Direct Analysis of Silica Gel Extracts from Immunostained

Anal. Chem. 2004, 76, 2248-2255

Direct Analysis of Silica Gel Extracts from Immunostained Glycosphingolipids by Nanoelectrospray Ionization Quadrupole Time-of-Flight Mass Spectrometry Iris Meisen, Jasna Peter-Katalinic´, and Johannes Mu 1 thing*

Institute for Medical Physics and Biophysics, University of Mu¨nster, Robert-Koch-Strasse 31, D-48149 Mu¨nster, Germany

A combined strategy of preparative high-performance thinlayer chromatography overlay assay and mass spectrometry was established for the structural characterization of immunostained glycosphingolipids (GSLs) in silica gel extracts. Crude chloroform/methanol/water (30/60/8, v/v/v) extracts of immunostained TLC bands were analyzed by nanoelectrospray low-energy CID mass spectrometry without further purification. The GSL species investigated were isomeric monosialogangliosides of the neolacto series from a ganglioside preparation of human granulocytes, the disialoganglioside GD3 from a human melanoma lipid extract, and ganglio series Gg3Cer of a neutral GSL preparation from murine lymphoreticular MDAY-D2 cells. For the specific detection of lipid-bound oligosaccharides, polyclonal chicken IgY, murine monoclonal IgG3, and IgM antibodies were used. The resulting mass spectra show that only analytical quantities of ∼1 µg of a single GSL within a complex mixture are required for the structure determination of immunostained GSLs by MS and MS/MS. All species investigated were detected as singly charged deprotonated molecular ions, and neither buffer-derived salt adducts nor coextracted contaminants from the immunostaining procedure or the silica gel layer were observed. This effective HPTLC-MSjoined procedure offers a wide range of applications for any carbohydrate binding agents such as bacterial toxins, plant lectins, and others. Glycosphingolipids (GSLs) consist of a hydrophilic oligosaccharide chain and a hydrophobic component named ceramide.1 The long-chain aminodiol sphingosine (4-sphingenine) serves as the lipid backbone in most vertebrate GSLs. Fatty acids of varying chain lengths are attached via amide bonds to the amino group of sphingosine, forming the GSL’s ceramide moiety. The oligosaccharide chains of GSLs are binding sites for carbohydraterecognizing lectins,2 viruses and bacteria,3 bacterial toxins,4 and * Corresponding author. Telephone: +49-251-8355192. Fax: +49-251-8355140. E-mail: [email protected]. (1) Mu ¨ thing, J. In Carbohydrate analysis by modern chromatography and electrophoresis; El Rassi, Z., Ed.; Elsevier Science: Amsterdam, The Netherlands, 2002; Vol. 66, pp 423-482. (2) Feizi, T. Immunol. Rev. 2000, 173, 79-88.

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antibodies.5 Gangliosides, the most complex group of GSLs, are characterized by the presence of one or more sialic acid moieties (N-acetylneuraminic acid and its derivatives) in the oligosaccharide chain. Human neutrophil neolacto-series gangliosides with Neu5AcR2-3Galβ1-4GlcNAc-residues are specifically recognized by the neutrophil-activating protein of Helicobacter pylori.6 The isomeric gangliosides with Neu5AcR2-6Galβ1-4GlcNAc-residues are found to be differently expressed by human B and T cells.7 Recently, they have been identified as receptors of the recombinant mistletoe lectin I8 being currently in several clinical-phase I-trials for the treatment of cancer.9 The aberrant or enhanced expression of certain GSLs is a ubiquitous feature common to numerous types of tumors. Examples are the overexpression of neolacto-series gangliosides in human hepatocellular tumors,10 ganglioside GD3 in human melanoma cells,11 and gangliotriaosylceramide (Gg3Cer) in cell lines from patients with Hodgkin’s disease.12 High-performance thin-layer chromatography (HPTLC) is routinely used for the separation and partial characterization of GSLs in mixtures.13,14 The analytes can be detected either by the nondestructive agent primuline15 or by other lipophilic fluoro(3) Mu ¨ thing, J. In Glycoscience: Chemistry and Chemical Biology, Vol 3: Glycolipids; Fraser-Reid, B., Tatsuta, K., Thiem, J., Eds.; Springer: Heidelberg, Germany, 2001; pp 2183-2252. (4) Lingwood, C. A.; Boyd, B.; Nutikka, A. Methods Enzymol. 2000, 312, 459473. (5) Kannagi, R. Methods Enzymol. 2000, 312, 160-179. (6) Teneberg, S.; Miller-Podraza, H.; Lampert, H. C.; Evans, D. J., Jr.; Evans, D. G.; Danielsson, D.; Karlsson, K. A. J. Biol. Chem. 1997, 272, 1906719071. (7) Kniep, B.; Scha¨kel, K.; Nimtz, M.; Schwartz-Albiez, R.; Schmitz, M.; Northoff, H.; Vilella, R.; Gramatzki, M.; Rieber, E. P. Glycobiology 1999, 9, 399406. (8) Mu ¨ thing, J.; Burg, M.; Mo¨ckel, B.; Langer, M.; Metelmann-Strupat, W.; Werner, A.; Neumann, U.; Peter-Katalinic´, J.; Eck, J. Glycobiology 2002, 12, 485-497. (9) Habeck, M. Drug Discovery Today 2003, 8, 52-53. (10) Taki, T.; Yamamoto, K.; Takamatsu, M.; Ishii, K.; Myoga, A.; Sekiguchi, K.; Ikeda, I.; Kurata, K.; Nakayama, J.; Handa, S.; Matsumoto, M. Cancer Res. 1990, 50, 1284-1290. (11) Pukel, C. S.; Lloyd, K. O.; Travassos, L. R.; Dippold, W. G.; Oettgen, H. F.; Old, L. J. J. Exp. Med. 1982, 155, 1133-1147. (12) Kniep, B.; Monner, D. A.; Burrichter, H.; Diehl, V.; Mu¨hlradt, P. F. J. Immunol. 1983, 131, 1591-1594. (13) van Echten-Deckert, G. Methods Enzymol. 2000, 312, 64-79. (14) Yu, R. K.; Ariga, T. Methods Enzymol. 2000, 312, 115-134. (15) Skipsi, V. P. Methods Enzymol. 1975, 35, 396-425. 10.1021/ac035511t CCC: $27.50

