ESI QTOF MS and Tandem MS for Separation

and Development Ltd., Prilaz baruna Filipovića 29, 10000 Zagreb, Croatia ..... Alternatively, the detection limit of the nano-HPLC/ESI QTOF MS me...
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Anal. Chem. 2008, 80, 4711–4722

On-Line Nano-HPLC/ESI QTOF MS and Tandem MS for Separation, Detection, and Structural Elucidation of Human Erythrocytes Neutral Glycosphingolipid Mixture Stephan Kirsch,† Mostafa Zarei,† Mario Cindric´,‡ Johannes Mu ¨ thing,† Laura Bindila,*,† and ,† Jasna Peter-Katalinic ´* Institute for Medical Physics and Biophysics, University of Mu¨nster, Robert Koch Strasse 31, D-48149 Mu¨nster, Germany, and Pliva Research and Development Ltd., Prilaz baruna Filipovic´a 29, 10000 Zagreb, Croatia A superior approach involving nano-high-performance liquid chromatography (nano-HPLC) in on-line conjunction to electrospray ionization quadrupole time-of-flight mass spectrometry (ESI QTOF MS) and tandem MS for screening and structural characterization of complex mixtures of neutral glycosphingolipids (GSLs) is here described. Neutral GSLs purified from human erythrocytes were efficiently separated according to the differences in carbohydrate chain length by an optimized nanoHPLC protocol and flow-through detected by ESI QTOF MS at the low femtomole level. Additionally, GSL species were accurately distinguished from the accompanying lipids in the mixture, thus permitting the determination of detailed structural characteristics by data-dependent analysis for identification of GSL constitution within single experiments. An alternative nano-HPLC/ESI QTOF MS approach was designed for dissection of unsaturation/ saturation degree of the ceramide moieties defining the hydrophobic portion of GSLs and subsequent localization by nano-HPLC/ESI QTOF MS/MS of the -CHdCHwithin the ceramide regions. The method is fast, highly sensitive, and high-throughput amenable and is highlighted as a new and valuable analytical dimension in glycolipidomics. A hydrophilic oligosaccharide chain and a hydrophobic ceramide moiety are characteristic features of amphipathic glycosphingolipids (GSLs). The oligosaccharides vary considerably in number and type of monosaccharides and their anomeric configuration, whereas the ceramide shows less structural heterogeneity. In mammalian cells, the ceramide part is typically built up from the long-chain aminoalcohol sphingosine (d18:1), which is linked with a fatty acid differing in chain length from C16 to C24 and in degree of unsaturation. The oligosaccharides of GSLs interact with the cellular environment and serve as identification tags, whereas the ceramide operates as an intracellular regulator upon binding * Corresponding authors. E-mail: [email protected] (J.P.-K.), [email protected] (L.B.). Phone: +492518352308(J.P.-K.), +492518355127(L.B.). Fax: +492518355140 (J.P.-K.), +492518355121 (L.B.). † University of Mu ¨ nster. ‡ Pliva Research and Development Ltd. 10.1021/ac702175f CCC: $40.75  2008 American Chemical Society Published on Web 05/21/2008

of extracellular ligands.1,2 Because they undergo specific changes in their pattern in an age-dependent as well as disease-specific manner, GSLs represent potential biomarkers for various diseases including cancer.3–5 There is an entire arsenal of well-established analytical tools for their characterization, relying mostly on the use of chromatographic techniques.6 Mass spectrometry (MS) emerged as a dedicated mean for GSLs determination;7–10 however, it is usually preceded by extensive sample handling and multiple chromatographic steps.6–10 Consequently, the complete analytical flow becomes very complex, laborious, and time-consuming.11,12 Even though a great deal of lipid can be removed by the now classical preparation procedure, still interference of lipid contaminants and/or derived side products within a GSL MS profile occurs by covering a rather broad range of m/z values. This is considerably detrimental for sensitive detection of GSLs as well as for curative structural characterization. This is due to the fact that an accurate extraction of GSL components from any biological matrix is hardly achievable, since invariably a variety of lipids accompany and quantitatively dominate the GSLs. A complete depletion of lipid content in a GSL mixture is not feasible so far, since lipids and GSLs exhibit similar physicochemical and migration properties in many chromatographic steps and/or a quantitative removal by alkaline or enzymatic treatment of lipids is difficult to achieve. In an alternative approach, the enzymatic cleavage of the oligosaccharide chain from the corresponding ceramide portion is frequently used, i.e., by ceramidase treatment.13 However, this way, the ceramide and glycan chains are independently characterized, and the corresponding separation/ (1) Hannun, Y. A.; Obeid, L. M. Trends Biochem. Sci. 1995, 20, 73–77. (2) Mu ¨ thing, J. In Glycosciences III; Fraser-Reid, B., Tatsuta, K., Thiem, J., Eds.; Springer: Berlin, 2001; pp 2220-2237. (3) Hakomori, S. Cancer Res. 1985, 45, 2405–2414. (4) Zhang, X.; Kiechle, F. L. Ann. Clin. Lab. Sci. 2004, 34, 3–13. (5) Feizi, T. Nature 1985, 314, 53–57. (6) Mu ¨ thing, J. Methods Enzymol. 2000, 312, 45–63. (7) Peter-Katalinic´, J.; Egge, H. Methods Enzymol. 1990, 193, 713–733. (8) Egge, H.; Peter-Katalinic´, J. In Mass Spectrometry in the Health and Life Sciences; Burlingame, A. L., Castagnoli, N., Jr., Eds.; Elsevier Science Publishers: Amsterdam, 1985; pp 401-423. (9) Costello, C. E.; Juhasz, P.; Perreault, H. Prog. Brain Res. 1994, 101, 45– 61. (10) Bindila, L.; Peter-Katalinic´, J. Mass Spectrom. Rev., 2008, in press. (11) Levery, S. B. Methods Enzymol. 2005, 405, 300–369. (12) Schnaar, R. Methods Enzymol. 1994, 230, 348–370. (13) Hansson, G. C.; Li, Y. T.; Karlsson, H. Biochemistry 1989, 28, 6672–6678.

