Characterization of glycosphingolipids from gastrointestinal stromal tumours

Gastrointestinal stromal tumours (GISTs) are the major nonepithelial neoplasms of the human gastrointestinal tract with a worldwide incidence between 11 and 15 per million cases annually. In this study the acid and non-acid glycosphingolipids of three GISTs were characterized using a combination of thin-layer chromatography, chemical staining, binding of carbohydrate recognizing ligands, and mass spectrometry. In the non-acid glycosphingolipid fractions of the tumors globotetraosylceramide, neolactotetraosylceramide, and glycosphingolipids with terminal blood group A, B, H, Lex, Lea, Ley and Leb determinants were found. The relative amounts of these non-acid compounds were different in the three tumour samples. The acid glycosphingolipid fractions had sulfatide, and the gangliosides GM3, GD3, GM1, Neu5Acα3neolactotetraosylceramide, GD1a, GT1b and GQ1b. In summary, we have characterized the glycosphingolipids of GISTs and found that the pattern differs in tumours from different individuals. This detailed characterization of glycosphingolipid composition of GISTs could contribute to recognition of new molecular targets for GIST treatment and sub-classification.

Characterization of total acid GIST glycosphingolipids. LC-ESI/MS. The native total acid glycosphingolipids fractions from GIST I and II were analyzed by LC-ESI/MS (exemplified in Fig. 3). In both cases the major molecular ions were from the GM3 ganglioside with d18:1-16:0 ceramide (m/z 1151) and d18:1-24:1 ceramide (m/z 1261). In the case of GIST II there were also molecular ions of the GD3 ganglioside with d18:1-24:1 ceramide (m/z 776) and the GD1a ganglioside with d18:1-24:1 ceramide (m/z 959). A minor ion at m/z 794 was found by a search for molecular ions of sulfatide, and MS 2 of this ion gave a characteristic sulfatide spectrum (Supplemental Figure S1) 15 .
Chromatogram binding assays. The binding of a number of carbohydrate binding ligands to the total acid glycosphingolipids fractions from GIST I and II were next examined (Fig. 1). Here the monoclonal antibodies directed against the Neu5Acα3Galβ4GlcNAc sequence recognized several slow-migrating compounds in the acid fractions from the two GISTs (Fig. 1C, lanes 4 and 5), indicating the presence of complex gangliosides with neolacto (Galβ4GlcNAc) core chains. Presence of the GM1 ganglioside was indicated by the binding of cholera toxin B-subunits (Fig. 1D, lanes 4 and 5). The monoclonal antibodies directed against the GD1a ganglioside also bound to the acid fractions from the two GIST tumours (Fig. 1E, lanes 4 and 5), whereas there was no binding of anti-GD3 antibodies or antibodies directed against the Neu5Acα6Galβ4GlcNAc sequence (not shown).

Separation of the acid glycosphingolipids of GIST II. Since very little information was obtained from
these LC-ESI/MS analyses the total acid glycosphingolipid fraction of GIST II was further separated on an Iatrobeads column eluted with increasing volumes of methanol in chloroform. This gave one fraction containing slow-migrating compounds (18 mg), and this fraction was further separated into subfractions that were pooled according to their mobility on thin-layer chromatograms. This gave five subfractions which were denoted fractions A-1 to A-5 (Fig. 4A, lanes 2-6).  www.nature.com/scientificreports/ The base peak chromatogram obtained from fraction A-3 was weak and only had one [M − 2H + ] 2− ion at m/z 777 corresponding to the GD3 ganglioside and one [M − 2H + ] 2− ion at m/z 960 corresponding to the GD1a ganglioside, both with d18:1-24:0 ceramide (data not shown).

