Progress in Detection and Structural Characterization of

Article pubs.acs.org/ac

Progress in Detection and Structural Characterization of Glycosphingolipids in Crude Lipid Extracts by Enzymatic Phospholipid Disintegration Combined with Thin-Layer Chromatography Immunodetection and IR-MALDI Mass Spectrometry Ivan U. Kouzel, Alexander Pirkl, Gottfried Pohlentz, Jens Soltwisch, Klaus Dreisewerd, Helge Karch, and Johannes Müthing* Institute for Hygiene, University of Münster, Robert-Koch-Strasse 41, D-48149 Münster, Germany S Supporting Information *

ABSTRACT: In order to proceed in detection and structural analysis of glycosphingolipids (GSLs) in crude lipid extracts, which still remains a challenge in glycosphingolipidomics, we developed a strategy to structurally characterize neutral GSLs in total lipid extracts prepared from in vitro propagated human monocytic THP-1 cells, which were used as a model cell line. The procedure divides into (1) extraction of total lipids from cellular material, (2) enzymatical disintegration of phospholipids by treatment of the crude lipid extract with phospholipase C, (3) subsequent multiple thin-layer chromatography (TLC) overlay detection of individual GSLs with a mixture of various anti-GSL antibodies, and (4) structural analysis of immunostained GSLs directly on the TLC plate using infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry (IR-MALDI-o-TOF MS) in combination with collision-induced dissociation (CID). Whereas GSLs were mostly undetectable in untreated crude lipid extracts, pretreatment with phospholipase C resulted in clear-cut mass spectra. MS1 and MS2 analysis gave similar results when compared to those obtained with a highly purified neutral GSL preparation of THP-1 cells, which served as a control. We could demonstrate in this study the feasibility of simultaneous multiple immunodetection of individual neutral GSLs in one and the same TLC run and their structural characterization in crude lipid extracts after phospholipase C treatment, thereby avoiding laborious and long-lasting sample purification. This powerful combinatorial technique allows for efficient structural characterization of GSLs in small tissue samples and takes a step forward in the emerging field of glycosphingolipidomics.

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Although the neutral GSL lactosylceramide (Lc2Cer) plays a key role in the biosynthesis of all core structure families, its cellular function has begun to emerge only recently.7,10 The neutral GSL globotriaosylceramide (Gb3Cer) became prominent due to its accumulation in Fabry disease, a lysosomal GSL storage disease caused by defective α-galactosidase A,11,12 and as tumor-associated GSL in various types of cancer such as colon and pancreatic malignancies.13−15 Gb3Cer and globotetraosylceramide (Gb4Cer) represent the highly and less effective receptors of Shiga toxins,16 the major toxins of enterohemorrhagic Escherichia coli (EHEC). EHEC are greatly feared pathogens as documented by the 2011 German outbreak caused by an EHEC strain of O104:H4 serotype.17−19 Human endothelial cells of the kidney and the brain predominantly

lycosphingolipids (GSLs) are built up by a hydrophilic glycan chain, which is attached to a hydrophobic Nacylated sphingoid base termed ceramide, the latter anchoring the amphiphilic molecule in the outer leaflet of the plasma membrane.1,2 GSLs play a key role in membrane lateral organization and are integral parts of membrane microdomains as highly ordered supramolecular structures referred to as “lipid rafts”.3,4 GSLs are not just structural elements of the “fluid mosaic” of cell membrane lipids,5 but also participate in transmembrane signaling despite restriction to the outer bilayer leaflet.6 The glycan headgroups act as primary receptors for carbohydrate binding proteins, while the different lipoforms of GSLs may play a significant, but still largely unknown, functional role.7 Mammalian GSLs can be categorized into neutral and charged GSLs, which in turn can be largely subcategorized into one of the most prominent core structure families referred to as ganglio-, globo-, isoglobo-, neolacto-, and lacto-series.8,9 © 2013 American Chemical Society

