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THE ,JOURNAI. OF BIOLOT.ICAI. CHEMISTRY Vol. 259, No. 1, Issue of January 10, p. 481-466 1964 hated in ti.S A. Gas Chromatography/Mass Spectrometry Analysis of Oligosaccharides from Neutral Glycosphingolipids of Murine B Cell Hybridomas* (Received for publication, August 1, 1983) Maciej Ugorski, Bo Nilsson, Kenneth Schroer, Jo Anne Cashel, and David Zopf$ From the Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205 Monoclonal B cell hybridoma cell lines express gly- colipids characteristic of both the myeloma and normal lymphocyte parents. The neutral glycolipids from hybridoma cell lines metabolically radiolabeled with [‘4C]galactose plus [‘4C]glucosamine separate by high performance thin layer chromatography into patterns that may reflect differences in glycolipid expression among B cell subsets. Gas chromatography/mass spec- trometry of oligosaccharides released by trifluoroace- tolysis from neutral glycolipids extracted from lo9 clonally expanded hybridoma cells reveals the carbo- hydrate composition of the major glycolipid compo- nents detected by thin layer chromatography. Glyco- lipids differentially expressed among six cell lines ana- lyzed include monohexosylceramide, lactosylceramide, globotriaosylceramide, globoside, asialo-GMz, and 2’fucosyllactosylceramide. The latter compound has not been described previously in cells from the mouse. Monoclonal B cell hybridoma lines express glycolipids char- acteristic of both the myeloma and normal lymphocyte par- ents (1). When splenocytes are fused with the myeloma cell line SP2/0, which produces only mono- and dihexosylceram- ides, many of the resulting hybridomas express neutral gly- colipids containing three or more monosaccharides. The pat- tern expressed by each hybridoma is stable in cell culture for several weeks. We are comparing B cell hybridoma cell lines derived from selected preparations of splenocytes to deter- mine whether functionally distinct B cell subsets express different glycolipids. In this report we present analyses of the total neutral glycolipids from six cell lines. Oligosaccharides released by trifluoroacetolysis from a mixture of total neutral glycolipids extracted from lo9 cells are analyzed by gas chro- matography/mass spectrometry (2). The oligosaccharides de- tected correspond to the major bands detected by autoradiog- raphy of metabolically labeled glycolipids separated by thin layer chromatography. One of the hybridomas studied con- tains 2’-fucosyllactosylceramide, a glycolipid not previously reported in murine cells. EXPERIMENTAL PROCEDURES’ RESULTS For each cell line described below, total neutral glycosphin- golipids purified from 10H cells were subjected to acid hydrol- ysis and the total sugar content was determined by gas chro- * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby matography of monosaccharides as alditol acetates. Hybrido- mas 3525.1 (22 pg) and 39.5.1 (19 pg) contain about lo-fold more sugar in their neutral glycolipids than SP2/0 (2 pg), the myeloma from which they were derived. Hybridoma 4.6.10 (78 pg) contains about 7-fold more sugar in neutral glycolipids than its parent myeloma, 45.6.TG (11 pg). All glycolipid mixtures contained glucose and galactose as major compo- nents plus trace amounts of fucose, N-acetylglucosamine, and N-acetylgalactosamine. Molar proportions of monosaccha- rides were subject to as much as 50% variation on repeated analyses because the quantities determined were near the limits of sensitivity of the method. SP2/0 Cells-Glycosphingolipids of the murine cell line SP2/0 radiolabeled for 48 h with [‘“Clgalactose plus [“Cl glucosamine migrate during HPTLC’ as two sets of double bands in positions that correspond to CMH and CDH (Fig. 1A). Separation of glycosphingolipids with identical carbo- hydrate chains into two or three closely migrating bands has been shown to be due to heterogeneity in their ceramide moieties (15,16). GC/MS analysis of oligosaccharides released by trifluoroacetolysis from total neutral glycolipids of 10’ SP2/0 cells shows a single peak whose relative retention time (Fig. 2) and mass spectrum (data not shown) are the same as for permethylated lactitol. Hybridoma 35.25.1-Hybridoma 35.25.1 was derived from the products of fusion of BALB/c mouse splenocytes with SP2/0 cells and secretes an IgMk antibody (2). HPTLC analysis of neutral glycosphingolipids from 35.25.1 cells met- abolically radiolabeled with [%]galactose and [“‘C]glucosa- mine (Fig. 1B) shows 3 major components that co-migrate with CMH, CDH, and CTH. A minor band migrates between CDH and CTH in approximately the same position as re- ported for 2’-fucosyllactosylceramide from rat intestinal epi- thelial cells (3). When material in the minor band is eluted from the chromatogram and treated with cu-fucosidase, it migrates on subsequent HPTLC analysis as a single band marked “aduertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact. j To whom all correspondence should be sent. ’ Portions of this paper (including “Experimental Procedures” and Figs. 2-12) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard maenifvinq glass. Full size photocopies are available from the Journal-of “Biological Chemistry, 9650 Rockville Pike. Bethesda. MD 20814. Rearrest Doc- ument No. 83M-2183, cite the authors, and include a check or money order for $6.00 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press. ’ The abbreviations used are: HPTLC, high performance thin layer chromatography; NTF, N-trifluoroacetylated; GC/MS, gas chroma- tography/mass spectrometry; CMH, ceramide monohexosides; CDH, ceramide dihexosides; CTH, ceramide trihexosides; GH~, 113-N-acetyl- neuraminosylgangliotriaosylceramide. 481 by guest on March 2, 2020 http://www.jbc.org/ Downloaded from

