (scan 226). This mass increment could not be attributed to the eicosasphingosine moiety from these spectral data alone. However, this assignment was achieved by replacing ammonia as a protonating CI reagent gas in favor of more energetic processes, as shown below. The separation of GM1 into components differing by two methylene units suggested that ganglioside resolution by numbers of neuraminyl groups would be equally effective by SFC. Presented in Fig. 2 is the total ion chromatogram obtained from a brain ganglioside sample that was permethylated and analyzed under similar conditions. A review of these data indicate three sets of molecular-weight-related ions between scans 380 and 437. Each profile was obtained by scanning a mass interval that included the protonated molecular ion and its 28-Da increment. The resultant ion chromatograms are presented to the right of the total-ion plot, and labeled G,, , GDla,b, and G,,, . The similarity of the totalion profile to that obtained using flame ionization detection (11) indicates that the SFC-MS interface

GLYCOSPHINGOLIPID

STRUCTURE

BY

CHROMATOGRAPHY-MASS

SPECTROMETRY

27

226

ton

a

Profile G Ml

222

(MH)+A

1826

lee!

-

Scan

Al-200

#222

80~3 4

b

240 Number

5e-

1573

)

(MNH,)+

1854 ‘S ,m d

(MW *

Scan

c

+r

#226

50, WW+

488

688

888

ieee

1288

1466

1688

18BB

m/z as CI reagent gas. (a, inset) Ion profile of each component; relative intensity vs FIG. 1. SFC-CIMS of permethylated G,, using NH,/CO, scan number. Scan range 1810 to 1830 and 1840 to 1860, respectively. Chromatography: SB-phenyl-5 column, with a pressure gradient from m/z 1826. Spectra were acquired by exponential down scan, 3000 to 115 to 415 atm, at 5 atm/min. (b) Mass spectrum taken at scan 222; (MH)+, 400 amu, at 10 s/decade, with l-s interscan time, with an instrument resolution of 1000. (c) Mass spectrum taken at scan 226; (MH)‘, m/z 1854. Spectra were acquired by exponential down scan, 3000 to 400 amu, at 10 s/decade, with l-s interscan time, with an instrument resolution of 1000.

maintains chromatographic resolution and molecular integrity. As observed for G,, and the brain ganglioside samples (Figs. 1 and 2, respectively), the two peaks obtained for each component can be attributed to an approximately equal concentration of sphingoid analogues sphingosine (C,,) and eicosasphingosine (C,,). The mass interval (375 Da) between each of the three major constituents provide the ganglioside class designation. Thus, the components in the sample can be considered to have one, two, and three neuraminyl groups, with molecular-weight-related ions at m/z 1826/1854, 21871 2215, and 2548/2576, respectively. The relationship between components of this mixture can also be observed in the G,, ion profile and the trailing peaks that coeluting with GDla,band G,,, (scans 400 to 430, Fig. 2). This is due to a facile loss of neuraminyl residue(s) resulting in a fragment ion isobaric with the GM,, homologue. That fragment with the loss of one and two is, GDlab and (&lb neuraminyl residues to yield ions isobaric with G,, . Recent GSL studies (3-5) have correlated both hydrophilic and hydrophobic structural elements with specific biological functions. A particularly difficult problem has been the identification of alkane molecular heterogeneity (i.e., changes in the amide fatty acid or sphingoid residue), which becomes impossible with limited amounts of material. Mass spectral fragments generated by FAB, DCI, or CID have been successful in the

characterization of intramolecular structure, and CI, using charge-exchange reagent gas mixtures, have identified a unique a-cleavage fragment within the sphingosine residue that differentiates the aliphatic heterogeneity (11). This fragment increases in ion abundance with CO, (charge-exchange CI processes) and decreases with a protonating reagent gas (e.g., NH,, MeOH, H,O). The protonating gases provide molecular-weight information and such CO,-methanol mixtures have been used to unravel the heterogeneity of two novel cu-galactosyl analogues of the ganglioside Gmb (5). Using a similar approach, the permethylated brain ganglioside sample was re-examined with a CO,-methanol mixture added directly to the ion source during SFC-CIMS. The results are demonstrated in Figs. 3 and 4 for each of the peaks obtained for G,, (Fig. 3a, scan 222; Fig. 3b, scan 227) and Gnla,b (Fig. 4a, scan 233; Fig. 4b, scan 239). G,, provided an abundant molecular-weight-related ion for each peak, (MH)+, at m/z 1826 and 1854, and four or five other fragments of important structural value (Table 1). A mass difference of 28 Da was again observed between the GM1 doublet ions (MH)+, an interval observed for two other fragments, mlz 1451/1479 and 576/ 604. The m/z 145111479 fragments, [(MH) - 375]+ indicates the loss of neuraminic acid, and the other lower mass fragments, m/z 576/604, can be considered a ceramide-related ion. Three remaining fragments occur in both spectra, and their invariance suggests structures