LC-ESI/MS of fractions A1-A5. LC-ESI/MS of fraction
[M − 2H + ] 2− ions at m/z 777 and at m/z 960 corresponding to the GD3 and GD1a gangliosides were also found by LC-ESI/MS of fraction A-4 (Fig. 4D). Here the major compound gave a [M − 2H + ] 2− ion at m/z 1105.5. MS 2 of m/z 1105.5 demonstrated a ganglioside with three Neu5Ac, three Hex and one HexNAc and d18:1-24:0 ceramide (Fig. 5A). The ion at m/z 581 demonstrated a Neu5Ac-Neu5Ac sequence, while the ions at m/z 1920,  www.nature.com/scientificreports/ m/z 1629 and m/z 1338 were due to loss of one, two and three Neu5Ac from the molecular ion. MS 3 of m/z 960 also gave ions at m/z 1629 and m/z 1338 caused by loss of one and two Neu5Ac, and also an ion at m/z 972 due to loss of a Hex and a HexNAc (Fig. 5B). Taken together this demonstrated a GT1b ganglioside with d18:1-24:0 ceramide (Fig. 5C). In the same manner the GT1b ganglioside with d18: www.nature.com/scientificreports/ The gangliosides characterized in in the acid glycosphingolipid fraction of GIST II are summarized in Table 2.

Characterization of total non-acid GIST glycosphingolipids. LC-ESI/MS of native glycosphingolip-
ids. To get an overview of the ceramide composition the native non-acid glycosphingolipid fractions of GIST I and GIST II were first analyzed by LC-ESI/MS using a polyamine column (exemplified in Fig. 7). Thereby dihexosylceramide, trihexosylceramide, and a tetraosylceramide with one HexNAc and three Hex, all with mainly sphingosine and non-hydroxy fatty acids with 16 and 24 carbon atoms were identified.

LC-ESI/MS of glycosphingolipid-derived oligosaccharides.
Thereafter the non-acid glycosphingolipid fractions were hydrolyzed with endoglycoceramidase II from Rhodococcus sp., and the oligosaccharides thereby obtained were characterized by LC-ESI/MS using a graphitized carbon column. By LC-ESI/MS of oligosaccharides on graphitized carbon columns a resolution of isomeric oligosaccharides is obtained, and by MS 2 a series of C-type ions is obtained which gives the carbohydrate sequence 16 . Furthermore, the MS 2 spectra of oligosaccharides with a Hex or HexNAc substituted at C-4 have diagnostic cross-ring 0,2 A-type fragment ions which allow identification of linkage positions 16,17 . Thus, MS 2 spectra of oligosaccharides with globo (Galα4Gal) or type 2 (Galβ4GlcNAc) core structures have such 0,2 A-type fragment ions, but not the spectra obtained from oligosaccharides with isoglobo (Galα3Gal) or type 1 (Galβ3GlcNAc) core chains.    Fig. 8 and Supplementary Fig. S2). A prominent 0,2 A 2 fragment ion at m/z 281 demonstrating a terminal Hex-HexNAc sequence with a 4-substituted HexNAc, i.e. a type 2 core chain (Galβ4GlcNAc) was present in the MS 2 spectrum of the ion at m/z 706 (Fig. 8B). There was also a C 2 ion at m/z 382 and a C 3 ion at m/z 544, and taken together this demonstrated a neolacto tetrasaccharide (Galβ4GlcNAcβ3Galβ4Glc).
The MS 2 spectrum of the ion at m/z 852 at retention time 19.5 min (Fig. 8C) had a fragment ion at m/z 364. This type of fragment ion is caused by a double glycosidic cleavage of the 3-linked branch at C 3 and Z 3 β, and is diagnostic for an internal 4-linked GlcNAc with a Fuc at 3-position 17 . The MS 2 spectrum also had a C 2 ion at m/z 528 and a C 3 ion at m/z 690. Taken together these spectral features identified a Le x pentasaccharide (Galβ4(Fucα3)GlcNAcβ3Galβ4Glc). MS 2 of the molecular ion at m/z 852 eluting at 26.0 min (Fig. 8D) identified a Fuc-Hex-HexNAc-Hex-Hex pentasaccharide. This was conluded from the series of C-type fragment ions (C 2 at m/z 325, C 3 at m/z 528, and C 4 at m/z 690. 4-substitution of the HexNAc, i.e. a type 2 core chain (Galβ4GlcNAc) was indicated by the 0,2 A 3 -H 2 O fragment ion at m/z 409 and the 0,2 A 3 fragment ion at m/z 427. Taken together this identified a H type 2 pentasaccharide (Fucα2Galβ4GlcNAcβ3Galβ4Glc).