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detection is optimized with respect to the specific class of components. Yet, the information on the heterogeneity in the ceramide portion occurring to a certain glycan chain becomes inaccessible, thereby drastically compromising the eventual understanding of the functional roles of GSLs.14 Therefore, concerted efforts were invested in developing analytical and preparative methods for GSL/lipid separation, mainly involving high-performance thin-layer chromatography (HPTLC) and liquid chromatography (HPLC).15 Depending on the separation basis, normal and reversed-phase-based chromatography have been employed with the detection primarily carried out by UV absorbance, fluorescence emission, evaporative light scattering (ELS),16 and/or staining-based methods as for HPTLC.17,18 HPLC with UV or fluorescence detection methods invariably requires the derivatization of the GSLs to confer to them chromophoric groups for their eventual identification and/or to enhance either the hydrophilic or the hydrophobic properties of these amphiphilic molecules to render them amenable for a certain chromatographic separation and detection criterion.19–21 Yet, the information gained upon GSL components by these tools has to be extensively completed with relevant structural details, obtained additionally by biochemical means or by off-line hyphenation to MS.22–24 Besides, the sensitivity level exhibited by these approaches frequently does not suffice for the identification of minor species of particular relevance in a biological mixture. On-line hyphenation of HPLC with MS was successfully introduced so far for identification of neutral GSLs at the microlevel, in particular GlcCer and LacCer.25 Most recently, separation of a ganglioside mixture containing GM1, GD1, and GT1 species on the basis of the sialylation degree and their subsequent on-line MS detection was proved to be feasible on a silica column (PVA-Sil).26 However, very few reports on applicability of hyphenated separation-MS methods for analysis of complex GSL mixtures of biological origin exist, while to our knowledge a method for separation/detection of a neutral GSL mixture at the nanoscale level was not reported so far. Consequently, the immediate need to down-scale the analytical flow for GSL mapping to accomplish their separation and detection at the nano level becomes obvious. We present here for the first time a method for the nanoscale HPLC/electrospray ionization quadrupole time-of-flight (ESI QTOF) (14) Kannagi, R.; Nudelmann, E.; Hakomori, S. Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 3470–3474. (15) Kannagi, R.; Watanabe, K.; Hakomori, S. Methods Enzymol. 1987, 138, 3– 12. (16) Roy, S.; Gaudin, K.; Germain, D. P.; Baillet, A.; Prognon, P.; Chaminade, P. J. Chromatogr., B 2004, 805, 331–337. (17) Svennerholm, L. J. Neurochem. 1956, 1, 42–53. (18) Izumi, K.; Sawada, M. T. Lipids 2001, 36, 97–101. (19) McCluer, R. H.; Evans, J. E. J. Lipid Res. 1973, 14, 611–617. (20) Suzuki, A.; Handa, S.; Yamakawa, T. J. Biochem. 1977, 82, 1185–1187. (21) Neville, D. C. A.; Coquard, V.; Priestman, D. A.; te Vruchte, D. J. M.; Sillence, D. J.; Dwek, R. A.; Platt, F. M.; Butters, T. D. Anal. Biochem. 2004, 331, 275–282. (22) Peter-Katalinic´, J. Mass Spectrom. Rev. 1994, 13, 77–98. (23) Dreisewerd, K.; Mu ¨ thing, J.; Rohlfing, A.; Meisen, I.; Vukelic´, Zˇ.; PeterKatalinic´, J.; Hillenkamp, F.; Berkenkamp, S. Anal. Chem. 2005, 77, 4098– 4107. (24) Meisen, I.; Peter-Katalinic´, J.; Mu ¨ thing, J. Anal. Chem. 2004, 76, 2248– 2255. (25) Merill, A. H., Jr.; Sullards, M. C.; Allegood, J. C.; Kelly, S.; Wang, E. Methods Enzymol. 2005, 36, 207–224. (26) Sommer, U.; Herscovitz, H.; Welty, F. K.; Costello, C. E. J. Lipid Res. 2006, 47, 804–814.