Received: October 28, 2013 Accepted: December 19, 2013 Published: December 19, 2013 1215

dx.doi.org/10.1021/ac4035696 | Anal. Chem. 2014, 86, 1215−1222

Analytical Chemistry

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as phospholipids and triglycerides) were saponified by alkaline treatment. Neutral GSLs were isolated by anion-exchange chromatography, followed by silica gel column chromatography.41 Neutral GSLs were further purified as peracetylated derivatives on a Florisil column according to Saito and Hakomori42 as described in detail by Li and co-workers.43,44 This preparation represents the control of highly purified neutral GSLs, and the term “purified neutral GSL fraction” is used for it throughout this paper. The nomenclature follows the IUPAC-IUB recommendations,45 and the symbolic representation system according to Varki46 and the Consortium for Functional Glycomics47 is used for illustration of GSL structures. Preparation of Crude Lipid Extract and Phospholipase C Treatment. Total lipids from THP-1 cells were consecutively extracted with methanol and chloroform/ methanol mixtures with increasing ratios of chloroform as described in the previous paragraph. The extracts were pooled, dried in vacuo, and dissolved in chloroform/methanol (2/1, v/ v). This preparation is referred to as “crude lipid extract”, and aliquots equivalent to 2 × 106 cells were employed for TLC immunostaining. Aliquots of the crude lipid extract, corresponding to 4 × 106 cells, were dried under a stream of nitrogen and solubilized under short sonication in 400 μL of ultrapure water. A volume of 150 μL of Tris buffer (40 mM Tris, 40 mM CaCl2, pH 7.5) was added to the aqueous solution, and the enzyme reaction was started by adding of 2.5 U of phospholipase C (PLC) from Bacillus cereus (Sigma-Aldrich, Steinheim, Germany; cat. no. P6621). After incubation for 16 h at 37 °C, salts were removed by dialysis against deionized water at 4 °C for 3 days. After lyophilization the sample was solubilized under short sonication in 100 μL of chloroform/methanol (2/1, v/v), and 50 μL (equivalent to 2 × 106 cells) of this “phospholipase-treated crude lipid extract” (as named throughout the following text) was applied for TLC immunostaining. Primary Antibodies, Antibody Mixture, and Secondary Antibody. Polyclonal chicken antibodies against Lc2Cer, Gb3Cer, Gb4Cer, and neolactotetraosylceramide (nLc4Cer) were used for TLC immunostaining. Their specificities have been demonstrated in previous publications.15,48−50 Primary antibodies were used in TLC overlay assays for single detection of respective GSL. As a novelty of this study, we prepared an antibody mixture composed of identical aliquots of the four mentioned antibodies and used this antibody cocktail in the same way like the individual single antibodies. Alkaline phosphatase (AP)-conjugated rabbit antichicken IgY antibody (Dianova, Hamburg, Germany; code 303-055-033) served as secondary antibody for both single and multiple GSL detection. High-Performance Thin-Layer Chromatography Immunostaining. The TLC immunostaining procedure has been described previously in detail.51,52 Briefly, after chromatography the silica gel of TLC plates was fixed with poly(isobutyl methacrylate) (Plexigum P28; Röhm, Darmstadt, Germany) to prevent detachment of the silica gel layer. Single primary antiGSL antibodies and the antibody mixture as well as secondary AP-labeled antibodies were used in 1:2000 dilutions. Bound antibodies were visualized by color development using 0.05% (w/v) 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (BCIP; Roth, Karlsruhe, Germany) in glycine buffer. Details about standard TLC are provided in the Supporting Information.