A pentasaccharide with HexNAc-Hex-HexNAc-Hex-Hex sequence was identified by the C-type fragment ion series (C 2 at m/z 382, C 3 at m/z 585, and C 4 at m/z 747) obtained by MS 2 of the molecular ion at m/z 909 (Fig. 8E). Here the 0,2 A 3 fragment ion at m/z 484, and the 0,2 A 3 -H 2 O ion at m/z 466, demonstrated a 4-substituted internal HexNAc. These features of the MS 2 spectrum allowed identification of an x 2 pentasaccharide (GalNAc β3Galβ4GlcNAcβ3Galβ4Glc).
The MS 2 spectrum obtained from the ion at m/z 998 (Fig. 8F) had a prominent fragment ion at m/z 348. This type of ion is diagnostic for an internal 3-linked GlcNAc substituted with a Fuc at C-4 17 , and is a double glycosidic cleavage of the 3-linked branch at C 3 and Z 3 α. C-type fragment ions were present at m/z 674 (C 3 ) and m/z 836 (C 4 ), and taken together this indicated a Le b hexasaccharide (Fucα2Galβ3(Fucα4)GlcNAcβ3Galβ4Glc).
The series of C-type fragment ions (C 2 at m/z 528, C 3 at m/z 731, and C 4 at m/z 893) from MS 2 of the ion at m/z 1055 (Fig. 8G) demonstrated a HexNAc-(Fuc)Hex-HexNAc-Hex-Hex sequence. A type 2 core chain (Galβ4GlcNAc), i.e. substitution of the internal HexNAc at C4, was demonstrated by the 0,2 A 3 fragment ion at m/z 630, and the 0,2 A 4 -H 2 O fragment ion at m/z 612. Thus, a blood group A type 2 hexasaccharide (GalNAcα3(Fucα2) Galβ4GlcNAcβ3Galβ4Glc) was identified.
A neolacto hexasaccharide (Galβ4GlcNAcβ3Galβ4GlcNAcβ3Galβ4Glc) was characterized by MS 2 of the ion at m/z 1071 (Fig. 8H). This was deduced from the C-type fragment ion series (C 2 at m/z 382, C 3 at m/z 544, C 4 at m/z 747, and C 5 at m/z 909), demonstrating a Hex-HexNAc-Hex-HexNAc-Hex-Hex carbohydrate sequence, along with the 0,2 A 4 fragment ion at m/z 646, which demonstrated 4-substitution of the innermost HexNAc. . Molecular ion profile from LC-ESI/MS of the total non-acid glycosphingolipid fraction from GIST II. The identification of glycosphingolipids was based on their retention times, determined molecular masses and subsequent MS 2 sequencing. The data were processed using the Xcalibur software (version 2.0.7, Thermo Scientific, www.therm ofish er.com). In the shorthand nomenclature for fatty acids and bases, the number before the colon refers to the carbon chain length and the number after the colon gives the total number of double bonds in the molecule. Fatty acids with a 2-hydroxy group are denoted by the prefix h before the abbreviation e.g. h16:0. For long chain bases, S designates sphingosine (d18:1) and P phytosphingosine (t18:0). *marks a nonganglioside contaminant. www.nature.com/scientificreports/ MS 2 of the molecular ion at m/z 1201 (Fig. 8I) gave a spectrum that was very similar to the MS 2 spectrum obtained from reference blood group type 1/ALe b heptasaccharide 18 . The series of C-type fragment ions (C 2 at m/z 528, C 3 at m/z 877, and C 4 at m/z 1039) demonstrated a HexNAc-(Fuc-)Hex-(Fuc-)HexNAc-Hex-Hex sequence. The spectrum also had an ion at m/z 656, caused by a double glycosidic cleavage of the 3-linked branch at C 3 and Z 4 α 17 , and thus signifying an internal 3-linked GlcNAc substituted with a Fuc at C-4. Thus, this demonstrated a blood group A type 1/ALe b heptasaccharide (GalNAcα3(Fucα2)Galβ3(Fucα4)GlcNAcβ3Galβ4Glc).
The series of B-type and C-type fragment ions (C 2 at m/z 528, B 4 at m/z 875, B 5 at m/z 1078, and C 6 at m/z 1258) obtained by MS 2 of the ion at m/z 1420 (Fig. 8J) indicated a HexNAc-(Fuc)Hex-HexNAc-Hex-HexNAc-Hex-Hex sequence. The 0,2 A 3 fragment ion at m/z 630, and the 0,2 A 5 fragment ion at m/z 995, demonstrated that the two internal HexNAcs were substituted at C4. Taken together this indicated a blood group A type 2 octasaccharide (GalNAcα3(Fucα2)Galβ4GlcNAcβ3 Galβ4GlcNAcβ3Galβ4Glc).