© 2004 American Chemical Society Published on Web 03/19/2004

chromes.16,17 More detailed information can be obtained by solidphase binding assay (also named TLC overlay or TLC immunoassay) using specific lectins or antibodies.18 A further development of these techniques was the transfer of TLC-separated GSLs to nitrocellulose19 and poly(vinylidene difluoride) membranes,20 followed by GSL detection on the membrane with lectins for example for the screening of R2-3- and R2-6-linked sialic acids in mixtures of gangliosides.21 Mass spectrometry (MS) is routinely used for defining the primary structure of GSLs in mixtures.22-24 The general procedure for combining preparative HPTLC with mass spectrometric characterization is the staining of GSLs with primuline followed by extraction of the UV-visible bands from the TLC plate.25,26 Alternative techniques are the direct desorption of separated GSLs from thin-layer chromatograms by fast atom bombardment mass spectrometry,27 matrix-assisted secondary ion mass spectrometry (SI-MS),28 and direct matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and SI-MS of GSLs from TLC plates or from membranes after blotting transfer.29,30 Recently, the coupling of an overpressure TLC instrument to an electrospray ionization quadrupole time-of-flight (ESI-QTOF) mass spectrometer has been reported,31 using acidic glycolipids as examples for on-line TLC/ESI-MS. However, in most approaches described so far, MALDI-MS was used for structural characterization of TLCseparated and fluorochrome-stained GSLs due to the higher tolerance toward sample impurities and salts in comparison to ESI-MS. Up to now, only one mass spectrometric investigation of immunostained GSLs has been reported.29 In this study, TLCseparated GSLs were detected by a specific antibody, transferred to membranes, and analyzed by MALDI-TOF-MS. In conclusion, improved ESI-MS techniques for the structural characterization of TLC-separated GSL mixtures without preceding column chromatographic separation steps are still required. In this contribution, we present a combined strategy for the structural characterization of GSLs based on reaction with specific antibodies, GSL extraction from silica gel, and structural assignment of the immunostained GSLs in crude extracts by means of ESI-MS/ MS. (16) Mu ¨ thing, J.; Heitmann, D. Anal. Biochem. 1993, 208, 121-124. (17) Mu ¨ thing, J.; Kemminer, S. Anal. Biochem. 1996, 238, 195-202. (18) Mu ¨ thing, J. In Methods in Molecular Biology, Vol. 76: Glycoanalysis Protocols; Hounsell, E., Ed.; Humana Press Inc.: Totowa, NJ, 1998; pp 183-195. (19) Towbin, H.; Schoenenberger, C.; Ball, R.; Braun, D. G.; Rosenfelder, G. J. Immunol. Methods 1984, 72, 471-479. (20) Ishikawa, D.; Taki, T. Methods Enzymol. 2000, 312, 145-157. (21) Johansson, L.; Johansson, P.; Miller-Podraza, H. Anal. Biochem. 1999, 267, 239-241. (22) Peter-Katalinic´, J.; Egge, H. Methods Enzymol. 1990, 193, 713-732. (23) Peter-Katalinic´, J. Mass Spectrom. Rev. 1994, 13, 77-98. (24) Metelmann, W.; Mu ¨ thing, J.; Peter-Katalinic´, J. Rapid Commun. Mass Spectrom. 2000, 14, 543-550. (25) White, T.; Bursten, S.; Fedeighi, D.; Lewis, R. A.; Nudelman, E. Anal. Biochem. 1998, 258, 109-117. (26) Hildebrandt, H.; Jonas, U.; Ohashi, M.; Klaiber, I.; Rahmann, H. Comp. Biochem. Physiol. B 1999, 122, 83-88. (27) Karlsson, K. A.; Lanne, B.; Pimlott, W.; Teneberg, S. Carbohydr. Res. 1991, 221, 49-61. (28) Kushi, Y.; Rokukawa, C.; Handa, S. Anal. Biochem. 1988, 175, 167-176. (29) Guittard, J.; Hronowski, X. L.; Costello, C. E. Rapid Commun. Mass Spectrom. 1999, 13, 1838-1849. (30) Taki, T.; Ishikawa, D.; Handa, S.; Kasama, T. Anal. Biochem. 1995, 225, 24-27. (31) Chai, W.; Leteux, C.; Lawson, A. M.; Stoll, M. S. Anal. Chem. 2003, 75, 118-125.