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MS analysis of complex mixture of neutral GSLs purified from human erythrocytes without any additional need for prior derivatization and/or enzymatic treatment. With the use of an amidophase column the human erythrocytes GSLs were successfully separated according to their sugar chain length and unsaturation degree of the ceramide anchor, whereas by ESI QTOF MS and tandem MS the identity and structures of the individual GSL species were straightforwardly discerned. The method is highlighted as a dedicated analytical tool for high-throughput glycolipidomic studies. MATERIALS AND METHODS Reagents. Methanol, water, formic acid (98-100%), isopropyl alcohol (100%) of HPLC analytical grade, ethanol (99%), acetic acid (98%), ammonia (32%), and ammonium acetate were purchased from Merck (Darmstadt, Germany) and used without further purification. HPLC grade acetonitrile was delivered by SigmaAldrich (Seelze, Germany). For GSL sample preparation, distilled chloroform and methanol (Merck) were used. Sample Preparation. Outdated blood bottles (blood group AB, rh. pos.), each of 320 mL concentrated human erythrocytes, were subjected to GSL extraction according to standard procedures.27 Neutral GSLs were isolated by anion-exchange chromatography on DEAE Sepharose CL-6B (Amersham Pharmacia Biotech AB, Uppsala, Sweden) as described elsewhere28 and further purified by chloroform/methanol gradient chromatography on silica gel 60. The chloroform/methanol (40/60, v/v) fraction was peracetylated and separated on Florisil (100-200 mesh, Fluka Chemie AG, Buchs, Switzerland) as described by Saito and Hakomori.29 Peracetylated neutral GSLs were eluted with dichloroethane/acetone (1/1, v/v), saponified, desalted by dialysis, and freeze-dried. The resulting amount of about 20 mg of neutral GSL mixture for each blood bottle was finally taken up in chloroform/ methanol (2/1, v/v) and adjusted to a concentration of 5 µg/µL. For MS a stock solution of GSL mixture was prepared in methanol at a concentration of 0.5 µg/µL, and further working aliquots for LC/MS experiments were prepared by dissolving the sample in the desired solvent for injection to a concentration of 2 pmol/µL (calculated for an average molecular mass of 1000 Da). INSTRUMENTATION High-Performance Thin-layer Chromatography. Neutral GSLs were applied to glass-backed silica gel 60 precoated HPTLC plates (no. 1.05633.0001, Merck, Darmstadt, Germany) with an automatic applicator (Linomat IV, CAMAG, Muttenz, Switzerland) and separated in chloroform/methanol/water (120/70/17, v/v/ v). Neutral GSLs were visualized with orcinol and quantified with a CD60 scanner (Desage, Heidelberg, Germany). Bands were scanned in reflectance mode at 544 nm with a light beam slit of 0.02 mm × 3 mm. Nano-High-Performance Liquid Chromatography. GSLs were separated by use of an Ultimate nano-LC system equipped with a Famos autosampler (Dionex/LC Packings, U.S.A.) and a nanoscale TSK-Gel Amide 80 column (5 µm, 75 µm × 100 mm) purchased from Alltech Grom GmbH (Rottenburg, Germany). (27) Ledeen, R. W.; Yu, R. K. Methods Enzymol. 1982, 83, 139–191. (28) Mu ¨ thing, J.; Egge, H.; Kniep, B.; Mu ¨ hlradt, P. F. Eur. J. Biochem. 1987, 163, 407–416. (29) Saito, T.; Hakomori, S.-I. J. Lipid Res. 1971, 12, 257–259.

Two chromatographic methods were optimized. Method 1: the first solvent system consisted of solvent B, an aqueous solution of 50 mM formic acid adjusted with ammonia to pH 4.4, and solvent A, 20% solvent B in acetonitrile. For this chromatographic method the working aliquots were dried and reconstituted in solvent A to a concentration of ∼2 pmol/µL for injection. The separation method employed with this solvent system is 30 min 70% solvent A followed by decreasing over the next 10 min the polarity of the mobile phase to 90% A and maintaining at 90% solvent A until completion of the LC run, meaning 110 min. The used flow rate was 200 nL/min. The second method involved a solvent system composed of solvent A, acetonitrile/methanol/acetic acid (99.7/0.2/0.1, v/v/ v), and solvent B, methanol/acetic acid (99.9/0.1, v/v), both containing 5 mM ammonium acetate. In this case, the working aliquots were diluted in acetonitrile/methanol (8/2, v/v) to a final concentration of ∼2 pmol/µL for injection. For the separation method the LC system was equilibrated and washed for 3 min after injection with 100% solvent A. Subsequently, the ratio of solvent B in the mobile phase was increased over 20 min to 100% in a linear way and kept to 100% B for 4 min. After that, over 2 min the solvent A was increased to 100% and maintained so for reequilibration until completion of the run, meaning after 90 min. The flow rates were set to 200 and 300 nL/min, respectively. Nano-ESI/QTOF MS. The nano-HPLC instrument was connected to an orthogonal time-of-flight instrument (Micromass, U.K.) equipped with an ESI ion source. For data acquisition and processing the MassLynx software (Micromass, U.K.) was used. The HPLC/ESI QTOF MS coupling was realized via an in-housemade sheathless interface involving coated silica emitters (New Objective, U.S.A.) with an i.d. of 15 µm. The mass spectrometer was operated in positive ion mode at electrospray potentials between 1700 and 1900 V and sampling cone potentials within 40-75 V. Tandem MS experiments were performed in datadependent analysis (DDA) fashion employing collision energy values between 50 and 75 eV, whereas the collision gas pressure was set to 15 psi for all MS/MS experiments. The acquisition of the MS and MS/MS signal followed immediately the sample injection. All mass spectra were externally calibrated using a NaI solution. The assignment of the GSL-derived fragment ions follows the rules established by Domon and Costello.30 The nomenclature of GSLs follows the 1997 IUPAC-IUBM recommendations.31 Triplicate nano-HPLC/ESI QTOF MS experiments were conducted within a day to study the reproducibility of the method on a run-to-run basis, and a variation of ±0.1-0.3 min in the retention time of the GSL species has been deduced from the TIC MS profile, while in the same range, i.e., ±0.1-0.3 min lies the retention time variation on a month-to-month basis. RESULTS AND DISCUSSION Nano-HPLC/ESI QTOF MS and Tandem MS Analysis of GSLs. The MS detection and structural characterization of individual neutral GSLs in mixtures of biological origin at the low picomole or femtomole level is drastically jeopardized by the presence in the extract of glycerol-based neutral lipids and phospholipids, as readily observed in a direct MS screening of (30) Domon, B.; Costello, C. E. Glycoconjugate J. 1988, 287, 253–257. (31) Chester, M. A. Glycoconjugate J. 1999, 16, 1–6.