Finally, the MS 2 spectrum of the ion at m/z 1436 (Fig. 8K) gave a tentative identification of a neolacto octasaccharide (Galβ4GlcNAcβ3Galβ4GlcNAcβ3Galβ4GlcNAcβ3Galβ4Glc). Here the lower mass region of the spectrum was weak, but had similarities with the spectrum in Fig. 8H. Thus, the C 4 ion at m/z 747, the C 5 ion at m/z 909 and the C 6 ion at m/z 1112, allowed a tentative identification of an octasaccharide with Hex-HexNAc-Hex-HexNAc-Hex-HexNAc-Hex-Hex sequence, and 4-substitution of the two innermost HexNAcs was demonstrated by with the 0,2 A 4 fragment ion at m/z 646 and the 0,2 A 6 fragment ion at m/z 1011.
Thus, the oligosaccharide derived from the non-acid glycosphingolipids of GIST I had mainly type 2 core chains, with neolactotetraose (Galβ4GlcNAcβ3Galβ4Glc) as the predominant compound. There were also several other minor compounds with neolacto core chain as the Le x and x 2 pentasaccharides, the blood group A type 2 hexa-and octasaccharides, and neolacto hexa-and octasaccharides. Minor oligosaccharides with type 1 core chains (Le b hexasaccharide, and blood A type 1 heptasaccharide) were also present.
The base peak chromatogram from LC-ESI/MS of the oligosaccharides obtained from GIST II also had molecular ions ranging from m/z 706 to m/z 1201 ( Supplementary Fig. S3A). Here MS 2 showed that the GIST II oligosaccharides had a globo tetrasaccharide in addition to the neolacto tetrasaccharide, a Le a pentasaccharide in addition to the Le x hexasaccharide, a Le y hexasaccharide in addition to the Le b hexasaccharide (exemplified in Supplementary Fig. S3B-G), and a blood group B heptasaccharide in addtion to the blood group A heptasaccharide (see further below). The x 2 and blood group H type 2 pentasaccharides, the blood group A type 2 hexasaccharide, and the neolacto and blood group A type 2 octasaccharides were not detected in the GIST II sample.
A globo trisaccharide, globo and neolacto tetrasaccharides, Le x , x 2 and blood group H type 2 pentasaccharides, and Le b and Le y hexasaccharides were characterized by LC-ESI/MS of the oligosaccharides derived from GIST III (data not shown). The glycosphingolipid-derived oligosaccharides from GIST I, GIST II and GIST III characterized by LC-ESI/MS are summarized in Table 3.
Chromatogram binding assays. In order to extend the structural information obtained by mass spectrometry the binding of carbohydrate binding ligands to the non-acid glycosphingolipid fractions of GISTI and GIS-TII was next examined in a chromatogram binding assay. Thereby, a distinct interaction of the Galβ4GlcNAc binding lectin from E. cristagalli 19 was obtained, confirming the presence of neolactotetraosylceramide in both tumors (Fig. 2B, lanes 3 and 4). The lectin binding to the glycosphingolipids of GIST I (lane 3) was more distinct than the binding to the glycosphingolipids of GIST II (lane 4), in line with the higher amount of neolactotetraosylceramide in GIST I. The presence of glycosphingolipids with blood group A determinants in the GISTs was confirmed by the binding of anti-A antibodies (Fig. 2C, lanes 3 and 4).

Separation of the non-acid glycosphingolipids of GIST II.
After these studies the remaining material in the non-acid glycosphingolipid fraction from GIST II was separated on Iatrobeads columns, in order to enrich the slow-migrating glycosphingolipids. After pooling three glycosphingolipid-containing fractions were obtained (denoted fractions N-I to N-III).  (Fig. 9D,F). There was a C-type fragment ion series (C 2 at m/z 528, C 3 at m/z 877, and C 4 at m/z 1039) identifying a HexNAc-(Fuc-)Hex-(Fuc-) HexNAc-Hex-Hex sequence (Fig. 9E), and a C 3 /Z 4 α ion at m/z 656, identifying an internal 3-linked GlcNAc substituted with a Fuc at C-4 17 was also obtained. This MS 2 spectrum was very similar to the MS 2 spectrum of reference a blood group A type 1/ALe b heptasaccharide 18 .