express globo-series neutral GSLs and are preferentially targeted by Shiga toxin. GSL-mediated binding and internalization of the toxin result in endothelial cell damage20,21 followed by severe extraintestinal complications in vivo and, finally, at its worst, in death. Recent advances in the analysis of the mammalian sphingolipidome by liquid chromatography tandem mass spectrometry show that eukaryotic cells contain hundreds of sphingolipids and sphingolipid metabolites.22 The compounds that can be readily analyzed are free long-chain (sphingoid) bases, sphingoid base 1-phosphates, and more complex species such as ceramides, ceramide 1-phosphates, sphingomyelins, mono- and dihexosylceramides, and sulfatides.23 The analysis of GSLs with longer oligosaccharide chains seems difficult to accomplish within sphingolipidomic approaches. Moreover, the determination of linkage positions of sugars and their anomeric configuration as well as the conformation of sugar rings of the oligosaccharide moieties of GSLs remains challenging for future (glyco)sphingolipidomics.2,8 Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) can be applied to all known lipid classes for determination of lipid structural characteristics from cells, tissues, and body fluids, and analysis of their lipidomes, although different lipid classes are detected with different sensitivities.24−26 Though thin-layer chromatography (TLC) is a widely employed technique for lipid analysis, MS profiling is indispensable for gaining structural details about the various molecular species within a lipid class.27,28 TLC is a robust method for separating complex lipid mixtures, and the combination between TLC and MALDI MS offers a powerful tool to investigate the identity of TLC spots in exceptional detail. Combining TLC with MALDI MS allows the spatially resolved detection of lipids directly on a TLC plate with a higher sensitivity than common staining protocols.29−31 The analytical potential of TLC separation of GSLs combined with MALDI MS has been shown by us32 and others.33−35 This strategy has been subsequently improved by combining TLC immunodetection (using specific anti-GSL antibodies, which define the glyco-epitope) with MALDI MS.36−38 Since GSLs are in most tissues minor components of cellular membranes, their detection by MS techniques in crude lipid extracts of cells, tissues, or organs is still a challenge. Low ionization of minor GSLs compared to highly abundant phospholipids and other lipids might be the reason for failure in their detection. To this end, we developed a strategy based on pretreatment of crude cellular lipid extracts with phospholipase C for disintegration of phospholipids using human monocytic THP-1 cells as model cell line. This sample pretreatment resulted in enormously enhanced detection efficiency of GSLs and allowed for their structural assignment by infrared (IR)-MALDI MS1 and MS2 directly on the TLC plate combined with preceding glyco-epitope identification of individual GSLs using a cocktail of various anti-GSL reactive antibodies.

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EXPERIMENTAL SECTION Preparation of Highly Purified Neutral GSLs. Neutral GSLs were purified from THP-1 cells as previously described.39,40 For details of THP-1 cells and their cultivation refer to the Supporting Information. Briefly, total lipids from THP-1 cells were extracted with methanol and chloroform/ methanol in (1/2, v/v), (1/1, v/v), and (2/1, v/v) ratios. Pooled extracts were evaporated, and coextracted lipids (such 1216



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Figure 1. TLC immunodetection of individual GSLs in the purified neutral GSL fraction (A) and in the crude lipid extract (B) of THP-1 cells without and with preceding PLC treatment. (A) GSL amount of the purified neutral GSL fraction applied for reference orcinol stain (a) corresponds to 1.25 × 107 cells; GSL amounts of purified neutral GSLs used for single detection of GSLs in the anti-Lc2Cer (b), anti-Gb3Cer (c), anti-Gb4Cer (d), and anti-nLc4Cer (e) antibody overlay assay and those employed for simultaneous multiple GSL detection (f) in one and the same chromatogram with an antibody mixture, which contains the four mentioned antibodies, equal 2 × 106 cells, respectively. (B) GSL amounts used for simultaneous multiple GSL detection in the crude lipid extract before (a) and after PLC treatment (f) and those employed for single detection of GSLs in the PLC-pretreated crude lipid extract with anti-Lc2Cer (b), anti-Gb3Cer (c), anti-Gb4Cer (d), and anti-nLc4Cer (e) antibodies are equivalent to 2 × 106 cells, respectively.