the purified GSL mixture (data not shown). Besides, fatty acids resulting after an enzymatic/alkaline treatment of a GSL-containing mixture, which would cover lower m/z range in MS, might reaggregate in solution to larger sizes, thereby still affecting a direct MS detection of neutral GSL components.32 These aspects reasoned most probably the use of relatively high amounts of GSL samples for direct MS profiling and structural determination, typically in the range of 10-50 pmol/µL, or analysis of individual TLC bands.24,33 A considerable gain in sensitivity of the direct MS screening was obtained in our study by nanochip-based infusion ESI/QTOF MS and MS/MS, where for 1 pmol/µL an in-depth representation of mixture heterogeneity and the structural characterization by fragmentation study could be achieved (data not shown). Still interference of products other than GSLs does occur, compromising the structure identification of minor species in particular. Thus, the requirement for a sensitive and efficient separation tool on-line coupled with MS becomes evident. The well-established HPTLC technique in conjunction with orcinol and immunostaining for structural characterization of GSLs is partially informative with respect to mixture composition, i.e., heterogeneity within the ceramide portions of components, and frequently requires several subfractionation steps. Although the HPTLC data can be complemented by MS analysis, this is usually accomplished by removal and subsequent analysis of individual TLC bands.24,33,34 The overall analytical flow of such an investigation is, therefore, time-consuming and less sensitive. From this perspective, the development of a reliable on-line nano-HPLC/MS protocol for qualitative analysis of GSLs would address a majority of these aspects by providing a nanoscale fractionation and structural identification of components by fragmentation within single experiments. The GSL mixture selected for investigation in this study contains basically monohexosylceraminde (Glc/Galβ1-1Cer or MHC), lactosylceramide (Galβ1-4Glcβ1-1Cer or LacCer or Lc2Cer), and globo-series GSLs: globotriaosylceramide (GalR14Galβ1-4Glcβ1-1Cer or Gb3Cer) and globotetraosylceramide (GalNAcβ1-3GalR1-4Galβ1-4 Glcβ1-1Cer or Gb4Cer). In Figure 1 the orcinol-stained thin-layer chromatogram of the purified GSL mixture is displayed, where the four expected components are clearly identified. A densitometric estimation of the orcinolstained HPTLC bands roughly indicates that Gb4Cer accounts for 61%, Gb3Cer for 26%, LacCer for 11%, and MHC about 2% of the total mixture, if considering that MHC, LacCer, Gb3Cer, and Gb4Cer account together for 100% (data not shown). This evaluation concurs with our quantitative data obtained for a similarly prepared GSL mixture from human erythrocytes,35 as well as with the estimation of other groups.36 A number of additional TLC bands (Figure 1) related to species exhibiting slower migration properties are visible as well and are assumed to correspond to GSLs species with larger carbohydrate chains than Gb4Cer, most probably consistent with the AB blood group associated GSL species. The identification of these components (32) Han, X. Front. Biosci. 2007, 12, 2601–2615. (33) Metelmann, W.; Peter-Katalinic´, J.; Mu ¨ thing, J. J. Am. Soc. Mass Spectrom. 2001, 12, 964–973. (34) White, T.; Bursten, S.; Federighi, D.; Lewis, R. A.; Nudelman, E. Anal. Biochem. 1998, 258, 109–117. (35) Meisen, I.; Friedrich, A. W.; Karch, H.; Witting, U.; Peter-Katalinic´, J.; Mu ¨ thing, J. Rapid Commun. Mass Spectrom. 2005, 19, 3659–3665. (36) Suzuki, A.; Kundu, S. K. J. Lipid Res. 1980, 21, 473–477.

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Figure 1. Orcinol stain of HPTLC-separated neutral GSLs from human erythrocytes. The amount of 5 µg of GSL mixture was applied and separated in chloroform/methanol/water (120/70/17, v/v/v).

in human erythrocytes by HPTLC followed by immunostaining and MS-based techniques is the subject of an ongoing project. To establish a nano-HPLC/ESI QTOF MS and tandem MS protocol for GSL profiling and sequencing, the choice of the amido-phase chromatographic column was guided by the basic fact that the separation criterion resides primarily on the hydrogen bonding between available hydroxyl groups of the monosaccharides and amido groups of the stationary phase.37,38 Though a similar retention mechanism is provided by amino-phase columns it is apparent that amido columns essentially exhibit better stability and deliver lower background chemical noise, which certainly increase sensitivity of MS detection.38 The separation capability of amido columns for carbohydrate analysis was already proved on analytical ones, but in an MS-hyphenated mode one publication39 describes its application for characterization of an N-glycan mixture, enzymatically released from glycoproteins, and most recently two chromatographic methods were successfully developed and applied for glycosaminoglycans investigation.40,41 Accordingly, in the present study a separation of the individual GSL species on the basis of their hydrophilic chain length was expected on one hand, and a separation of lipids and fatty acids from the GSL content on the other. However, for LC/MS analysis of GSLs the most critical parameter to compel with is the choice of the LC solvent system. The opposing character of the hydrophilic carbohydrate chain and hydrophobic nature of ceramide moiety invariably adds the need for a very fine balance between the proportions of organic solvents (37) D’Amboise, M.; Noel, D.; Hanai, T. Carbohydr. Res. 1980, 79, 1–10. (38) Churms, S. C. In Carbohydrate Analysis by Modern Chromatography and Electrophoresis; El Rassi, Z., Ed.; Elsevier Science: Amsterdam, 2002; pp 121-163. (39) Wuhrer, M.; Koeleman, C. A. M.; Deelder, A. M.; Hokke, C. H. Anal. Chem. 2004, 76, 833–838. (40) Naimy, H.; Leymarie, N.; Bowman, M.; Zaia, J. Biochemistry 2008, 47, 3155– 3161. (41) Hitchcock, A.; Costello, C.; Zaia, J. Proteomics 2008, 8, 1384–1397.