MATERIALS AND METHODS Nomenclature. Abbreviations and corresponding structures of neutral GSLs and gangliosides used in this study follow: MHC or monohexosylceramide or Glcβ1-1Cer; Lc2 or lactosylceramide or Galβ1-4Glcβ1-1Cer; Gg3Cer or asialo GM2 or GalNAcβ14Galβ1-4Glcβ1-1Cer; Gg4Cer or Galβ1-3GalNAcβ1-4Galβ14Glcβ1-1Cer; GM3 or Neu5AcR2-3Galβ1-4Glcβ1-1Cer; GD3 or Neu5AcR2-8Neu5AcR2-3Galβ1-4Glcβ1-1Cer; IV3/6nLc4Cer or Neu5AcR2-3/6Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1Cer; VI3/6nLc6Cer or Neu5AcR2-3/6Galβ1-4GlcNAcβ1-3Galβ14GlcNAcβ1-3Galβ1-4Glcβ1-1Cer. Reference GSLs. Neolacto-Series Gangliosides from Human Granulocytes. A mixture of human granulocyte gangliosides (HGGs) containing GM3(d18:1, C16:0/C24:1) and neolacto-series gangliosides IV3nLc4Cer(d18:1, C16:0/C24:1), IV6nLc4Cer(d18: 1, C16:0/C24:1), VI3nLc6Cer(d18:1, C16:0/C24:1), and VI6nLc6Cer(d18:1, C16:0/C24:1) as major components was isolated as already described.32 GD3 from Bovine Buttermilk. Gangliosides from bovine buttermilk were isolated according to standard procedures33 and purified by adsorption chromatography on Iatrobeads 6RS-8060 (Macherey-Nagel, Du¨ren, Germany) as described by Ueno et al.34 GD3 was further isolated using TMAE-Fractogel chromatography35 and finally purified by adsorption HPLC on silica gel (Nucleosil 50-7, 250 mm × 20 mm, Macherey-Nagel) with a linear polarity gradient from 85/15 to 0/100 chloroform/methanol (v/ v). GSL Mixture from Human Melanoma. The melanoma used in this study represented the part of a sample that remained after pathohistological diagnosis. The tumor has been classified as Breslow level V and Clark level IV.36 The cancer tissue was homogenized in distilled water, and the slurry was freeze-dried. The dried homogenate was extracted with chloroform/methanol/ water (30/60/8, v/v/v) and chloroform/methanol (2/1, v/v). The extracts were filtrated, rotary evaporated, und incubated with 1 N aqueous NaOH for 2 h at 37 °C. After neutralization with 10 N HCl, the solution was dialyzed against distilled water and freezedried. The desalted extract was dissolved in chloroform/methanol (2/1, v/v) and adjusted to a defined concentration corresponding to 1 mg of tissue wet weight/µL. Ganglio-Series Neutral GSLs from Murine MDAY-D2 Cells. The neutral GSL fraction from the murine lymphoreticular MDAY-D2 cell line contained as major compounds lactosylceramide and the ganglio-series neutral GSLs Gg3Cer and Gg4Cer, all of them appearing as doublet bands in TLC runs due to substitution of the sphingosine moiety with C24 and C16 fatty acids (upper and lower bands, respectively).37,38 High-Performance Thin-Layer Chromatograhy. GSLs were separated on glass-backed silica gel 60 precoated high-performance thin-layer chromatography plates (HPTLC plates, size 10 (32) Mu ¨ thing, J.; Unland, F.; Heitmann, D.; Orlich, M.; Hanisch, F.-G.; PeterKatalinic´, J.; Kna¨uper, V.; Tschesche, H.; Kelm, S.; Schauer, R.; Lehmann, H. Glycoconjugate J. 1993, 10, 120-126. (33) Ledeen, R. W.; Yu, R. K. Methods Enzymol. 1982, 83, 139-191. (34) Ueno, K.; Ando, S.; Yu, R. K. J. Lipid Res. 1978, 19, 863-871. (35) Mu ¨ thing, J.; Unland, F. J. Chromatogr., B 1994, 658, 39-45. (36) Kemminer, S. E.; Conradt, H. S.; Nimtz, M.; Sˇ agi, D.; Peter-Katalinic´, J.; Diekmann, O.; Drmic´, I.; Mu ¨ thing, J. Biotechnol. Prog. 2001, 17, 809-821. (37) Schwartz, R.; Kniep, B.; Mu ¨ thing, J.; Mu ¨ hlradt, P. F. Int. J. Cancer 1985, 36, 601-607. (38) Mu ¨ thing, J.; C ˇ acˇic´, M. Glycoconjugate J. 1997, 14, 19-28.