Infrared Matrix-Assisted Laser Desorption/Ionization Orthogonal Time-of-Flight Mass Spectrometry (IRMALDI-o-TOF MS). Immunostained TLC plates were dipped three times for 3 min in distilled chloroform to remove plexigum fixative and cut into 3 × 4 cm2 pieces prior to measurements to fit into the MALDI MS TLC holder. IRMALDI mass spectrometry was performed on an orthogonal time-of-flight mass spectrometer (QSTAR pulsar i, AB SCIEX) equipped with an oMALDI 2 ion source using glycerol as a matrix.32,36 The beam of an Er:YAG laser (Speser, Spektrum Laser, Berlin, Germany, emission wavelength 2.94 μm, pulse duration 150 ns, pulse repetition rate 2 Hz) was focused by an infrasil lens and entered the fine vacuum ion source through a CaF2 window. The laser port replaced the standard fiber port of the oMALDI 2 ion source.53 The laser spot size at sample position was about 110 × 220 μm2 (1/e2-definition). Ions were generated in an N2 buffer gas environment of ∼0.7 mbar. For low-energy collision-induced dissociation (CID) of selected precursor ions Ar was used as collision gas. All presented mass spectra were recorded in positive ion mode.

hamper reliable MS analysis. The aim of this study was to sensitively analyze and structurally characterize GSLs in crude lipid extracts, to reduce sample purification to a minimum, and to perform analysis in a single chromatogram using the TLC overlay assay combined with IR-MALDI MS1 and MS2 analysis. The procedure divides into (1) extraction of total lipids from cellular material, (2) enzymatical disintegration of phospholipids by sample treatment with PLC, an enzyme that cleaves the polar head groups of phospholipids, (3) subsequent use of this preparation for simultaneous multiple detection of GSLs with a mixture of various anti-GSL antibodies (antibody cocktail) in one and the same TLC run, and (4) further structural analysis of GSLs by means of direct IR-MALDI MS in combination with CID. We used purified neutral GSLs from THP-1 cells as reference39 in this study in comparison to crude lipid extracts and PLC-treated crude lipid extracts, whereby the latter preparation was employed to develop the novel strategy for GSL analysis. TLC Immunodetection of GSLs in a Purified Neutral GSL Fraction of THP-1 Cells. An aliquot of purified neutral GSLs of THP-1 cells was separated by TLC and stained with orcinol (Figure 1A, lane a) indicating the presence of monohexosylceramide (MHC), Lc2Cer, Gb3Cer, Gb4Cer, and nLc4Cer in this mixture. GSLs separate as double bands due to ceramide heterogeneity caused by long (C22−C24, upper band) and short (C16, lower band) fatty acids, whereas sphingosine (d18:1) represents the constant portion of the

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RESULTS So far it is hardly feasible, if at all, to detect and structurally characterize GSLs in total lipid extracts, because coextracted non-GSL “impurities” such as phospholipids, triglycerides, etc. predominate over minor GSLs and considerably disturb GSL separation and detection by means of TLC and, in particular, 1217