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to concomitantly meet three criteria: efficient solubilization, high resolving power chromatographic separation, and promotion of efficient ionization.42 Applying the first nano-HPLC/ESI QTOF MS method, involving a solvent system composed of acetonitrile, water, formic acid, and ammonium formate (cf., Materials and Methods) for the human erythrocytes-GSL mixture, a good separation of the LacCer from Gb3Cer and Gb4Cer species, e.g., about 4 min difference in retention time (data not shown), accompanied by a considerably high ionic yield and signal-to-noise (S/N) ratio of the species was achieved. This demonstrates a fairly selective hydrogen bonding between hydroxyl groups of GSL-oligosaccharides and the amido groups of the stationary phase. However, this is valid only for GSL species differing by one Hex residue like LacCer and Gb3Cer. The Gb3Cer and Gb4Cer were not resolved under these solvent/gradient conditions. Instead, a significantly high separation efficiency of the saturated from unsaturated ceramide/GSL species was accomplished as documented in Figure 2A, where the extracted ion chromatogram effected for the ions at m/z 1338.5 and 1340.5, along with the corresponding spectra, are displayed. Gb3Cer and Gb4Cer species of various ceramide portions, but exhibiting identical degree of unsaturation, i.e., one double bond, eluted at min 25 (Figure 2A, upper panel), while their homologous species in their saturated form were detected at min 70 after signal acquisition (Figure 2A, lower panel). At min 9, formally overlapping isobaric species related to lipidlike products were exclusively detected, as also supported by tandem MS/MS fragmentation where no diagnostic ions for the presence of glycolipids were evidenced (data not shown). The ions at m/z 1134.86 related to Gb3Cer(d18:1/24:1) coeluted together with the ions at m/z 1337.90 at min 25 and were separated from the coeluting ions at m/z 1136.88 and 1339.92 assigned to Gb3Cer(d18:1/24:0) and Gb4Cer(d18:1/24:0) at min 70. Irrespective of any further adjustment of the method in terms of gradient and solvent composition the resolving power of Gb3Cer from Gb4Cer species could not be improved. This chromatographic behavior directly implies that the difference in polarity induced by the presence of the double bond within the ceramide chain accounts for a higher selectivity on the amido-phase column than that exhibited by a HexNAc-differing species, as Gb3Cer and Gb4Cer. Due to the high polarity of the solvent system used for this method, this observation is at the first glance surprising. However, we hypothesize that the presence of water in the solvent system renders a hydrophobic effect, which minimizes the contact between hydrocarbon parts with the aqueous phase, exposing thus the unpolar site of GSLs to the stationary phase. In this model, secondary interactions with the carbamoyl groups of the stationary phase are more pertinent to consider as the basis of affinity. Similar evidence was reported on various solid phases like aminopropyl-modified silica, where seemingly three types of interactions can occur: as primary interaction is the hydrogen bonding of the analyte with the amino groups, hydrophobic interactions of the analyte with an acyl chain as well as with the accessible OH groups of the silanol.43 From the spectra in Figure 2A the level of unsaturation characterizing the Gb3Cer and Gb4Cer species can be clearly assessed by molecular mass (42) Jemal, M. Biomed. Chromatogr. 2000, 14, 422–429. (43) Moors, M.; Massart, D. L.; McDowall, R. D. Pure Appl. Chem. 1994, 66, 277–304.

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Figure 2. (A) Extracted ion chromatogram for the ions at m/z 1338.5 and 1340.5 effected within a 0.5 amu window and the corresponding (+)-nano-HPLC/ESI QTOF mass spectra derived by combining across the TIC MS peak at min 25 min (upper panel) and 70 min (lower panel) after MS signal acquisition. Inset in upper panel: list of GSL species eluting at min 25 and their corresponding assignment showing mainly unsaturated species. Inset in lower panel: list of GSL species eluting at min 70 and their corresponding assignment showing mainly saturated species. The nano-HPLC chromatographic method 1 was used (cf., Materials and Methods). Electrospray potential, 1900 V; sampling cone voltage, 75 V. (+)-Nano-HPLC/ESI QTOF DDA MS2 of (B) Gb4Cer(d18:1/24:1) at m/z 1337.90 and (C) Gb4Cer(d18:1/24:0) at m/z 1339.92. Collision energy was set to 65 eV for Gb4Cer species and 50 eV for Gb3Cer (cf., the Supporting Information), respectively; collision gas pressure, 15 psi; sample injection, 2 pmol of GSL mixture.

calculation (cf., the table in Figure 2A); however, the location of the double bond within the ceramide portion is not accessible. Though not typical for this GSL species, additional double bonds can be located either in the sphingosine part or in the fatty acid chain. Therefore, an on-line nano-HPLC/ESI QTOF DDA fragmentation analysis was carried out for the ion species corresponding to Gb3Cer and Gb4Cer each with (d18:1/24:1) and (d18:1/ 24:0) ceramide in their composition, to derive diagnostic ions for location of additional double bond within the ceramide portion. In Figure 2, parts B and C, the corresponding fragmentation spectra of the Gb4Cer species with (d18:1/24:0) eluting at min 25 (Figure 2A) and Gb4Cer with (d18:1/24:1) eluting at min 70 (Figure 2A) are depicted. The successive cleavage of the glycosidic bonds of the precursor ions, represented by the Z3-, Z2-, Z1-, and Z0-type ions along with their counterparts, B1, B2, and B3 ions 4716

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unambiguously define the Gb4 oligosaccharide chain of both Gb4Cer species. The Z0-type fragment ions at m/z 630.63 (Figure 2B) and 632.22 (Figure 2C) corresponding to Cer(d18:1/24:1) and Cer(d18:1/24:0), respectively, directly report on the length and overall unsaturation degree of the lipid anchor. Moreover, the fragment ions retaining the reducing end that arise from glycosidic bond cleavages, i.e., Z3-, Z2-, Z1-, and Z0-like ions detected in the spectra of both Gb4Cer species (Figure 2, parts B and C) differ by 2 amu between the two Gb4Cer isoforms, consistent for a double bond, therefore further indicating the different degree of unsaturation. The reporter ions for location of the additional double bond in the Gb4Cer species (Figure 2B) are the fragment ions at m/z 264.27 (N′′), arising from sphingosine moiety of d18:1 type. The presence of two double bonds in the sphingosine moiety would give rise to formation of N′′ fragment ions of a nominal