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cm × 10 cm, thickness 0.2 mm, no. 5633; Merck, Darmstadt, Germany). Neutral GSLs were chromatographed in chloroform/ methanol/water (120/70/17, v/v/v) and gangliosides in chloroform/methanol/water (120/85/20, v/v/v) for 20 min, respectively, each supplemented with 2 mM CaCl2. For analytical purpose, GSLs were stained with orcinol.39 The TLC plate was dipped for 10 s in 0.3% (w/v) orcinol in 3 M H2SO4 and then transferred to a preheated heating plate (110 °C) until the appearance of pinkishviolet spots. For preparative purposes, GSLs were stained with primuline.15 The TLC plate was dipped for 10 s in 0.02% (w/v) primuline (Sigma, Deisenhofen, Germany; no. P-7522) in acetone/ water (4/1, v/v). Visualization was performed under long-wave UV light at 366 nm. Lipids were detected as light blue or yellowish bands on the dark, blue-violet background. GSL-Specific and Secondary Antibodies. Two polyclonal chicken anti-Neu5AcR2-3Galβ1-4GlcNAc-R (AB2-3) and antiNeu5AcR2-6Galβ1-4GlcNAc-R (AB2-6) antibodies were generated according to the method of Kasai et al.40 as previously described.41 The antibodies were used in 1:1000 dilutions in 1% (w/v) bovine serum albumin in phosphate-buffered saline (PBS; ) solvent A) and detected with a secondary rabbit anti-chicken IgY antibody labeled with alkaline phosphatase (Dianova, Hamburg, Germany), 1:2000 diluted in solvent A. The original hybridoma cell line R24 was obtained from the American Type Culture Collection (ATCC, Bethesda, MD; HB 8445). The cell line was produced by fusing murine myeloma cells with spleen cells from mice that had been immunized with the SK-MEL-28 human melanoma cell line.42 This mouse IgG3 monoclonal antibody (mab) was later shown to specifically bind to ganglioside GD3.11 Cell culture supernatant43 was used 1:20 diluted in solvent A. Monoclonal mouse IgM anti-Gg3Cer (asialo GM2) antibody was produced with the hybridoma cell line 2D4 (ATCC, TIB-185) cultivated in RPMI 1640 supplemented with 10% fetal calf serum (v/v). The cell line was generated by fusing murine myeloma cells with spleen cells from mice that had been immunized with Gg3Cer noncovalently adsorbed to naked Salmonella minnesota.44 Original cell culture supernatant was used applying an 100-µL aliquot per cut lane (1.5 cm × 10 cm) of chromatographed GSLs, a procedure designated as “microscale method” as previously described.45,46 Both murine mabs, R24 (IgG3) and 2D4 (IgM), were detected with a secondary goat anti-mouse IgG and IgM antibody labeled with alkaline phosphatase (Dianova), 1:2000 diluted in solvent A. TLC Immunostaining. The TLC immunodetection procedure, which has been published in two reviews,18,47 was used with minor (39) Svennerholm, L. Neurochemistry 1956, 1, 42-53. (40) Kasai, M.; Iwamori, M.; Nagai, Y.; Okumura, K.; Tada, T. Eur. J. Immunol. 1980, 10, 175-180. (41) Meisen, I.; Peter-Katalinic´, J.; Mu ¨ thing, J. Anal. Chem. 2003, 75, 57195725. (42) Dippold, W. G.; Lloyd, K. O.; Li, L. T. C.; Ikeda, H.; Oettgen, H. F.; Old, L. J. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 6114-6118. (43) Mu ¨ thing, J.; Kemminer, S. E.; Conradt, H. S.; Sˇ agi, D.; Nimtz, M.; Ka¨rst, U.; Peter- Katalinic´, J. Biotechnol. Bioeng. 2003, 83, 321-334. (44) Young, W. W., Jr.; MacDonald, E. M. S.; Nowinski, R. C.; Hakomori, S.-I. J. Exp. Med. 1979, 150, 1008-1019. (45) Mu ¨ thing, J.; Spanbroek, R.; Peter-Katalinic´, J.; Hanisch, F.-G.; Hanski, C.; Hasegawa, A.; Unland, F.; Lehmann, J.; Tschesche, H.; Egge, H. Glycobiology 1996, 6, 147-156. (46) Duvar, S.; Peter-Katalinic´, J.; Hanisch, F.-G.; Mu ¨ thing, J. Glycobiology 1997, 7, 1099-1109. (47) Mu ¨ thing, J. J. Chromatogr., A 1996, 720, 3-25.

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modifications. Briefly, after TLC of GSLs, the silica gel was fixed with poly(isobutyl methacrylate) (Plexigum P28, Ro¨hm, Darmstadt, Germany) followed by incubation of the plates in PBS at 37 °C overnight. All the following steps were performed at ambient temperature. Blocking of unspecific antibody binding was performed in solvent A for 15 min. The plates were incubated with primary anti-GSL antibodies (see above) for 1 h and then washed three times with 0.05% (v/v) Tween 21 in PBS () solvent B). After 2-h incubation of the plates with the respective secondary antibodies (see above), the plates were washed three times with solvent B and twice with glycine buffer (0.1 M glycine, 1 mM ZnCl2, 1 mM MgCl2, adjusted to pH 10.4 with NaOH). Bound antibodies were visualized by color development using 0.05% (w/ v) 5-bromo-4-chloro-3-indolyl phosphate (BCIP; Biomol, Hamburg, Germany) in glycine buffer. If not otherwise mentioned, the reaction was stopped by washing the plates twice with glycine buffer. Additionally, final washing of the plates for 2 min in distilled water was performed as mentioned in the Results section. The plates were dried and stored at -20 °C. Extraction of Gangliosides from TLC Plates. The TLC plates with immunodetected GSL bands were disposed of the silica gel fixative (Plexigum) by dipping the plates in distilled chloroform. When the silica gel became transparent, the plates were removed and dried at ambient temperature in a fume hood. The silica gel of an antibody positive spot was scraped off the plate with a scalpel and transferred into small conical screw-capped glass tubes. The GSLs were extracted from the silica gel three times with 100 µL of chloroform/methanol/water (30/60/8, v/v/ v) under sonication in an ultrasound bath (30 s). The slurries were centrifuged for 1 min, and the pooled supernatants were dried in a sample concentrator under a stream of nitrogen at 37 °C. The primuline positive bands were marked under UV light visualization; the silica gel was scraped off the plate and extracted as described for antibody stained GSLs. The crude extracts were analyzed by ESI-QTOF-MS without any further purification. Mass Spectrometry. The extracted and dried GSL samples were dissolved in 20 µL of pure distilled methanol (Merck) to an estimated concentration of ∼0.05 µg/µL. Electrospray mass spectrometry was carried out on a QTOF instrument equipped with a nanospray manipulator (Micromass, Manchester, U.K.) essentially as described before.41,48 Negative ion mode was applied exclusively for the mass spectrometric investigation of the extracted GSLs; nitrogen was used as desolvation and nebulizer gas. Capillaries were made in-house from borosilicate glass (Hilgenberg, Malsfeld, Germany) using a vertical pipet puller (model 720, David Kopf Instruments, Tujunga, CA). High voltage was applied via a steel wire to the sample solution. The capillary voltage was set to 1.1 kV, and the voltage on the counter electrode was set to 140 V. For MS/MS experiments, the singly charged precursor ions were selected with the first quadrupole by increasing the dc/ac ratio. Collision-induced dissociation (CID) was performed using argon as collision gas, whereby the pressure in the collision cell was ∼3.5 × 10-5 mbar. The collision energy applied was set to values reducing the (48) Metelmann, W.; Peter-Katalinic´, J.; Mu ¨ thing, J. Am. Soc. Mass Spectrom. 2001, 12, 964-973.