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THE ,JOURNAI. OF BIOLOT.ICAI. CHEMISTRY Vol. 259, No. 1, Issue of January 10, p. 481-466 1964 hated in ti.S A.

Gas Chromatography/Mass Spectrometry Analysis of Oligosaccharides from Neutral Glycosphingolipids of Murine B Cell Hybridomas*

(Received for publication, August 1, 1983)

Maciej Ugorski, Bo Nilsson, Kenneth Schroer, Jo Anne Cashel, and David Zopf$ From the Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205

Monoclonal B cell hybridoma cell lines express gly- colipids characteristic of both the myeloma and normal lymphocyte parents. The neutral glycolipids from hybridoma cell lines metabolically radiolabeled with [‘4C]galactose plus [‘4C]glucosamine separate by high performance thin layer chromatography into patterns that may reflect differences in glycolipid expression among B cell subsets. Gas chromatography/mass spec- trometry of oligosaccharides released by trifluoroace- tolysis from neutral glycolipids extracted from lo9 clonally expanded hybridoma cells reveals the carbo- hydrate composition of the major glycolipid compo- nents detected by thin layer chromatography. Glyco- lipids differentially expressed among six cell lines ana- lyzed include monohexosylceramide, lactosylceramide, globotriaosylceramide, globoside, asialo-GMz, and 2’fucosyllactosylceramide. The latter compound has not been described previously in cells from the mouse.

Monoclonal B cell hybridoma lines express glycolipids char- acteristic of both the myeloma and normal lymphocyte par- ents (1). When splenocytes are fused with the myeloma cell line SP2/0, which produces only mono- and dihexosylceram- ides, many of the resulting hybridomas express neutral gly- colipids containing three or more monosaccharides. The pat- tern expressed by each hybridoma is stable in cell culture for several weeks. We are comparing B cell hybridoma cell lines derived from selected preparations of splenocytes to deter- mine whether functionally distinct B cell subsets express different glycolipids. In this report we present analyses of the total neutral glycolipids from six cell lines. Oligosaccharides released by trifluoroacetolysis from a mixture of total neutral glycolipids extracted from lo9 cells are analyzed by gas chro- matography/mass spectrometry (2). The oligosaccharides de- tected correspond to the major bands detected by autoradiog- raphy of metabolically labeled glycolipids separated by thin layer chromatography. One of the hybridomas studied con- tains 2’-fucosyllactosylceramide, a glycolipid not previously reported in murine cells.

EXPERIMENTAL PROCEDURES’

RESULTS

For each cell line described below, total neutral glycosphin- golipids purified from 10H cells were subjected to acid hydrol- ysis and the total sugar content was determined by gas chro-

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby

matography of monosaccharides as alditol acetates. Hybrido- mas 3525.1 (22 pg) and 39.5.1 (19 pg) contain about lo-fold more sugar in their neutral glycolipids than SP2/0 (2 pg), the myeloma from which they were derived. Hybridoma 4.6.10 (78 pg) contains about 7-fold more sugar in neutral glycolipids than its parent myeloma, 45.6.TG (11 pg). All glycolipid mixtures contained glucose and galactose as major compo- nents plus trace amounts of fucose, N-acetylglucosamine, and N-acetylgalactosamine. Molar proportions of monosaccha- rides were subject to as much as 50% variation on repeated analyses because the quantities determined were near the limits of sensitivity of the method.