Thus, fraction N-III from GIST II was a complex mixture of blood group A and B heptaosylceramides with both type 1 and type 2 core chains, and also Le b and Le y hexaosylceramides.

Discussion
GISTs are the most common primary mesenchymal tumour of the human alimentary tract, counting for less than 1% of all gastrointestinal tumours and about 5% all sarcomas. GISTs originate from the interstitial cells of Cajal in the muscularis propria of the gastrointestinal tract. These cells are specialized smooth muscle cells, which receive inputs from nerve cells within the myenteric and submucosal plexuses, and also communicate electrically with smooth muscle cells in the longitudinal and circular muscle layers 20,21 , and thereby contribute to peristalsis which facilitates propulsion of intestinal contents, and small intestine segmentation which facilitates absorption of nutrients 22 .
The interstitial cells of Cajal and the intestinal circular smooth muscle cells both originate from a common, mesodermally derived mesenchymal pluripotential precursor 22,23 . These mesenchymal progenitors express both the receptor tyrosine kinase c-Kit and smooth muscle myosin heavy chain. Cells that will differentiate into interstitial cells of Cajal maintain the expression of c-Kit, while the cells destined to become smooth muscle cells up-regulate the synthesis of myofilament proteins and down-regulate Kit 24,25 .
It is obvious that the glycosphingolipids of the Cajal cells can not be isolated for structural characterization, and studies of glycosphingolipids of human smooth mucle are rare. However, by binding studies using a battery of monoclonal antibodies, Gillard et al. 26 found that the major non-acid glycosphingolipids of cultured smooth muscle cells, derived from human umbilical cord veins, were lactosylceramide, globotri-and globotetraosylceramide. There were also small amounts of neolactotetraosylceramide, Le x pentaosylceramide and globopentaosylceramide/SSEA-3 (Galβ3GalNAcβ3Galα4Galβ4Glcβ1Cer). GM3 and Neu5Acα3-neolactotetraosylceramide were the major gangliosides, whereas no GD3 ganglioside was detected. Most of the long chain gangliosides had a neolacto core (as e.g. Neu5Acα3-dimeric-Le x nonaosylceramide (Neu5Acα3Galβ4(Fucα4)GlcNAcβ3Galβ4(Fucα4) GlcNAcβ3Galβ4Glcβ1Cer)), or globo core (as e.g. Neu5Acα3-globopentaosylceramide/SSEA-4 (Neu5Acα3Galβ 3GalNAcβ3Galα4Galβ4Glcβ1Cer)). There was no binding of a monoclonal anti-H type 2 antibody to the smooth muscle cell glycosphingolipids, and binding anti-A or anti-B antibodies was not tested.
In this study, the non-acid and acid glycosphingolipids of three GIST tumours were characterised with the combination of thin-layer chromatography, binding of carbohydrate recognizing ligands and mass spectrometry. The acid fractions of GIST had sulfatide, and GM3 was the major ganglioside. In contrast to the previous study of smoth muscle cell glycosphingolipids, GD3 and the several gangliosides with ganglio core chain (GM1, GD1a, GT1b and GQ1b) were characterized. In the non-acid fractions of the tumours globotetraosylceramide, neolactotetraosylceramide, the x 2 pentaosylceramide, and glycosphingolipids with terminal blood group A, B, H, Le x , Le a , Le b and Le y determinants were characterized. The expression of blood group A, B, and H glycosphingolipids was in agreement with the ABO phenotype of the patients, and the relative amounts of the glycosphingolipids with blood group determinants were different in the three tumour samples. Most likely this is a reflection of the blood group determinants expressed by the cells of origin, i.e. the interstitial cells of Cajal.  