Figure 1. TLC stain and TLC overlay assay of human granulocyte gangliosides (HGGs). Lane a, orcinol stain; lane b, primuline stain; lane c, immunostain with anti-Neu5AcR2-3Galβ1-4GlcNAc-R (AB23) antibody; lane d, immunostain with anti-Neu5AcR2-6Galβ14GlcNAc-R (AB2-6) antibody. A 20-µg sample of HGGs was applied on each lane. The silica gel of primuline-stained and antibody-positive gangliosides, framed by dotted rectangles, were scraped off the TLC plate, extracted, and investigated by mass spectrometry (see Figures 2-4).

intensity of the precursor ion signal to one-third of its original intensity, at best (Elab ) 70-90 V). MS spectra were obtained by combining 80-101 scans per spectrum resulting in an acquisition time of ∼2-3 min. For MS/ MS spectra acquisition time was, depending on the relative abundance of the selected precursor ions, 6-13 min, and 228738 single scans were combined. RESULTS Nondestructive Detection of TLC Separated NeolactoSeries Gangliosides by Primuline Stain and Immunostain. The most abundant components of the ganglioside fraction from human granulocytes are besides GM3 (Neu5AcR2-3Galβ14Glcβ1-1Cer) neolacto-series monosialogangliosides with nLc4Cerand nLc6Cer-core structures as shown by orcinol stain (Figure 1, lane a). N-Acetylneuraminic acid may be attached in either R23- or R2-6-linkage to the terminal galactose residue of neolactoseries core structures, thus evoking different migration behaviors in TLC of isomeric IV3/6nLc4Cer and VI3/6nLc6Cer species, respectively. Due to the substitution of the sphingosine moiety (d18:1) with different fatty acids, mainly C24:1 and C16:0, gangliosides containing the same sugar moiety separate into double bands. On the basis of the different chromatographic properties of gangliosides in TLC, no further separation into pure isomeric fractions is necessary for the purpose of preparative TLC. Thus, GSLs can be extracted from the silica gel of scraped TLC bands after nondestructive detection and further investigated by mass spectrometry. In the first experiment, mass spectrometric investigation of neolacto-series monosialogangliosides from the silica gel extract, obtained from a TLC plate after staining with the nondestructive

fluorochrome primuline, was performed. The primuline-stained HGGs, visualized by UV light, are shown in lane b of Figure 1. The doublet band marked by a dotted rectangle, corresponding to IV6nLc4Cer(d18:1, C24:1) (upper band) and IV6nLc4Cer(d18: 1, C16:0) (lower band), was scraped off the TLC plate. The silica gel extract obtained by use of chloroform/methanol/water (30/ 60/8, v/v/v/) was investigated by mass spectrometry (see next section). Lane c of Figure 1 represents the TLC overlay detection of R2-3-sialylated neolacto-series gangliosides within the HGG fraction. Gangliosides with nLc4 and nLc6 cores were detected with a polyclonal anti-Neu5AcR2-3Galβ1-4GlcNAc-R antibody (AB2-3). Lane d of Figure 1 shows the TLC overlay assay obtained by immunostain of the HGG fraction with a polyclonal anti-Neu5AcR2-6Galβ1-4GlcNAc-R antibody (AB2-6). Gangliosides, with nLc4 and nLc6 cores carrying terminal neuraminic acid in R2-6-linkage, were detected. The antibody positive species considered for mass spectrometric investigation are framed by dotted rectangles. The marked ganglioside doublets were scraped off the TLC plate, extracted, redissolved in methanol after evaporation, and analyzed by nanoESI mass spectrometry without further purification (see below). Mass Spectrometric Investigation of the Silica Gel Extract Obtained from Primuline-Stained HGGs. The MS spectrum obtained from the silica gel extract of the two primuline-stained gangliosides IV6nLc4Cer(d18:1, C16:0/C24:1) and the MS/MS spectrum of IV6nLc4Cer(d18:1, C16:0) are shown in Figure 2A and B, respectively. It can be clearly deduced from the spectra that primuline and salts, remaining from the staining and extraction procedure, were tolerable in the ionization process, since both ganglioside species, scraped off the TLC plate and extracted from the silica gel, were detected as singly charged molecular ions without adducts (Figure 2A). By extraction of one TLC double band, structural characterization of the species was achieved, as shown by the low-energy CID spectrum of the IV6nLc4Cer(d18: 1, C16:0) species in Figure 2B. Full series of B- and Y-type ions, accompanied by ions generated by ring cleavages of the sugar moiety, allowed the clear-cut structure assignments. Additionally, the fragment ions 0,2X4 at m/z 1295.85, diagnostic for gangliosides carrying Neu5Ac in R2-6 linkage to galactose,41 were also detected beside the diagnostic fragment ions 0,4A2-CO2 (m/z 306.17) and 2,4A3-CO2 (m/z 468.25). The molecular structure and the corresponding fragmentation scheme of IV6nLc4Cer substituted with sphingosine (d18:1) and C16:0 fatty acid is shown in Figure 2C. The fragment ions are assigned according to the nomenclature of Domon and Costello.49 Coincident results were obtained by mass spectrometric structure determination of the species IV6nLc4Cer(d18:1, C24:1) (data not shown). Mass Spectrometric Investigation of Silica Gel Extracts Obtained after TLC Immunostain of HGGs with Polyclonal Antibodies. In the next step, the mass spectrometric characterization of silica gel extracts obtained by preparative TLC after detection of neolacto-series gangliosides with specific polyclonal antibodies was carried out. The procedure is based on TLC immunostain with the primary antibodies AB2-3 or AB2-6, followed by incubation with an alkaline phosphatase-conjugated (49) Domon, B.; Costello, C. E. Glycoconjugate J. 1988, 5, 397-409.