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ceramide moiety.39 Individual GSLs were detected in TLC runs (Figure 1A) with anti-Lc2Cer (lane b), anti-Gb3Cer (lane c), anti-Gb4Cer (lane d), and anti-nLc4Cer antibody (lane e). In addition to the single antibody-mediated detection, a mixture of these four antibodies was employed for cumulative immunodetection of the different GSLs in one and the same TLC run as shown in lane f of Figure 1A, permitting simultaneous multiple detection of individual GSLs and exhibiting a merge of the single antibody assays. TLC Immunodetection of GSLs in a Crude Lipid Extract of THP-1 Cells without and after PLC Treatment. Coextracted lipids such as phospholipids interfere with TLC separation and immunodetection of GSLs as illustrated for the crude lipid extract of THP-1 cells in Figure 1B (lane a). NonGSL spots disturb GSL chromatography resulting in squeezed GSL bands and disturbances in immunodetection as visible in case of Lc2Cer and at the position of Gb4Cer as well as beyond Gb4Cer and at the site of nLc4Cer. To get rid of phospholipids, we performed gentle enzymatic degradation of phospholipids by pretreatment of crude lipid extracts with PLC prior to TLC analysis of GSLs. TLC immunostains performed with single anti-Lc2Cer, anti-Gb3Cer, anti-Gb4Cer, and anti-nLc4Cer antibodies of the PLC-pretreated crude lipid extract are shown in Figure 1B, lanes b−e, respectively, exhibiting almost identical TLC overlay stains compared to those obtained with the highly purified neutral GSL fraction (see Figure 1A, lanes b−e, respectively). Furthermore, the TLC overlay stain of PLCpretreated crude lipid extract using the antibody cocktail is depicted in Figure 1B (lane f) illustrating the improvement in simultaneous immunodetection of several GSLs in one and the same TLC run. The obtained pattern was very similar to the TLC immunostain gained with the highly purified neutral GSL fraction of THP-1 cells (see lane f in Figure 1A). TLC-IR-MALDI MS Identification of GSLs in a Purified Neutral GSL Preparation, Crude Lipid Extract, and PLCTreated Crude Lipid Extract. The multiply immunostained chromatograms obtained with the antibody cocktail of (1) the purified neutral GSL fraction (see Figure 1A, lane f), (2) the untreated crude lipid extract (see Figure 1B, lane a), and (3) the crude lipid extract after PLC treatment (see Figure 1B, lane f) were subjected to direct TLC-IR-MALDI MS investigations. Lc2Cer Lipoforms. Figure 2 shows the direct TLC-IRMALDI MS1 spectra obtained from the laser-targeted upper band of Lc2Cer from the purified neutral GSL fraction (A), the lipid extract (B), and the lipid extract after PLC treatment (C). The major Lc2Cer species in the sample of purified neutral GSLs (Figure 2A) were detected as monosodiated ions at m/z 996.73 (d18:1, C24:0) and m/z 994.72 (d18:1, C24:1), accompanied by minor Lc2Cer species at m/z 968.70 (d18:1, C22:0). No GSL-derived signals were detectable in the untreated lipid extract of THP-1 cells by laser targeting of the corresponding upper band of Lc2Cer (Figure 2B). In the PLC-pretreated lipid extract (Figure 2C) the same lipoforms of Lc2Cer, identified as monosodiated ions at m/z 996.73 (d18:1, C24:0), 994.71 (d18:1, C24:1), and 968.70 (d18:1, C22:0), were detected as in the purified neutral GSL fraction (Figure 2A) with similar IR-MALDI MS detection capability. Major Lc2Cer lower band species were identified in the mixture of purified neutral GSLs and the PLC-pretreated crude lipid extract of THP-1 cells as Lc2Cer (d18:1, C16:0) and Lc2Cer (d18:1, C18:0) by m/z 884.60 and 912.64, respectively, whereas only extremely low signal intensities of the same Lc2Cer species were obtained from the crude lipid extract

Figure 2. Direct TLC-IR-MALDI MS1 spectra of the upper band of antibody-detected Lc2Cer species in the purified neutral GSL fraction (A), the crude lipid extract (B), and the PLC-pretreated crude lipid extract (C) of THP-1 cells. MS1 spectra of Lc2Cer (arrowheads) were obtained from multiply immunostained TLC lanes of the purified neutral GSL fraction (A, see lane f in Figure 1A), the crude lipid extract without PLC treatment (B, see lane a in Figure 1B), and after PLC treatment (C, see lane f of Figure 1B).

indicating a significant improvement in detection of short-chain Lc2Cer lipoforms after PLC pretreatment of the crude lipid extract (Figure S-1 in the Supporting Information). We conclude that enzyme exposure allows for distinct detection of Lc2Cer species free of interference by phospholipids. The list of monosodiated ([M + Na]+) ions, which were detected in the lipid extract of THP-1 cells after PLC treatment, and proposed structures of Lc2Cer lipoforms are presented in Table S-1 of the Supporting Information. Gb3Cer Lipoforms. The MS1 spectra of the Gb3Cer species present in the lower band of Gb3Cer immunopositive double bands are exemplarily shown in Figure 3. Gb3Cer (d18:1, C16:0) and Gb3Cer (d18:1, C18:0) were identified by molecular ions at m/z 1046.66 and 1074.69, respectively, as the two main species of the purified neutral GSL preparation (Figure 3A). Only trace quantities of the same ion species were 1218