Figure S2 for interpretation formulas. The data were processed using the Xcalibur software (version 2.0.7, Thermo Scientific, www.therm ofish er.com). The oligosaccharides identified in the chromatograms were: Le b -6, Fucα2Galβ3(Fucα4)GlcNAcβ3Galβ4Glc; A6-2, GalNAcα3(Fucα2)Galβ4GlcNAcβ3Galβ4Glc; Le x -5, Galβ4(Fucα3)GlcNAcβ3Galβ4Glc; A7-1, GalNAcα3(Fucα2)Galβ3(Fucα4)GlcNAcβ3Galβ4Glc; nLc4, Galβ4GlcNAcβ3Galβ4Glc; A8-2, GalNAcα3(Fucα2)Galβ4GlcNAcβ3Galβ4GlcNAcβ3Galβ4Glc H5-2, Fucα2Galβ4GlcNAcβ3Galβ4Glc; x 2 , GalNAcβ3Galβ4GlcNAcβ3Galβ4Glc; nLc6,Galβ4GlcNAcβ3Galβ4GlcNAc β3Galβ4Glc; nLc8, Galβ4GlcNAcβ3Galβ4GlcNAcβ3Galβ4GlcNAcβ3Galβ4Glc. *marks a non-oligosaccharide contaminant. www.nature.com/scientificreports/ Overall the glycosphingolipid patterns of the three gastric GISTs had a high similarity with the glycosphingolipids of the human stomach 18,27 . The non-acid glycosphingolipids of human stomach had a high degree of structural complexity, and the composition of glycosphingolipids differed for individuals of separate blood groups. The major complex glycosphingolipids of blood group O(Rh-)P stomach were neolactotetraosylceramide, the Le x , Le a , and H type 2 pentaosylceramides, and the Le y hexaosylceramide. The A(Rh +)P, and A(Rh +) p stomachs had lactotetraosylceramide, the x 2 and the H type 1 pentaosylceramides, neolactohexaosylceramide the Le b hexaosylceramide and the A type 1/ALe b heptaosylceramide 18 . The acid human stomach glycosphingolipids characterized were sulfatide and the gangliosides GM3, GD3, GM1, Neu5Acα3-neolactotetraosylceramide, Neu5Acα3-neolactohexaosylceramide and Neu5Acα3-neolactooctaosylceramide, GD1a and GD1b 27 . The major difference observed was the relatively low levels of sulfatide in the GIST tumours compared to the human stomach 27 .
Several gangliosides are overexpressed in cancers, and a substantial number of cancer immunotherapies targeting such gangliosides have been developed 28,29 . Already in 1985, a phase I clinical trial with an anti-GD3 antibody demonstrated regression of tumours, activation of T-cells, antibody-dependant-cell-cytotoxicity, and complement dependent cytotoxicity in malignant melanoma patients 30 . More recently an anti-GD2 monoclonal antibody Dinutuximab (Unituxin) has been approved by the Food Drug Administration (FDA) and European Medicines Agency (EMA) for the treatment of high-risk neuroblastomas 31 . Dinutuximab is used in combination with interleukin-2, granulocyte-macrophage colony-stimulating factor, and isotretinoin (13-cis-retinoic acid) for maintenance treatment of pediatric patients with high-risk neuroblastoma who has achieved at least a partial response to first-line multiagent, multimodality therapy 32 . Dinutuximab gave an increased the 2-year event-free survival and overall survival when compared to standard treatment in phase III trials 33 .
This study is the first structural characterization of GIST glycosphingolipids, and demonstrates the presence of glycosphingolipids with blood group ABO and Lewis determinants, along with several complex gangliosides, in these tumors derived from the interstitial cells of Cajal, which are specialized smooth muscle cells. Further studies will be needed to clarify the potential value of these findings for tumour biology, response to therapy and patient survival.

Methods
Glycosphingolipids preparations. The study was conducted according to the tenets of the Declaration of Helsinki, and was approved by the Regional Ethics Committee of Gothenburg (No. 621-18 (decision 2018-09-19)). All patients provided written informed consents before enrollment in the study.
Three gastric GIST tumours (denoted GIST I, GIST II and GIST III) were collected at the Sahlgrenska University Hospital. The tumours were kept at − 70 °C. The isolation of total acid and total non-acid glycosphingolipids was done by the method described by Karlsson 14 . The tumours were lyophilized, followed by Soxhlet extraction with mixtures of chloroform and methanol (2:1 and 1:9, by volume). The resulting material was pooled and subjected to mild alkaline hydrolysis followed by dialysis. Thereafter non-polar compounds were removed by chromatography on a silicic acid column. Acid and non-acid glycosphingolipids were separated by ion change chromatography on a DEAE-cellulose column. In order to separate the non-acid glycosphingolipids from alkalistable phospholipids, the non-acid fractions were then acetylated and separated on a second silicic acid column, followed by deacetylation and dialysis. Final purifications are performed by chromatography on DEAE-cellulose and silicic acid columns.