m/z value of 262. Yet, no traces of such fragment ions could be detected under any experimental circumstances, indicating that the additional double bond must necessarily reside within the fatty acid chain. Similarly, accurate data sets for identification of Gb3Cer species differing in their ceramide portions by one double bond were obtained by fragmentation analysis, where, again, the diagnostic ions at m/z 264.27 (N′′) as well as m/z 282.29 (N′) precisely attest the unsaturation of the fatty acid chain in the Gb3Cer, confirming the validity of the ceramide assignment as (d18:1/24:1) (cf., Supporting Information). By this method, besides the dominating C16 and C24 fatty acid chain, the presence of Gb3Cer and Gb4Cer species with C22 was clearly evidenced, whereas an unsaturation level from 0 to 3 in their fatty acid chain were here detected, substantially supplementing the data obtained by HPTLC (Figure 1) and direct MS screening (data not shown). The chain length and unsaturation degree of the lipids exert a direct influence on their spatial organization, defining the thickness and ordering of the hydrophobic region of the cell membrane. It has been recently recognized that structural differences in the microdomain lipids, both the lipids facing the cytoplasm and the glycosylated ones exposed to extracellular environment, act in concert with membrane deformation to modulate the activity of the transmembrane channels and peripheral membrane binding proteins.44 In this context, the nano-HPLC/ESI QTOF MS method proposed here is highlighted as well suited for gaining insights into the dynamics of the cell membrane by accurate dissection of the unsaturation degree and occurrence within the ceramide moiety of the GSL species. To explore the potential of the nano-HPLC/ESI QTOF MS technique for resolving/detection of all the individual GSLs on the basis of their carbohydrate chain length a different analytical approach has been designed. Basically, a nonaqueous solvent system was expected to prevent the hydrophobic effect and, thus, the secondary interactions of the GSLs with acyl chains of the stationary phase. The second solvent system was adjusted to contain acetonitrile, methanol, acetic acid, and ammonium acetate (cf., Materials and Methods). Various proportions of the organic solvents and several gradients were tested. The best results in terms of separation of GSLs according to the length of the hydrophilic chain were obtained when increasing the methanol ratio over a linear gradient from 0.2% to 99.9% as described above (see Materials and Methods). The base peak chromatogram obtained by nano-HPLC/ESI QTOF MS analysis of GSL mixture, displayed in Figure 3A asserts already a high separation efficiency and resolving power of the three GSL species, LacCer, Gb3Cer, and Gb4Cer, by the corresponding differences in retention times, ∼4.50 min between LacCer and Gb3Cer and ∼1.8 min between Gb3Cer and Gb4Cer. The narrower time window between Gb3Cer and Gb4Cer as compared to the one between LacCer and Gb3Cer might be attributable to the lower number of OH- groups available on a HexNAc residue than on a Hex one. Thus, elongation of the sugar chain by a hexose apparently increases the affinity to the stationary phase much more efficiently than the addition of an N-acetylated aminohexose. A trace of MHC could not be detected at these operating parameters, reasoned (44) Janmey, P. A.; Kinnunen, P. K. J. Trends Cell Biol. 2006, 16, 539–546.

by its extremely low content in the total mixture, i.e., less than 2%, which would correspond to a total of 10 fmol of MHC. A similar rationalization applies for the TLC bands in Figure 1 that correspond to polyglycosylceramides. Due to the longer carbohydrate chains, they exhibit darker bands than MHC species, but concurrently lower molar amounts. These minor species appear to be present in the GSL mixture analyzed by nano-HPLC/ESI QTOF MS most probably in the attomole range, therefore beyond the limit of detection of this method in particular and MS of carbohydrates in general. On the other hand, the extraction procedure used in this study was intended for the GSL species with shorter carbohydrate chains, i.e., up to Gb4Cer species, and thus, the abundance of the polyglycosylceramides deducible from the TLC analysis (Figure 1) is lower than could be actually calculated. Unfortunately, there are presently no established protocols for extraction of entire short GSLs and polyglycosylceramides in a single step. Rather, appropriate sequential procedures have to be applied specifically with respect to the polarity of GSLs. Such a sample preparation protocol45 will be addressed in future studies for nano-HPLC ESI QTOF MS characterization of polyglycosylceramides. Alternatively, the detection limit of the nanoHPLC/ESI QTOF MS method might be further improved to accomplish MHC and polyglycosylceramides detection by using lower i.d. electrospray emitters or low-flow chip-ESI. The spectra resulting by combining across the individual peaks corresponding to LacCer, Gb3Cer, and Gb4Cer are depicted in Figure 3B-D, respectively. As visible here, the optimized method gives rise to not only an accurate separation of individual GSL species but, concurrently, to their fairly high ionization efficiency and S/N ratio of detection. The MS pattern of individual chromatographic peaks (see also the inset in Figure 2A) are in agreement with the HPTLC data in what regards the distribution and relative abundances of the fatty acid chains within the GSL species: for LacCer the dominant species is the one containing a C16 chain at m/z 884.58 (Figure 3B), whereas for Gb3Cer and Gb4Cer the C24 chain is predominant as revealed by the ions at m/z 1158.78 (Figure 3C) and 1361.83 (Figure 3D), respectively. Mentionable here is that a similar variety in the unsaturation degree as well as in ceramide length with Gb3Cer and Gb4Cer (Figure 2A) was not observed for LacCer species, where almost exclusively fatty acid chains of 16:0 and 24:0 were detected. Only the C24 chain with one double bond was visible at m/z 994.75 (Figure 3B), whereas as a minor species the LacCer(d18:1/26:0) could be observed. This solvent system is seemingly promoting superior ionization efficiency and sensitivity of detection than the aqueous solvent system employed previously (Figure 2A). This is attested by the higher heterogeneity revealed in the spectra in Figure 3B-D to include the detection of the homologous LacCer, Gb3Cer, and Gb4Cer containing a C26 fatty acid chain in their ceramide moiety. It is in this context also important to mention that a prevalent ionization of sodiated species over the protonated ones, as compared to Figure 2, is observed and can be explained by the higher pKa of the acetic acid versus formic acid used (cf., Materials and Methods). An interesting feature arising from the data in Figure 3 is the existence of baseline-separated peaks (45) Hanfland, P.; Kordowicz, M.; Niermann, H.; Egge, H.; Dabrowski, U.; PeterKatalinic´, J.; Dabrowski, J. Eur. J. Biochem. 1984, 145, 531–542.