SP2/0 Cells-Glycosphingolipids of the murine cell line SP2/0 radiolabeled for 48 h with [‘“Clgalactose plus [“Cl glucosamine migrate during HPTLC’ as two sets of double bands in positions that correspond to CMH and CDH (Fig. 1A). Separation of glycosphingolipids with identical carbo- hydrate chains into two or three closely migrating bands has been shown to be due to heterogeneity in their ceramide moieties (15,16). GC/MS analysis of oligosaccharides released by trifluoroacetolysis from total neutral glycolipids of 10’ SP2/0 cells shows a single peak whose relative retention time (Fig. 2) and mass spectrum (data not shown) are the same as for permethylated lactitol.

Hybridoma 35.25.1-Hybridoma 35.25.1 was derived from the products of fusion of BALB/c mouse splenocytes with SP2/0 cells and secretes an IgMk antibody (2). HPTLC analysis of neutral glycosphingolipids from 35.25.1 cells met- abolically radiolabeled with [%]galactose and [“‘C]glucosa- mine (Fig. 1B) shows 3 major components that co-migrate with CMH, CDH, and CTH. A minor band migrates between CDH and CTH in approximately the same position as re- ported for 2’-fucosyllactosylceramide from rat intestinal epi- thelial cells (3). When material in the minor band is eluted from the chromatogram and treated with cu-fucosidase, it migrates on subsequent HPTLC analysis as a single band

marked “aduertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact.

j To whom all correspondence should be sent. ’ Portions of this paper (including “Experimental Procedures” and

Figs. 2-12) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard maenifvinq glass. Full size photocopies are available from the Journal-of “Biological Chemistry, 9650 Rockville Pike. Bethesda. MD 20814. Rearrest Doc- ument No. 83M-2183, cite the authors, and include a check or money order for $6.00 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

’ The abbreviations used are: HPTLC, high performance thin layer chromatography; NTF, N-trifluoroacetylated; GC/MS, gas chroma- tography/mass spectrometry; CMH, ceramide monohexosides; CDH, ceramide dihexosides; CTH, ceramide trihexosides; GH~, 113-N-acetyl- neuraminosylgangliotriaosylceramide.

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r..

B C D

Glycolipids of B

E F

FIG. 1. Thin layer chromatogram of metabolically radiola- beled total neutral glycosphingolipids from the following cell lines. A, SP2/0; H, 35.25.1; C', 39.5.1; I ) , 45.6.TG; E, 4.6.10; h', 1Al. The procedures for radiolabeling with ["C]galactose plus ["C]gluco- samine and purification of glycosphingolipids are described in the text. For each cell line, an aliquot of total neutral glycosphingolipids containing 15.000 cpm is applied to a plate coated with Silica Gel 60 and the chromatogram is developed with chloroform/methanol/water, 60::35:8 (v/v/v). After drying, the plate is exposed to Kodak X-Omat AR film, XAR5 at room temperature for 48 h. Neutral glycosphin- golipid standards containing 1, 2, 3, or 4 simple sugars (not shown) migrate in the regions of the chromatogram indicated by vertical bars on the kft .

with the same mobility as CDH (Fig. 3). HPTLC analysis after metabolic radiolabeling with ['Tlfucose (Fig. 3) shows a pair of hands, the faster migrating at the position of the minor band in Figs. 1 and 4, top, and the slower co-migrating with the faster hand of CTH.

GC/MS analysis of the permethylated oligosaccharide al- ditols from total neutral glycosphingolipids of 35.25.1 cells shows three peaks (Fig. 4). Two of these have relative reten- tion times (Fig. 4) and mass spectra (data not shown) identical with permethylated oligosaccharide alditols released from standard CDH and CTH. The third has the same retention time (Fig. 4) and mass spectrum (Fig. 5) as standard permeth- ylated 2'-fucosyllactitol.

Hybridoma 395.1-Hybridoma 39.5.1 was derived as de- scribed above for 35.25.1 and secretes a IgG2bk antibody." TLC of metabolically radiolabeled neutral glycosphingolipids shows bands in positions that correspond to CMH, CDH, CTH, and glohosides (Fig. 1). GC/MS analysis of oligosac- charides shows three peaks whose relative retention times and mass spectra are characteristic of the permethylated alditols of lactose and globotriaose and the permethylated NTF alditol of globo-N-tetraose (Fig. 6). The mass spectrum of material with the same relative retention time as the permethylated NTF alditol of globo-N-tetraose (Fig. 7) agrees with the spectrum obtained for the authentic standard except that it lacks ions above m / z 518.