After the first characterization by binding assays and LC-ESI/MS, the acid glycosphingolipids from GIST II were separated on an Iatrobeads column eluted with increasing volumes of methanol in chloroform. The Table 3. Glycosphingolipid-derived oligosaccharides from the total non-acid GIST fractions identified by LC-ESI/MS. www.nature.com/scientificreports/ fractions obtained were analyzed by thin-layer chromatography and anisaldehyde/resorcinol detection, and thereafter pooled according to their mobility on thin-layer chromatograms, resulting in five subfractions, which were denoted fractions A-1 to A-5. The non-acid glycosphingolipids from GIST II were separated on an Iatrobeads column eluted with eluted with chloroform:methanol:water (60:35:8, by volume), 29 × 1 ml. The fractions were analysed by thin-layer chromatography, and after pooling according to their mobility on thin-layer chromatograms, three glycosphingolipidcontaining fractions were obtained (denoted fractions N-I to N-III).
Reference glycosphingolipids. Total acid and non-acid glycosphingolipid fractions were isolated as described 14 . Individual glycosphingolipids were isolated by repeated chromatography on silicic acid columns and by HPLC, and identified by mass spectrometry 16,34 and 1 H-NMR spectroscopy 35 . Chromatogram binding assays. The carbohydrate binding ligands and dilutions used in the chromatogram binding assays are given in Table 4. Binding of antibodies to glycosphingolipids separated on thin-layer chromatograms was performed as described by Barone et al. 38 . After elution, the dried thin-layer chromatograms plates were treated with a mixture of 0.5% polyisobutylmethacrylate (w/v) in diethylether/n-hexane (1:5 v/v) for 1 min, and then air-dried. Thereafter followed a 2 h incubation at room temperature with PBS (phosphate-buffered saline, pH 7.3) containing 2% (w/v) bovine serum albumin, 0.1% (w/v) NaN 3 and 0.1% (w/v) Tween 20 (BSA/PBS/TWEEN) to reduce unspecific binding. Then, the chromatograms were incubated for 2 h at room temperature with suspensions of monoclonal antibodies diluted in BSA/PBS/TWEEN. The chromatograms were washed with PBS, and the chromatograms were thereafter incubated for 2 h with 125 I-labeled (labeled by the IODO-GEN method according to the manufacturer's instructions (Pierce/Thermo Scientific)) rabbit antimouse antibodies (Pierce/Thermo Scientific) diluted in BSA/PBS/TWEEN. Finally, the chromatograms were washed with PBS, dried and autoradiographed overnight using x-ray films (Carestream; 8,941,114). Binding of 125 I-labeled Erythrina cristagalli lectin (Sigma-Aldrich), and cholera toxin B-subunits (List Laboratories), to glycosphingolipids on thin-layer chromatograms was done as described 19,39 . LC-ESI/MS of native glycosphingolipids. The native glycosphingolipid fractions were analyzed by LC-ESI/MS as described 18,40 . Aliquots of the glycosphingolipid fractions were dissolved in methanol:acetonitrile 75:25 (by volume) and separated on a 200 × 0.250 mm column, packed in-house with 5 µm polyamine II particles (YMC Europe GmbH, Dinslaken, Germany). An autosampler, HTC-PAL (CTC Analytics AG, Zwingen, Switzerland) equipped with a cheminert valve (0.25 mm bore) and a 2 µl loop, was used for sample injection. An Agilent 1100 binary pump (Agilent Technologies, Palo Alto, CA) delivered a flow of 250 µl/min, which was splitted down in an 1/16″ microvolume-T (0.15 mm bore) (Vici AG International, Schenkon, Switzerland) by a 50 cm × 50 µm i.d. fused silica capillary before the injector of the autosampler, allowing approximately 2-3 µl/ min through the column. Samples were eluted with an aqueous gradient (A:100% acetonitrile to B: 10 mM ammonium bicarbonate). The gradient (0-50% B) was eluted for 40 min, followed by a wash step with 100% B, and equilibration of the column for 20 min. The samples were analyzed in negative ion mode on a LTQ linear quadrupole ion trap mass spectrometer (Thermo Electron, San José, CA), with an IonMax standard ESI source equipped with a stainless steel needle kept at − 3.5 kV. Compressed air was used as nebulizer gas. The heated capillary was kept at 270 °C, and the capillary voltage was − 50 kV. Full scan (m/z 500-1800, two microscans, maximum 100 ms, target value of 30,000) was performed, followed by data-dependent MS 2 scans (two microscans, maximun 100 ms, target value of 10.000) with normalized collision energy of 35%, isolation window of 2.5 units, activation q = 0.25 and activation time 30 ms). The threshold for MS 2 was set to 500 counts.