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Figure 3 4718

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Figure 3. (A) (+)-Nano-HPLC/ESI QTOF BPI MS acquired by use of chromtographic method 2 (cf., Materials and Methods). (+)-Nano-HPLC/ ESI QTOF mass spectra corresponding to the BPI peaks of (B) LacCer at min 33, (C) Gb3Cer at min 38, and (D) Gb4Cer at min 39. ESI potential, 1900 V; sampling cone potential, 70 V; sample injection, 2 pmol of GSL mixture.

corresponding to LacCer (Figure 3A) and Gb3Cer (Figure 3, parts C and D). Though isomeric species of LacCer and Gb3Cer are known, there is no biological evidence to assign these double peaks to any isomeric species. Rather, as also observed by Costello’s group, this peculiarity is attributable to an incomplete protonation/cationization of the neutral GSL species,26 resulting in a mixture of protonated, sodiated, and neutral species already present in solution, giving rise to a differential retention time on the column. This concept is also supported by the difference in ionic yield of the protonated versus sodiated LacCer and Gb3Cer species between the baseline-separated double peaks. The protonated LacCer species at m/z 862.69 was exclusively detected in the first BPI peak at min 33.58, whereas in Figure 3D mainly protonated Gb3Cer species were detected as compared to Figure 3C. The ion species assigned to dehydrated LacCer, Gb3Cer, and Gb4Cer in Figure 3B-D, respectively, as well as in Figure 2A (cf., also the inset table) are considered to derive exclusively insource by neutral loss of one water molecule from their corresponding precursors and not be originally present in the mixture. This premise is further supported by the fact that dehydrated GSL species were observed only to accompany their protonated forms, whereas no loss of water was observed from the sodiated ones. Additionally, attempts to decrease the yield of in-source decomposition by applying lower values for the sampling cone voltage resulted in the considerable decrease of the dehydrated species abundance in mass spectra. Apart from the separation efficiency of individual GSL species enabled by the optimized nano-HPLC chromatographic method, the GSL species were well-resolved from any lipid or lipid derivatives content, as compared to Figure 2A, where ion species in the range of m/z 1190-1240 designated to lipid-like species are visible. Any of such products eluted by the use of the second chromatographic method between min 5 and 23 so that major but also minor GSL species could be accurately distinguished from the mixture. The structural elucidation of the molecular structures of major GSL species was conducted by nano-HPLC/ESI QTOF MS2 in

DDA fashion (Figure 4A). The ions at m/z 884.66, 1158.78, and 1361.83 corresponding to LacCer(d18:1/16:0), Gb3Cer(d18:1/24: 0), and Gb4Cer(d18:1/24:0), respectively, were selected for automatic switching MS/MS, and the resulting TIC MS/MS chromatogram is depicted in Figure 4A. The spectrum obtained by combining across the TIC MS/MS peaks corresponding to LacCer species at m/z 884.58 (Figure 4B) displays as the major fragmentation products the ions corresponding to glycosidic bond cleavages. The fragment ions at m/z 722.54 (Y1) and 560.48 (Y0) along with their counterparts B1 (m/z 185.02) and B2 (m/z 347.08) directly evidence the lactosyl moiety as the core disaccharide attached to the ceramide portion. The type of the ceramide is readily deducible from the ions at m/z 560.48 (Y0) consistent with (d18:1/16:0) and its dehydrated form at m/z 542.50 (Z0), whereas the ions at m/z 264.27 clearly define the sphingosine moiety of d18:1 type. In addition, a ring cleavage ion occurring within the inner Glc residue at the 0 and 2 linkages and preserving the nonreducing end was detected at m/z 305.07 (0,2A2). Similar fragmentation patterns, concerning the complete coverage of the glycosidic bond linkages, were obtained for the ions at m/z 1158.78 and 1361.83 corresponding to Gb3Cer(d18:1/24:0) and Gb4Cer(d18: 1/24.0), respectively. The series of Y2-, Y1-, and Y0-type ions detected in Figure 4C and Y3 to Y0 in Figure 4D, along with their counterpart ions of B type, unambiguously confirms the glycan chain composition of Gb3 (Figure 4C) and Gb4 (Figure 4D) type, respectively. The evidence on the sphingosine was obtained as well for both Gb3Cer and Gb4Cer species through the ions at nominal m/z 264 (N′′) detected at high S/N and abundance (Figure 4, parts C and D). It is apparent that the extent of ring cleavage formation is rather increasing with the size of the sugar chain. For Gb3Cer(d18:1/24:0) species two fragment ions arising from cleavage of the ring bonds within both terminal and subterminal monosaccharides were detected and assigned to 0,2X2 and 0,2X1 (Figure 4C). Fragmentation of Gb4Cer(d18:1/24:0) species gave rise to four ring cleavages, represented by the ions at m/z 245.15, 876.94, 1038.85, and 1212.51 attributable to 2,4A3/ B1-, 0,2X1-, 0,2X2-, and 1,4X3-type ions, respectively. Besides, for Analytical Chemistry, Vol. 80, No. 12, June 15, 2008