Myeloma 45.6. TG-Myeloma 45.6.TG is a spontaneous plasmacytoma from BALB/c mice that secretes IgG2bk an- tibodies (8). TLC analysis of metabolically radiolabeled neu- tral glycolipids from 45.6.TG shows major hands in positions that correspond to CMH, CDH, and CTH (Fig. 1). In addition, there are very faint bands in the position for globosides. GC/ MS analysis of oligosaccharides released from neutral glyco- lipids of 45.6.TG cells (Fig. 8) shows peaks identified as permethylated alditols of lactose and glohotriaose (Fig. 9). A

' Lundblad, A., Schroer, K., and Zopf, D., J. Immunol. Methods, in press.

Cell Hybridomas

trace peak with the same retention time as the permethylated NTF alditol of globo-N-tetraose is detected in the single ion trace for m/z 314, but the mass spectrum obtained (data not shown) is too weak to permit positive identification of the compound.

Hybridoma 4.6. IO-Hybridoma 4.6.10 was derived by fusion of myeloma 45.6.TG with splenocytes of BALB/c mice im- munized with pneumococcal polysaccharide Type 111 (8). It produces an IgAk antibody that binds the polysaccharide but does not secrete the IgG2bk antibody of the parental myeloma ceL4 The major radiolabeled glycosphingolipid in these cells migrates as a set of double bands in a position that corre- sponds to ceramide trihexosides (Fig. 1). Radiolabeled bands that correspond to ceramide monohexosides and ceramide dihexosides are much weaker. GC/MS analysis of oligosac- charides released from neutral glycosphingolipids of hybri- doma 4.6.10 (Fig. 10) shows a strong peak identified as the permethylated alditol of globotriaose and a much weaker peak identified as permethylated lactitol. In addition, there is a small peak identified by its retention time and mass spectrum as the permethylated NTF alditol of ganglio-N-triaose.

Lymphoma IAl-Lymphoma 1Al is a variant of L5178Y, a thy-1 bearing methyl-cholanthrene-induced lymphoma of DBA/2 origin, selected for susceptibility of NK cell-mediated lysis (9). TLC analysis of metabolically radiolabeled glyco- sphingolipids from 1Al (Fig. l) shows double sets of bands whose positions correspond to CMH and CDH, as well as a triple set of bands whose positions correspond to gangliotriao- sylceramides. Only trace amounts of more slowly migrating radiolabeled material are evident. GC/MS analysis of oligo- saccharides from neutral glycolipids of cell line 1Al (Fig. 11) shows two peaks, a minor peak identified as permethylated lactitol and a major peak whose retention time and mass spectrum are identical with those of the permethylated NTF alditol of ganglio-N-triaose (14) (Fig. 12).

DISCUSSION Hybridomas prepared by fusing normal lymphocytes with

myeloma cells express malignant properties of the myeloma parent cell line as well as characteristics of the differentiated normal lymphocyte parent, such as synthesis and secretion of immunoglobulins (6). The carbohydrate chains carried on glycolipids from different hybridoma cell lines vary in length and composition depending upon the source of parent lym- phocytes and the methods used to promote blast formation prior to fusion (1). We are analyzing the glycolipids produced by monoclonal B cell hybridoma lines in order to assess individual variation in expression of complex carbohydrates among B lymphocytes. SP2/0 is a suitable malignant cell line in these experiments because it expresses only glucosyl- and lactosylceramides (Fig. L4 and Fig. 2). As 45.6.TG expresses CTH and probably a small amount of globoside (Fig. 1D and Fig. 7), it is not suitable as a parent myeloma to study expression of these glycolipids in normal lymphocytes.