Data acquisition and processing were conducted with Xcalibur software version 2.0.7 (Thermo Scientific, Waltham, MA). Manual assignment of glycosphingolipid sequences was done with the assistance of the Glycoworkbench tool (Version 2.1), and by comparison of retention times and MS 2 spectra of reference glycosphingolipids.
Endoglycoceramidase digestion and LC-ESI/MS. The non-acid glycosphingolipids were digested with endoglycoceraminidase and the oligosaccharides thereby obtained were analyzed by LC-ESI/MS 16,18,40 . The glycosphingolipids (50 µg) were resuspended in 100 µl 0.05 M sodium acetate buffer, pH 5.0, containing 120 µg sodium cholate, and sonicated briefly. Thereafter, 1 mU of endoglycoceramidase II from Rhodococcus spp. (Takara Bio Europe S.A., Gennevilliers, France) was added, and the mixture was incubated at 37 °C for 48 h. The reaction was stopped by addition of chloroform/methanol/water to the final proportions 8:4:3 (by volume). The oligosaccharide-containing upper phase thus obtained was separated from detergent on a Sep-Pak QMA cartridge (Waters). The eluant containing the oligosaccharides was dried under nitrogen and under vacuum.
The glycosphingolipid-derived oligosaccharides were resuspended in 50 µl water and analyzed by LC-ESI/ MS as described 16,18,40 . The oligosaccharides were separated on a column (100 × 0.250 mm) packed in-house with 5 µm porous graphite particles (Hypercarb, Thermo-Hypersil, Runcorn, UK). An autosampler, HTC-PAL www.nature.com/scientificreports/ (CTC Analytics AG, Zwingen, Switzerland) equipped with a cheminert valve (0.25 mm bore) and a 2 µl loop, was used for sample injection. An Agilent 1100 binary pump (Agilent technologies, Palo Alto, CA) delivered a flow of 250 µl/min, which was split down in an 1/16″ microvolume-T (0.15 mm bore) (Vici AG International, Schenkon, Switzerland) by a 50 cm × 50 µm i.d. fused silica capillary before the injector of the autosampler, allowing approximately 3-5 µl/min through the column. The oligosaccharides (3 µl) were injected on to the column and eluted with an acetonitrile gradient (A: 10 mM ammonium bicarbonate; B: 10 mM ammonium bicarbonate in 80% acetonitrile). The gradient (0-45% B) was eluted for 46 min, followed by a wash step with 100% B, and equilibration of the column for 24 min. A 30 cm × 50 µm i.d. fused silica capillary was used as transfer line to the ion source. The oligosaccharides were analyzed in negative ion mode on an LTQ linear quadrupole ion trap mass spectrometer (Thermo Electron, San José, CA). The IonMax standard ESI source on the LTQ mass spectrometer was equipped with a stainless steel needle kept at − 3.5 kV. Compressed air was used as nebulizer gas. The heated capillary was kept at 270 °C, and the capillary voltage was − 50 kV. Full scan (m/z 380-2 000, 2 microscans, maximum 100 ms, target value of 30 000) was performed, followed by data dependent MS 2 scans of the three most abundant ions in each scan (2 microscans, maximum 100 ms, target value of 10 000). The threshold for MS 2 was set to 500 counts. Normalized collision energy was 35%, and an isolation window of 3 u, an activation q = 0.25, and an activation time of 30 ms, was used. Selected fractions were also analyzed at m/z 1300-2000. Data acquisition and processing were conducted with version 2.0.7 (Thermo Scientific, Waltham, MA).
Manual assignment of glycan sequences was done on the basis of knowledge of mammalian biosynthetic pathways, with the assistance of the Glycoworkbench tool (Version 2.1), and by comparison of retention times and MS 2 spectra of oligosaccharides from reference glycosphingolipids 16 .