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Figure 2. NanoESI mass spectra in the negative ion mode obtained from the silica gel extract of the primuline-stained gangliosides IV6nLc4Cer(d18:1, C16:0/C24:1) (see Figure 1, lane b) and corresponding fragmentation scheme. (A) MS spectrum of IV6nLc4Cer(d18:1, C16:0/C24:1); (B) MS/MS spectrum of the ganglioside IV6nLc4Cer(d18:1, C16:0) with m/z 1516.97; (C) molecular structure and fragmentation scheme of the ganglioside IV6nLc4Cer(d18:1, C16:0).

secondary rabbit anti-chicken IgY antibody and visualization by the chromogenic substrate BCIP (see Figure 1, lanes c and d). The silica gel containing the immunostained double bands was scraped off the plate, extracted as described before and subsequently analyzed by ESI-QTOF mass spectrometry (see Figures 3 and 4). In the resulting mass spectra, the ganglioside doublets IV3Neu5Ac-nLc4Cer(d18:1, C16:0/C24:1) (Figure 3A) and IV6Neu5Ac-nLc4Cer(d18:1, C16:0/C24:1) (Figure 4A), carrying the antigenic determinants for AB2-3 and AB2-6, respectively, were detected unambiguously. The only species present in both mass spectra of the silica gel extracts were the singly charged deprotonated molecular ions of the antibody-detected gangliosides. Neither buffer-derived salt adducts, coextracted contaminants from the immunostain procedure, such as BCIP artifacts, nor the antibodies or antigen-antibody complexes were observed. In the low-energy CID MS/MS spectra obtained from IV3nLc4Cer and IV6nLc4Cer with C16:0 fatty acid (Figures 3B and 4B, respectively) and IV3nLc4Cer and IV6nLc4Cer with C24:1 fatty acid (Figures 2252 Analytical Chemistry, Vol. 76, No. 8, April 15, 2004

Figure 3. NanoESI mass spectra in the negative ion mode of the gangliosides IV3nLc4Cer(d18:1, C16:0/C24:1) obtained from the silica gel extract after immunodetection with the polyclonal antibody AB2-3 (see Figure 1, lane c). (A) MS spectrum; (B) MS/MS spectrum of IV3nLc4Cer(d18:1, C16:0) with m/z 1516.95; (C) MS/MS spectrum of IV3nLc4Cer(d18:1, C24:1) with m/z 1627.10.

3C and 4C, respectively), a full series of Y-type ions and a few B-type ions, enabling structural characterization of the sugar and the ceramide moieties of the immunostained gangliosides, were observed. By direct comparison of the low-energy CID spectra obtained from IV3nLc4Cer(d18:1, C16:0) (Figure 3B) and IV6nLc4Cer(d18:1, C16:0) (Figure 4B), the difference in fragmentation of gangliosides carrying Neu5Ac in R2-3- or R2-6-linkage, respectively, is obvious.41 The ions 0,2X4 (m/z 1295.67), 0,4A2-CO2 (m/z 306.14), and 2,4A3-CO2 (m/z 468.19) were detected within the ganglioside species with R2-6-linked Neu5Ac (Figure 4B) only. For assignment of the fragment ions, the fragmentation scheme in Figure 2C is referred to.

Figure 5. (A) TLC orcinol stain of a human melanoma extract and (B) the corresponding TLC overlay assay with the anti-GD3 monoclonal IgG3 antibody R24. Lanes a, melanoma extract; the amount applied corresponds to 15 mg of tissue wet weight (∼5 µg of GSLs); lanes b, 2 µg of purified GD3 from bovine buttermilk; lanes c, 20 µg of HGGs. The silica gel of antibody-positive GD3, framed by a dotted rectangle, was scraped off the TLC plate, extracted, and investigated by mass spectrometry (see Figure 6).

Figure 4. NanoESI mass spectra in the negative ion mode of the gangliosides IV6nLc4Cer(d18:1, C16:0/C24:1) obtained from the silica gel extract after immunodetection with the polyclonal antibody AB2-6 (see Figure 1, lane d). (A) MS spectrum; (B) MS/MS spectrum of IV6nLc4Cer(d18:1, C16:0) with m/z 1516.77; (C) MS/MS spectrum of IV6nLc4Cer(d18:1, C24:1) with m/z 1626.91.