28

MERRITT,

SHEELEY,

AND

REINHOLD

GM il

so a5 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5.-P--J--

Total

Ion

Plot

Scan

Number

FIG. 2. SFC-CIMS permethylated bovine brain gangliosides using NH,/CO, as CI reagent gas. Foreground: total ion profile obtained by scanning m/z 1000-3000 Da. Ion profiles for separate components obtained by a 30-Da scan range plotted to the right; G,, , Gmab, and GTlb. Data obtained from 20 ng total ganglioside injected on the SB-phenyl-5 column.

that do not include the source of heterogeneity. Thus, the interval between the molecular-weight-related ion (MH)+ and the first fragment represents loss of this alkyl moiety, [(MH) - 253]+ (Fig. 3a) and [(MH) - 281]+ (Fig. 3b), which leaves the isobaric a-cleavage fragment discussed above (511). This ion and the m/z 1047 fragment are present only under charge-exchange conditions and their abundance is very sensitive to the concentration and composition of the reagent gas. The mass of the m/z 1047 ion and absence of alkane heterogeneity suggests its structure to be a terminal tetrasaccharide fragment. These fragments, and their rationalized structures are summarized in Tables 1 and 2, and diagrammed at the top of Figs. 3 and 4. The molecular-weight-related ions and their increments in mass between different spectra provide a preliminary evaluation of their composition. Thus, the mass intervals between spectra (Figs. 3 and 4) indicate an additional neuraminyl residue, and an examination for this loss, [(MH) - 375]+, supports this conclusion with the fragments m/z 1812 and 1840 (Figs. 4a and 4b). The a-cleavage fragments, m/z 1934, (MH - 253)+ and (MH - 281)+, indicate an absence of heterogeneity in the amide-linked fatty acid, placing the 28-Da increment in the sphingoid residue, eicosasphingosine. The m/z 825 fragment provides an indication of oligomer sequence (Figs. 4a and 4b), which is consistent with a

terminal (NANA-Hex-HexNAc)+ pyranoxonium ion. Although not resolvable as a separate SFC peak, this brain ganglioside sample was reported to have additional amounts of G,,,. Two fragments support this composition: that of the terminal oligosaccharide residue, (Hex-HexNAc)+, m/z 464, and the dineuraminyl loss fragments, (MH - 736)+, m/z 1451 and 1479 (Fig. 4). This latter fragment can be considered only if the neuraminyl groups occur as a disaccharide unit, (736 = 376 + 361 - 1). Although an exact assignment of structure from these fragments can sometimes be misleading because of double cleavage; e.g., the primary m/z 825 fragment could undergo a secondary neuraminyl loss (with methyl group transfer) providing the ion with m/z 464. The absence of a fragment for (NANANANA-Hex-HexNAc)+, mlz 1186, indicates this disaccharide is not located terminally. Positional isomers are often resolved by a spectral comparison, and, in this case, SFC retention behavior on alternative columns may assist identification. A structural account of GDls (sphingosine analogue) fragmentation is presented at the top of Fig. 4. SFC-MS of Pentafluorobenzyl Derivatives

Trimethylsilyl

In addition to improved chromatographic resolution, a major feature of SFC-MS analysis is the absence of a

GLYCOSPHINGOLIPID

STRUCTURE

BY

CHROMATOGRAPHY-MASS

SPECTROMETRY

29

464

meg o=c

0’. \1047 0

me0 mea

GM~

11451

H ome c rime

2

(MH)+1826

a

I Scan

#222

b

400

600

880

1000

1288

1488

1600

1888

2000

m/z FIG. apex taken from

taken at 3. SFC-CIMS of permethylated brain gangliosides using CO,-methanol as a reagent gas; G M, peak area. (a) Mass spectrum of first peak in first doublet (scan 222; (MH)’ m/t 1826), corresponding to sphingoid containing G,, component. (b) Mass spectrum at apex of second peak in first doublet (scan 227; (MH)+, m/z 1854), corresponding to eicosasphingosine analogue of GM1. Data obtained 20 ng total ganglioside injected on the SB-phenyl-5 column.