In the hybridomas 35.25.1 (Fig. 1B) and 39.5.1 (Fig. lC), which were formed by fusion of splenic B cells with SP2/0, glycolipids with carbohydrate chains longer than lactosylcer- amide probably represent products of glycosyltransferases genetically regulated by the normal lymphocyte parent cell. The observation that a few glycolipid patterns such as those represented by hybridomas 35.25.1 and 39.5.1 (Fig. 1, B and C) recur frequently among large numbers of hybridomas pre- pared from similarly stimulated lymphocytes5 (1) suggests that there are major subpopulations of immunoresponsive B

K. Schroer, unpublished results. M. Ugorski, unpublished observations.

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Glycolipids of B Cell Hybridomas 483

cells that differ in expression of glycosyltransferase activities. Interpretation of the glycolipid patterns expressed by hybri- domas is complicated by the fact that genetic complementa- tion between malignant and normal lymphocyte parents could lead to carbohydrate structures that neither parent cell alone could produce (17). Alternatively, fusion with a malignant cell might lead to simplification of the carbohydrate chains of glycolipids as has been observed following other kinds of transforming events (18,19). For these and other reasons, the glycolipids expressed by a hybridoma cell line may not com- pletely represent those of the normal lymphocyte parent. Nevertheless, it is likely that some glycosphingolipids ex- pressed by hybridomas will prove useful as markers for differ- entiated subsets of immunologically responsive B cells.

To identify the carbohydrate chains in glycolipids from hybridomas, we have combined two complementary meth- ods-HPTLC and GC/MS. HPTLC of radiolabeled glyco- lipids from large numbers of cell lines permits a general comparison of the relative amounts of glycolipids of different carbohydrate chain lengths but provides only tentative as- signment of specific carbohydrate structures based upon mo- bilities of samples with respect to standards. Often, com- pounds of similar composition and molecular weight do not separate completely. In Fig. 1, for example, bands containing ceramide trihexosides from cell line 35.25.1 (lane B ) overlap with bands containing asialo-GwL from cell line 1Al (lane F ) . In the chromatogram of cell line 4.6.10 (Fig. lE ) , a relatively large amount of CTH obscures a much smaller amount of asialo-GM2, known to be present from GC/MS analysis (Fig. 9).

To further characterize the structures of carbohydrate chains of glycolipids, we have applied a recently developed GC/MS method. We have shown previously (2) that trifluo- roacetolysis liberates oligosaccharides quantitatively by spe- cific cleavage of the carbohydrate-ceramide linkage in glyco- lipids that contain 4-sphingenine. Carbohydrates linked to saturated bases (dihydrosphingosine or phytosphingosine) are not released. The carbohydrate mixture obtained after trifluo- roacetolysis of neutral glycolipid mixtures is reduced, per- methylated, and fractionated on silica gel to separate free permethylated NTF oligosaccharide alditols from the deriva- tized oligosaccharides still linked to a saturated base. Oligo- saccharide derivatives are identified according to their reten- tion times relative to an internal standard and by their mass spectra.

By the combination of methods outlined above, we have identified a glycolipid not previously reported in mouse cells with the probable structure:

Galfi1-4Glc-ceramide

I Fucotl

Evidence in support of this structure is as follows: 1) oligo- saccharides released from the purified total glycosphingolipids of cell line 35.25.1 include a compound whose retention time and mass spectrum are identical with those for authentic 2'- fucosyllactose (analyzed as the permethylated oligosaccharide alditol); 2) total neutral glycosphingolipids from the same cell line radiolabeled with ['4C]galactose and ['4C]glucosamine include a component that migrates on TLC in the same region as authentic 2'-fucosyllactosylceramide; 3) when scraped from the TLC plate, eluted from silica gel, and treated with beef kidney a-fucosidase, the labeled material is completely con- verted to a labeled compound that migrates with lactosylcer- amide; 4) metabolic labeling with ['4C]fucose gives two bands, the faster of which migrates in the same region as authentic

2'-fucosyllactosylceramide from rat intestine and the slower in a position overlapping CTH. 2'-Fucosyllactosylceramide has been described in rat intestinal epithelial cells (3), but we are not aware of any reports of this glycosphingolipid isolated from any other source, including lymphocytes (see "Note Added in Proof").

Our results establish the presence of CDH, CTH, globoside, and asialo-Gw2 in mouse B cells and confirm that these glycosphingolipids are differentially expressed in B cell hy- bridomas (20). Previous studies using metabolic radiolabeling techniques (10) demonstrated several neutral glycolipids in mouse B cells, including some that co-migrate with standard CMH, CDH, CTH, and globoside. Different patterns of radi- olabeled glycolipids were obtained from subfractions of sple- nocytes separated on nylon wool columns and by agglutina- tion with lectins, but no evidence for the chemical structures of the carbohydrate moieties was presented (10). Neutral glycosphingolipids from human lymphocytes contain carbo- hydrate chains similar to those in the mouse (21). The ratio of CTH to globoside differs markedly in lymphocyte subpop- ulations isolated from tonsils, thymus, and peripheral blood (21).