Mass Spectrometric Investigation of a Silica Gel Extract Obtained from Melanoma GD3 after TLC Immunostain with mab R24 (IgG3). Further antibody-based investigations were performed with respect to different types of GSLs and different immunoglobulin subclasses (IgG and IgM). The monoclonal IgG3 antibody R24, which binds to the tumor-associated disialoganglioside GD3, was chosen as a representative for a mab of the IgG subclass. A crude melanoma extract was separated by TLC and stained with orcinol as shown in Figure 5A (lane a). Two double bands representing GM3 and lactosylceramide, respectively, were detected as major components and a double band of the tumorassociated ganglioside GD3 as a minor constituent of the mela-

noma extract. A purified GD3 fraction, isolated from bovine buttermilk, was used as a reference (Figure 5A, lane b) and HGGs as a negative control (Figure 5A, lane c). The corresponding overlay assay obtained with mab R24 and an anti-mouse IgG/ IgM secondary antibody is shown in Figure 5B. A double band, corresponding to GD3(d18:1, C24:1) (upper band) and GD3(d18: 1, C16:0) (lower band), was detected in the melanoma extract (Figure 5B, lane a) and the buttermilk fraction (Figure 5B, lane b), whereas HGGs showed no reaction at all (Figure 5B, lane c). The silica gel, containing the two immunostained GD3 species of the melanoma extract (Figure 5B, lane a, framed dotted rectangle), was scraped off the TLC plate, extracted, and analyzed by mass spectrometry. The resulting nanoESI mass spectrum is shown in Figure 6A. The GD3 antigen is detected as doubly charged deprotonated species at m/z 720.94 (GD3(d18:1, C16:0)) and 776.00 (GD3(d18:1, C24:1)), respectively. The most abundant ions, detected at m/z 603.22, correspond to the sodiated B2 ions representing a disialic acid moiety due to in-source fragmentation of GD3 under the ionization conditions used. The singly charged molecular ions of GD3 are detected as sodium adducts at m/z 1464.85 (GD3(d18:1, C16:0)) and 1575.01 (GD3(d18:1, C24:1)), respectively. To reduce the amount of sodium adducts, i.e., to obtain singly charged deprotonated molecular ions, a second TLC overlay assay was performed using a slightly modified procedure: Prior to silica gel extraction of immunostained GD3, the TLC plate was washed twice with distilled water. In the resulting mass spectrum (Figure 6B), two dominant GD3 species could be detected unambiguously as singly charged deprotonated molecular ions at m/z 1443.01 (GD3(d18:1, C16:0)) and 1553.14 (GD3(d18:1, C24:1)), preferably selected as precursor ions for MS/ MS experiments. Additionally, minor GD3 variants substituted with C18:0 and C22:0 fatty acids (m/z 1471.03 and 1527.10, respectively) and the doubly deprotonated molecular ions of GD3(d18:1, C16:0) at m/z 721.00 and GD3(d18:1, C24:1) at m/z 776.06 were detected as well. Some fragment ions were observed under the ionization conditions used. As a result of the removal of salts through final washing of the TLC plate with water, the previously Analytical Chemistry, Vol. 76, No. 8, April 15, 2004

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Figure 6. NanoESI mass spectra in the negative ion mode of GD3 from human melanoma obtained from the silica gel extract after TLC overlay assay with the mab R24 (see Figure 5B, lane a). (A) MS spectrum obtained after final washing of the TLC plate with glycine buffer prior to GSL extraction; (B) MS spectrum obtained after washing of the TLC plate with glycine buffer followed by two final washings with water prior to GSL extraction.

high-abundant B2(Na) ions (Figure 6A) were now detected predominantly as deprotonated species at m/z 581.26 and as a specific decarboxylated species at m/z 537.29 (Figure 6B) as described before.23 Due to in-source fragmentation, the Y3 ions of GD3(d18:1, C16:0) at m/z 1151.88 and GD3(d18:1, C24:1) at m/z 1262.00 were detected. However, full structural information of the tumor-associated GD3 was obtained by low-energy CID even by fragmentation of the relatively low abundant doubly charged molecular ions (data not shown). Mass Spectrometric Investigation of the Silica Gel Extract Obtained after TLC Immunostain of Gg3Cer from MDAYD2 Cells with mab 2D4 (IgM). Mab 2D4, which binds to the neutral GSL Gg3Cer, was chosen as a representative for a mab of the IgM subclass. A mixture of ganglio-series neutral GSLs from MDAY-D2 cells served as reference. The orcinol stain and the corresponding immunostain of Gg3Cer are shown in the inset of Figure 7A. As documented by the immunostain (Figure 7A, inset lane b), the IgM antibody 2D4 is specific for the neutral GSL Gg3Cer, which can be substituted with different fatty acids. Other neutral GSLs, e.g., MHC, lactosylceramide, and Gg4Cer, present in the mixture of MDAY-D2 cells as visualized by orcinol stain (Figure 7A, inset lane a), were not detected. The mass spectrum obtained from the silica gel extract of the immunostained Gg3Cer species is shown in Figure 7A. A clear-cut series of Gg3Cer 2254 Analytical Chemistry, Vol. 76, No. 8, April 15, 2004

Figure 7. NanoESI mass spectra in the negative ion mode of the neutral GSL Gg3Cer obtained from the silica gel extract after immunodetection with the anti-Gg3Cer monoclonal IgM antibody 2D4. (A) MS spectrum of Gg3Cer; (B) MS/MS spectrum of Gg3Cer(d18:1, C16:0) with m/z 1063.91; (C) the corresponding molecular structure and fragmentation scheme. (A inset) TLC orcinol stain of 8 µg of neutral GSLs from MDAY-D2 cells (lane a) and the corresponding TLC immunostain of Gg3Cer with mab 2D4 (lane b). The silica gel of antibody-positive GSLs, framed by a dotted rectangle (lane b), was scraped off the TLC plate, extracted, and investigated by mass spectrometry.