matrix background so characteristic of spectra obtained by FAB ionization. Supercritical fluid injection, in addition, provides a “softer” ionization approach (diminished thermal energy of analyte as a consequence of the mobile phase cooling) that yields abundant molecularweight-related ions. The consequent high signal-tonoise ratios allow maximal signal amplification, but most importantly, these gains in sensitivity can be augmented with derivatization, using reagents of high electron affinity for sensitive negative ion detection. The work described below takes advantage of these factors and the extensive technology developed with gas chromatography coupled with electron-capture or NICIMS detection. Most recently, these techniques have been utilized to enhance detection of prostaglandins by esterification with PFB bromide (12-16); an analogue of this reagent has been synthesized and utilized for carbo-

hydrate analysis (23). This PFB moiety is particularly interesting because it imparts high electron affinity to the analyte, and during NICI, eliminates to leave an abundant anion. We have utilized the sialic acid carboxy1 group on gangliosides for PFB ester formation to enhance the sensitivity and specificity of detection for this class of GSLs. Initial studies of this esterification chemistry utilized neuraminyl lactose NANAL as a model compound and stearic acid as an internal standard. The reaction conditions reported for prostaglandins (16) and other fatty acids (24,25) were applied, followed by acetylation and product characterization by SFC. Although we were able to observe quantitative conversion of stearic acid to its PFB ester, these conditions led to extensive decomposition of NANAL. Reaction in acetonitrile with diisopropylethyl amine or in heterogeneous media with tet-

30

MERRITT,

SHEELEY,

AND

REINHOLD 1934

a (MW +

221s Scan

#239

(MW +

,464

b

I

604

1840

I

488 me

I

\

,

1934

888 1888 1268 1488 me isee 2eee 22ae 2488 m/z

FIG. 4. SFC-CIMS of permethylated brain gangliosides using CO,-methanol as reagent gas; G,,, or Gn,, peak area. (a) Mass spectrum taken at the apex of first peak in second doublet (scan 233; (MH)+, m/z 2187), corresponding to sphingosine analogue of Gma or Gnr,,. (b) Mass spectrum taken at the apex of second peak in second doublet (scan 239; (MH)+, m/z 2215), corresponding to eicosasphingosine analogue of Gn,, or Goit,. Data obtained from 20 ng total ganglioside injected on the SB-phenyl-5 column.

rabutylammonium salt (24) or 18-crown-6 ether (25) as catalysts proved unsuccessful. However, quantitative esterification was observed in acetonitrile using pyridine as a catalyst. If acetylation preceded esterification, these reactions did not yield the desired product. With these newly established conditions, GM,, was quantitatively converted to the corresponding PFB ester, but analysis by SFC-NICIMS indicated the acetylation reaction to be incomplete. Efforts to alter the reaction conditions to obtain quantitative acetylation were unsuccessful and trimethylsilylation was investigated. Conversion of the PFB ester to the trimethylsilyl ether derivative proved successful. The earlier work, however, allowed us to establish the conditions for conversion of G,, to PFB esters. Application of these conditions to the brain ganglioside sample provided the SFC data pre-

sented in Fig. 5a. The distribution of components in this chromatogram compared favorably with that produced by sample permethylation (11) (Fig. 2), which indicated a separation of major classes and ceramide heterogeneity within each class. As a chromatographic check, this brain ganglioside mixture was compared with purified samples (Figs. 5b-5d). The observed products had retention times identical with those of expected peaks in the mixture. From these chromatograms, it was judged that the commercial sample of GTla contained 30% G Dla,b 9 as indicated by the manufacturer. Although the PFB derivative would be expected to provide its greatest sensitivity under NICIMS conditions, it was possible to observe as little as 1 ng by flame ionization detection (on column injection, signal/noise, ca. ~10). The detection appeared linear between 1 and 400 pg.

GLYCOSPHINGOLIPID TABLE

STRUCTURE

BY

CHROMATOGRAPHY-MASS

1

Fragment Ion Structures

It is of some interest that we were not able to detect the (M - PFB)) ion for the G,,, analogue in this mixture. In related studies, using HPLC (26) to characterize 2,4-dinitrophenylhydrazides of gangliosides (27), it was found that both sialic acid residues of Gma were fully derivatized; whereas, only one sialic acid in GDlb and two sialic acids in GTlb were converted to dinitrophenylhydrazide. The authors suggested that the failure to form the dinitrophenylhydrazide derivatives at each sialic acid may have resulted from stearic hindrance. This does not appear likely in this case, for even one PFB group should provide the NICIMS sensitivity, albeit at 108 Da lower in mass for each missing PFB group (181 - 73 = 108).

GM,,

m/z 464

Me? m/z 576

Me;l$&;*oH

SUMMARY

+M~

Me ?