The major subclassifications of B lymphocytes are based upon differential expression of surface antigens (22) and selective responsiveness to carbohydrate-binding mitogenic lectins (23), antigens (24), and antibodies to cell surface immunoglobulin (25). Glycosphingolipids are known to func- tion in some cells as endogenously synthesized (26) or ac- quired antigens (27, 28) and as receptors for lectins (29) and toxins (30). Attempts to establish the presence or absence of glycolipid components on lymphocyte surface membranes us- ing antibodies and lectins have produced conflicting results (31-33). Interpretation of experiments that depend exclu- sively upon binding of antibodies and lectins to cell surfaces may be misleading due to cross-reactions between similar carbohydrate structures on glycoproteins and glycolipids (29) and unexplained variation in accessibility of membrane- bound glycolipids (16). We anticipate that further systematic chemical analysis of glycosphingolipids in monoclonal hybri- domas and lymphomas will provide useful information con- cerning the differential distribution of specific carbohydrate structures in functionally distinct lymphocyte subpopula- tions.

Note Added in Proof-After this manuscript was submitted, 2'- fucosyllactosylceramide was reported to occur in marine shellfish (Hayashi, A., and Matsubara, T . (1983) Proceedings of the 7th Inter- national Symposium on Glycoconjugates, Lund-Ronneby, July 17-23, 1983 (Chester, M. A,, Heinegird, D., Lundblad, A., and Svensson, S., eds) pp. 266-267, Rahms i Lund, Sweden).

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1691-1694

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Glycolipids of B Cell Hybridomas

SUPPLE*LITIUI V i l E R l A L TO

and 39.5.1 *ere derlred d l described prer laur ly2 . The 8alb/c w e l w 45.6.TG was k l n d l y t e l l Llner--SP210 c e l l s (61 were k l n d l y provided by Or. David Sa~hs. Hybr ldmas 35.25.1

provided by Dr. Matthew Scharff 1 7 ) . Mybr ldom 4.6.10 was der ived dr de~crlbed I n a pmv- lous pvb l iCdt ion *ere i t "as designated tC4.6 (8). 1A1 c e l l s ( 9 ) were k l n d l y prorlded by Or. Y ~ l l l a n Yaung.

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486 Glycolipids of B Cell Hybridomas

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RETENTION TIME IMINUTES)

rnIZ

Flgure 7. Mars spectrumof rnaterial eluted at 23.1 nln i n t h e gas ChraMto9rdm r h a n i n Fig. 6. The mass rpec- t r m i s s i m i l a r t o that obtained for the Oligosaccharide released by tr i f lu0roacetOlyrir frm authentic glob- ride. The ions mlz 314 and m/z 518 indicate the nowreducing terminal sequence hexNTF-her. The ion mlz 236 indicates d mOno-D-wbstituted hex-al-1-d a t t h e a l d i t o l terminal. lens frm pvimary cleavages tha t g i ve trisaccharide sequences are not observed. These ions are of la r e l a t i v e abundance i n the mss spectrum of standard globorlde (reference 21 and probably are absent i n the mass ~ p e n r m s h a n because of the -11 anaunt of material available for analyrir. The large re la t i ve abundance l>m)l o f m/z 187 a secondary fragnent that or ig inates frm m/z 518 by e l i n i n a t l o n O f trmminal hexNTF, ind icates that the l ink& a t the nowreducing t e r n l n a l i s hexNTFl-shex-: when the l inkage i s 1-2 1-4 or 1-6 the abundance O f m/z 187 i s a . The prerenee Of m/z 296 er tab l i rhe r t he presence Of I n i n t e r m i he; residue which i s not 3-CLwbrt i tuted. There findings support a t e t rasacchar ide a ld i t o l w th two in te rna l hex residues, a r t r u c t u y v h i c h i s also consistent with the GC retention time.

CDH CTH

RETENTION TIME Iminutml ~ "

I 219-7 423-7 CHDOMe 1

CDH CTH GnTR IM4 t t t

I

CDH GnTR i I

mlrZ35 u

B 3 U m

5 , h , . , , , , , , , , , , , I , , , / ,

Y

p m/z 314

I d 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 72 23 24 25 26 27

RETENTION TIME (minutes)

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M Ugorski, B Nilsson, K Schroer, J A Cashel and D Zopfglycosphingolipids of murine B cell hybridomas.

Gas chromatography/mass spectrometry analysis of oligosaccharides from neutral

1984, 259:481-486.J. Biol. Chem. 

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