carrying different fatty acid residues in the ceramide moiety was detected at m/z 1063.84, 1147.97, 1173.99, and 1176.00, corresponding to Gg3Cer(d18:1) substituted with C16:0, C22:0, C24:1, and C24:0 fatty acid, respectively. The low-energy CID spectrum of Gg3Cer(d18:1, C16:0) and the corresponding fragmentation scheme are shown in Figure 7B and C, respectively. A complete series of Y-type ions, accompanied by A ions generated by characteristic ring cleavages, allowed full structural characterization of the neutral GSL Gg3Cer(d18:1, C16:0). The ions at m/z 119.09, 143.09, 161.10, and 179.12, respectively, are assigned to hexose moieties. DISCUSSION High-performance thin-layer chromatography is the most commonly used tool in GSL analysis for analytical and preparative purposes because of its superior resolution. The “old-fashioned”

technique of TLC separation and subsequent extraction of GSLs from scraped silica gel is still a frequently used method. This procedure is in many cases advantageous due to its easy handling, whereas conventional purification of GSLs with repeated column chromatographies is a time-consuming task to obtain even a simple kind of purified GSL. The most sensitive nondestructive dye for lipid detection is primuline, which has become most popular in preparative HPTLC. The sample cleanup by use of RP18 cartridges and the removal of primuline from silica gel extracts prior to structural characterization of the respective GSLs has been reported, e.g., using a continuous or stepwise HPLC-based gradient elution with 2-propanol/hexane/water.50,51 Alternative techniques have been reported for preparative HPTLC of neutral GSLs and gangliosides using charged and uncharged fluorochromes, respectively,16,17 or for the isolation of GSLs and phospholipids by TLC blotting.52 However, the extracts of fluorochrome-stained GSLs still require purification steps such as anion-exchange chromatography and adsorption chromatography. The direct ESI-MS characterization of GM4 in a chloroform/ methanol (1/1, v/v) extract obtained by preparative TLC after primuline staining has been reported by Hildebrandt et al.26 Their spectrum obtained from a few micrograms of a crude sample revealed a series of peaks corresponding to a GM4-like monosialoganglioside species. In our first experiment, we investigated crude samples containing more polar neolacto-series gangliosides with nLc4Cer cores. After TLC separation and primuline stain, the silica gel was extracted using chloroform/methanol/water (30/60/8, v/v/v). In the resulting mass spectrum, the gangliosides were detected as singly charged deprotonated molecular ions, indicating that no further purification steps are required prior to structural investigation of silica gel extracts by mass spectrometry. Consequently, all investigations with respect to GSLs detected with specific antibodies were performed with chloroform/methanol/ water (30/60/8, v/v/v) extracts. Oligosaccharide moieties of GSLs are identified by immunostain, thus revealing the carbohydrate epitope. The complementary structural “hard data” of the sugar sequence and structural information of the ceramide moiety are provided by MS and MS/MS experiments at high sensitivity of the QTOF instrument. This combined strategy requires only analytical quantities of GSLs commonly used for routine overlay immunoassays. As a rule of thumb, ∼1 µg of a GSL species, revealing a clear antibody-positive band, is sufficient. The procedure was shown to be functional for polyclonal and monoclonal (50) Stroud, M. R.; Handa, K.; Salyan, M. E. K.; Ito, K.; Levery, S. B.; Hakomori, S. Biochemistry 1996, 35, 758-769. (51) Stroud, M. R.; Stapleton, A. E.; Levery, S. B. Biochemistry 1998, 27, 1742017428. (52) Taki, T.; Handa, S.; Ishikawa, D. Anal. Biochem. 1994, 221, 312-316.

antibodies as well, regardless of the GSL subclass, i.e., neutral GSLs (Gg3Cer) and mono- and disialogangliosides (IV3/6nLc4Cer and GD3, respectively). Moreover, the method is applicable for crude GSL extracts, as shown by the immunodetection and mass spectrometric characterization of GD3 in a total lipid extract from a human melanoma tissue. Up to now, only one direct MS investigation of immunostained GSLs has been reported by Guittard et al.29 GSLs containing Lewisx determinants that exhibited binding to a monoclonal antibody in an overlay assay on the TLC plate were transferred to a poly(vinylidene difluoride) membrane and analyzed by MALDI-TOF-MS without interference of the salts and buffers used during the binding and visualization steps. In the resulting MALDI mass spectrum in the positive ion mode, the expected GSLs were detected as sodium and potassium adducts. A limitation of this approach was the interference of the synthetic polymer used for the stabilization of the silica gel of TLC immunostained plates. The polymer signals were found to obscure those of the GSLs. However, as shown in this study, the fixative poly(isobutyl methacrylate) can be easily removed by dipping the immmunostained TLC plate in chloroform until the silica gel becomes transparent. The silica gel remains stable and the antibody-bound GSLs retain in the silica gel. To avoid sodium adducts, obtained only in the case of immunostaining of the disialoganglioside GD3 with mab R24, two short final washes of the TLC plate with distilled water after the substrate reaction and before removal of the silica gel fixative are sufficient to remove sodium salts and to obtain deprotonated molecular ions. Prolonged incubation with water results, however, in peeling off of the silica gel from the glass support. In summary, an efficient HPTLC-MS-joined strategy is described for structural characterization of immunostained GSLs in low-microgram scale. CONCLUSIONS The combined method of TLC overlay assay and nanoESI MS provides an excellent model for structural elucidation of GSL antigens in crude mixtures enabling a direct correlation of carbohydrate binding specificity and molecular structure. Crude GSL mixtures of, for example, tumor tissue extracts can be analyzed and several samples can be handled by immunodetection on a single HPTLC plate simultaneously. Due to an increasing number of carbohydrate-specific monoclonal antibodies5 and their at least partial commercial access, the procedure is suitable for a wide range of researchers working in this field. The described method should be applicable for any carbohydrate-binding agents such as bacterial toxins, plant lectins, and others. Received for review December 19, 2003. Accepted February 13, 2004. AC035511T

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