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Figure 4 4720

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Figure 4. (A) (+)-Nano-HPLC/ESI QTOF TIC MS/MS obtained by the use of the chromatographic method 2 (cf., Materials and Methods). (+)-Nano-HPLC/ESI QTOF MS/MS showing the fragmentation pattern of the ions at (B) m/z 884.58 attributed to LacCer(d18:1/16:0), (C) m/z 1158.78 related to Gb3Cer(d18:1/24:0), and (D) m/z 1361.88 assigned to Gb4Cer(d18:1/24:0). ESI conditions as in Figure 3. Collision gas pressure, 15 psi; collision energy, 50 eV for LacCer(d18:1/16:0), 65 eV for Gb3Cer(d18:1/24:0), and 75 eV for Gb4Cer(d18:1/24:0). Analytical Chemistry, Vol. 80, No. 12, June 15, 2008

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Gb3Cer(d18:1/24:0) and Gb4Cer(d18:1/24:0) species the ring cleavages occur within all monosaccharide residues except for the Glc residue proximal to the Cer moiety, in contrast to LacCer, where the immediate formation of ring cleavage ions arising from the core Glc was observed. These features might suggest that, in the case of linear GSLs, the cleavage of the ring bonds does not preferentially occur within the core residues, as in the case of other branched sugars,46 but rather appears within the extended glycan chain. Such an extensive and accurate fragmentation of the GSL species including a significant number of ring cleavage ions was only obtained by the nano-HPLC/ESI QTOF DDA method but could not be obtained by direct MS screening and subsequent fragmentation. A certain level of ions arising from an overlapping isobaric species of lipid type readily occurs in a direct admission, hindering the detection of minor but relevant fragment ions. This feature underlines the requirement for an efficient separation of GSL from the accompanying lipid-like products in a mixture on one hand, and validates the amenability of the proposed method for detailed heterogeneity and structural elucidation of biologically obtained GSL mixture, on the other. The presence of other components like Lc3Cer and nLc4Cer was not evidenced by on-line tandem MS experiments, even under consideration of their specific fragment ions that can be generated with the same efficiency as for Gb3Cer and Gb4Cer. Moreover, in a direct screening ESI QTOF MS and MS/MS analysis of sample concentration up to 10 pmol/µL (data not shown) the existence of such components was not deducible. This is attributable to the fact that the ratio of Gb4Cer and nLc4Cer abundance is about 103-104, calculated by densitometric analysis of HPTLC immunostained bands (data not shown), whereas the Lc3Cer is of even lower abundance than nLc4Cer. If roughly calculating that Gb4Cer species in Figure 3D correspond to approximately 1.2 pmol according to the quantitative estimation of the Gb4Cer in the 1 µL volume of 2 pmol/µL analyzed sample (see Materials and Methods), nLc4Cer abundance would be in the range of high attomole to very low femtomole. If further taking into account also the distribution of the nLc4Cer species over several homologous structures with different ceramide lengths and/or species exhibiting different cation adducts, the abundance of a single nLc4Cer homologue would roughly lie in the range of the low attomole level, which is far beyond the limit of detection of the nano-HPLC/ESI QTOF MS method and of MS of carbohydrates in general. Considering that 2 pmol of total mixture was injected into the column and the dynamic range proportions of the mixture it can be deduced that Gb4Cer accounts for about 1.2 pmol, which distributed over several isoforms (Figure 3D) results roughly in less than 200 fmol of detection per component, whereas in case of Gb3Cer and LacCer the detection limit for individual species was well below 50 fmol. Additionally, a number of additional structural details to confirm the identity of the species could be gathered by this high-sensitivity and high-efficiency fragmentation

study. Essentially, the two proposed nano-HPLC/ESI QTOF MS and MS/MS approaches are complementary, providing in concert extensive insights in the human erythrocytes GSL mixture: (a) detailed information on the ceramide heterogeneity occurring at single GSL glycan chains, (b) curative distinction of the GSL species from their lipid-containing environment, (c) detailed structural characteristics for the GSL glycan chain elucidation, (d) type of sphingosine, (e) the dissection of the unsaturation degree, and (f) residence of the additional double bonds within the fatty acid chain of the ceramide. While the applicability of the nano-HPLC/ESI QTOF MS and tandem MS method is demonstrated here for neutral GSL species containing up to four monosaccharide units, its feasibility for screening and sequencing of sialylated GSLs was documented in a recent study.47 Additionally, the potential for analysis of polyglucosyl GSL species is envisaged, if preceded by a specifically designed sample preparation protocol. The development of a unified protocol for efficient quantitative and qualitative extraction of all GSL species in a biological matrix is considered the first requisite for the implementation of the nano-HPLC/ESI MS approach for achieving a complete profile of GSLs in any given biological system in single experiments.

(46) Cancilla, M. T.; Penn, S. G.; Carroll, J. A.; Lebrilla, C. B. J. Am. Chem. Soc. 1996, 118, 6736–6745.

(47) Zarei, M.; Kirsch, S.; Mu ¨ thing, J.; Bindila, L.; Peter-Katalinic´, J. Anal. Bioanal. Chem. 2008, 391, 289–297.

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CONCLUSIONS A robust and reliable method for separation and structural characterization of complex mixtures of GSLs of biological origin at the nanoscale level involving hyphenated nano-HPLC and ESI MS was developed. Superior separation of individual neutral GSL species and of formally interfering lipid and fatty acid traces combined with efficient ionization/detection and fragmentation could be achieved by nano-HPLC/ESI QTOF MS and MS/MS within single experiments. The down-scaling of the overall analysis time on-line nano-HPLC/ESI QTOF MS to less than 40 min in combination with improving the analysis performances to the low femtomole level renders a fast, sensitive, and high-throughput amenable analytical approach for GSL analysis and a new analytical dimension in glycolipidomics. ACKNOWLEDGMENT We thank Ewald Kalthoff for expert technical assistance and Petra Hoffmann for densitometric quantification of GSLs. The study was financially supported by the European Union GLYFDIS Project No. LSHB-CT-2006-037661. The first two authors contributed equally to this study. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review October 22, 2007. Accepted April 15, 2008. AC702175F