31

SPECTROMETRY

oI

ml2 1047

AND

CONCLUSIONS

This report is an extension of an earlier study applying SFC to the separation of GSLs. The present work has discussed (i) a general approach for the separation and molecular-weight characterization of GSL mixtures using SFC-MS, (ii) CIMS techniques that enhance GSL fragmentation to provide a greater structural understanding, and (iii) a reagent labeling procedure for improved detection of ganglioside samples using NICIMS analysis.

NMe AC

RO 1 I ml.2 1573

6 R = glycan

Analysis of the PFB ester, TMS derivatized brain ganglioside sample (Fig. 5a) by SFC-NICIMS provided the data in Fig. 6. Figure 6a is a total ionization plot which showed improved chromatography compared to the permethylated sample (Fig. 2). A mass spectrum of the second eluting peak (scan 178, Fig. 6b) provided a single ion, m/z 2724, which corresponds to the trimethylsilylated eicosasphingosine analogue of G,,, (M - PFB)). Since only oxygen-bound hydrogens would be replaced during trimethylsilylation, and the carboxyl group of NANA was previously blocked with PFB, this would leave a total of 16 hydrogens, e.g., (16 X 72) + 1573 = 2725, to give the (M - PFB)) ion at m/z 2724. The third and fourth major peaks correspond to the Gnla,b area; a mass spectrum taken at scan 187 provided the data in Fig. 6c. These results, (M - PFB)), m/z 3411, are consistent with the eicosasphingosine analogue of GDle,b and correspond to a 760-Da increment over the G,, analogue. This increment corresponds to the anticipated mass shift for replacing one trimethylsilyl group with an additional PFB-NANA moiety.

20

10

30

40

MIN

b

32

=

40

d

I

36

44

36

44

MIN

FIG. 5. gliosides: column,

SFC analysis of TMS O-ether (a) bovine brain gangliosides; SB-phenyl-5 column.

PFB ester derivatives of gan(b) Grrrl; (c) GD,a,b; (d) GTlb;

32

MERRITT,

SHEELEY,

AND

TABLE

REINHOLD

2

Fragment Ion Structures

GDla,b

m/z a25

m/z 1029

AC

RO 1 I MeN

m/z

1934

+‘c

-CHs ! R = glycan

m/z

1812,

la40

The major feature of this report is the combination of on-line sample purification, detection, and molecularweight determination for the characterization of GSLs. These techniques allow the powerful manipulations that have so characterized GC-MS, e.g., single-ion monitoring, total-ion plots, background subtraction, library searches, and spectral reconstruction algorithms. An additional benefit of SFC interfacing is the utility of the mobile phase to also serve as the reagent gas, providing “soft” chemical ionization (low-energy thermal electrons) along with its demonstrated sensitivity (ion stability as a consequence of multiple, pressure-induced collisions). Moreover, mobile-phase decompression may contribute further to diminished fragmentation, as a consequence of analyte cooling. Thus, the resultant spectra provide molecular-weight information only with little or no spectral detail (Figs. 1 and 6). The relationship of biological function to a specific GSL molecular domain (3-5) demonstrates the need for a detailed structural understanding. As an example, the observed ceramide heterogeneity in a human neuroblastoma (aberrant fatty acid cY-hydroxylation) appears to be characteristic of ganglioside metabolism in the tumor (3,4). Thus, molecular weight data alone are insufficient

(MI-I)’

- NANA

for relating structure to biological function; consequently, techniques are needed that provide information on the individual components of GSL structure. Mass spectrometry using FAB (9), DC1 (28,29), and CID (30) experiments have proven adequate to address many of these structural problems. The most successful has been tandem MS coupled with CID, although the heterogeneity mentioned above is resolved only after sample permethylation and reduction. This structural problem of ceramide heterogeneity has proven to be most difficult and requires the formation of a ceramide fragment that would differentiate the two aliphatic chains and their component heterogeneity. The generation of such a fragment by direct analysis of an intact molecule was first realized using field desorption MS (31). This fragment was later characterized as the (Ycleavage fragment (5) (bond rupture (Y to the carbon bearing the heteroatom), where the odd, nonbonded electron residing in the nitrogen (produced by chargeexchange CI) triggers an elimination of the aliphatic terminal portion of the sphingoid moiety (Scheme I). More recent studies have indicated this same fragment could be induced by direct chemical ionization, using CO, as a charge-exchange reagent gas (32) or combined with

GLYCOSPHINGOLIPID

STRUCTURE

1881.

BY

CHROMATOGRAPHY-MASS

2724

98,

a

88,

3

Scan

f

7e-

;

6e-

2

58,

;

4e-

g

?a,

#178

b

m/z

FIG. 6. SFC-NICIMS analysis of brain gangliosides. (a) Total ion profile, insert, of a 20 ng bovine brain ganglioside sample prepared as the per (TMS) ether PFB ester derivative and collected by SFCNICIMS. Scan range, 1000-4500 Da using a SB-phenyl-5 column. (b) SFC-NICIMS spectrum taken at scan 178, m/z 2724 ((M - H)-)), characterized as the (TMS),, O-ether eicosasphingenine anion of G M,, ((M - H)))), (c) PFC-NICIMS spectrum taken at scan 187, m/z 3411 ((M - H)-), characterized as the (TMS),, O-ether PFB, ester eicosasphingosine anion of Gola,b, (M - H)-, corresponding to the loss of one PFB moiety from (TMS)i9 O-ether PFB, ester Goi+.

a reagent (PFB) with high electron affinity. Comparable techniques have been described for prostaglandin detection (13-16) and analogues of this reagent for enhancing carbohydrate sensitivity (23). The noteworthy features of this PFB group are its high electron avidity, transient anionic stability, and facile elimination to leave a single abundant molecular-weight-related carboxylate anion; this process is called dissociative capture. This sequence of events means that the total-ion current is directly related to the molecular weight, even though the ganglioside itself was uninvolved. The very high electron affinity of the PFB group, its facile elimination, and reported NICIMS sensitivity (16,23) suggest that (M - H)- ions are generated at a rate approaching the collision rate constant. Since the energy constraints of this resonance capture process limits fragmentation to the proximal ester only (not dispersed into alternative fragmentation pathways), the abundance of the ganglioside anion may be comparable to a molecule having the high electron affinity of a PFB group. The limits of NICI detection for PFB-derivatized gangliosides (Figs. 5 and 6) have not been determined, but it is anticipated that these procedures should provide subpicogram sensitivity considering the quantitative derivatization, abundant parent ions and limited fragmentation, and the documented sensitivity of related structural analogues using GC-NICIMS (16) and SFCNICIMS (23). In contrast to FABMS, SFC-MS is particularly suited for the characterization of mixtures and the detection of trace amounts of sample, where sensitivity is limited by the desorption matrix. Compared to the thermally driven process of DCIMS, fluid injection also avoids the pyrolytic and mass limitations imposed by that technique. The use of selective reagent gases dur-

0-CH, glycan-0-CH,-YH-CH-CH=FH

methanol to obtain additional protonated molecularweight information (11). Using the latter conditions, a series of fragments have been generated that provide considerable insight to all conjugating groups: carbohydrate, sphingoid, and fatty acid (see Tables 1 and 2 and Figs. 3 and 4 and structures therein). Although we report only the results obtained from ganglioside samples, these procedures are generally applicable to all neutral GSLs, and may be limited only by derivatization techniques that provide appropriate sample depolarization. The last aspect of this SFC-MS study applies to GSLs possessing a free carboxyl group and describes derivatization of gangliosides for enhanced NICI detection. This utilizes the neuraminic acid moiety to attach

33

SPECTROMETRY

CIMS

-

CB-MeOH

CH,--I+ I O=C-R

CH,

CI charge-exchange product

0 -CH, glycan

-

CH-CH-CH

0 -CH,-CH II

+

(!H,).

CH,---N+

I O=C-R

CH,

a-cleavage fragment

SCHEME

terminal sphingoid residue I

34

MERRITT,

SHEELEY,

ing chemical ionization provides considerable fragmentation flexibility to determine adequately the component structures comprising GSLs. The present study indicates that an on-line chromatographic MS technique can be achieved with SFC-MS, and when combined with PFB-labeling procedures and negative-ion chemical ionization, abundant molecularweight-related ions are available for maximizing detecting sensitivity. It is our present goal to adapt this phase of the study to CID fragmentation and tandem MS, which may provide the desired sensitivity along with adequate structural detail.

AND

REINHOLD

L. R., Ayers, C. R., Wills, M. R., and Savory, nun. Ch.em. Pathol. Pharmacol. 40,73-86. 14. Waddell, Spectrom.

K. A., Blair,

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P. H. (1979) J. (1987) C. E. (1988)

Adu. Enzymol. Mass

59,303-

Spectrom.

Biochemistry

19.

R. J., and Murphy,

Sheeley, 83-96.

Reu. 6, 27,

1534-

23. Caesar, them.

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25. Gylledhaal, 333.

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