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C22:0), whereas the untreated lipid extract gave poor results (Figure S-2 in the Supporting Information; for the list of detected [M + Na]+ ions after PLC treatment and the corresponding proposed Gb3Cer structures refer to Table S-1 in the Supporting Information). The clear-cut detection of Gb3Cer molecular ions ([M + Na]+) by direct IR-MALDI MS of immunopositive bands allowed us to proceed with CID fragmentation as exemplarily shown by MS2 spectra of the GSL lipoform Gb3Cer (d18:1, C16:0) derived from purified neutral GSLs and PLC-treated crude lipid extract in Figure S-3 of the Supporting Information. The MS2 spectra each revealed a full series of Y-type ions and complementary B2 and B3 as well as C2 and C3 ions, where the fragment ions are assigned according to the nomenclature of Domon and Costello,54,55 providing clear evidence for the proposed structure of Gb3Cer (d18:1, C16:0). Our results demonstrate that identical MS2 spectra were obtained regardless of whether the selected Gb3Cer precursor ions derived from purified neutral GSLs or from the PLC-treated crude lipid extract. Gb4Cer Lipoforms. Figure 4 displays MS1 spectra obtained from the respective lower band of the two top-down Gb4Cer immunopositive bands in the three approaches. In the purified neutral GSL fraction the two monosodiated species at m/z 1249.74 and m/z 1277.75 could be assigned to Gb4Cer (d18:1, C16:0) and Gb4Cer (d18:1, C18:0), respectively, and the doubly sodiated ions at m/z 1271.73 correspond to Gb4Cer (d18:1, C16:0) (Figure 4A). Both Gb4Cer species were undetectable in the lipid extract due to non-GSL lipid interference (Figure 4B), whereas PLC-mediated degradation of phospholipids in the lipid extract yielded the same distinct Gb4Cer profile (Figure 4C) as in purified neutral GSLs (Figure 4A). The m/z values of the Gb4Cer-derived [M + Na]+ ions detected in the PLC-pretreated lipid extract of THP-1 cells and proposed structures of the various Gb4Cer lipoforms are listed in Table S-1 of the Supporting Information. In addition, TLCIR-MALDI MS1 analysis of upper Gb4Cer bands yielded almost identical MS1 spectra from purified neutral GSLs and PLCpretreated lipid extract as demonstrated in Figure S-4, parts A and C, respectively, of the Supporting Information. The same signals for major and minor ions indicative for Gb4Cer species appeared in both spectra (for the list of detected [M + Na]+ ions after PLC treatment and proposed structures refer to Table S-1 of the Supporting Information). The m/z values of monosodiated ions at m/z 1359.83/1359.82 and at m/z 1361.86/1361.82 correspond to Gb4Cer (d18:1, C24:1) and Gb4Cer (d18:1, C24:0), respectively, the latter accompanied by doubly sodiated Gb4Cer (d18:1, C24:0) with m/z of 1383.84/ 1383.81, and minor signals at m/z 1333.84/1333.81 can be attributed to Gb4Cer (d18:1, C22:0). Again, the crude lipid extract gave unsatisfactory results due to failure in detection of any Gb4Cer-related signals (Figure S-4B of the Supporting Information). The [M + Na]+ ions of proposed Gb4Cer species were selected for CID experiments, and two examples of fragmentation spectra are shown in Figure S-5 of the Supporting Information for Gb4Cer (d18:1, C16:0) obtained from purified neutral GSLs (A) and the PLC-treated lipid extract (B) together with an assisting fragmentation scheme (C). Both MS2 spectra exhibit complete series of Y-type ions attained from Gb4Cer species precursor ions ([M + Na]+) at m/z 1249.74/1249.73 and ancillary B1, B2, and B4 as well as C4 ions, which evidence the proposed structure of Gb4Cer (d18:1,

Figure 3. Direct TLC-IR-MALDI MS1 spectra of the lower band of antibody-detected Gb3Cer species in the purified neutral GSL fraction (A), the crude lipid extract (B), and the PLC-pretreated crude lipid extract (C) of THP-1 cells. MS1 spectra of Gb3Cer (arrowheads) were obtained from multiply immunostained TLC lanes of the purified neutral GSL fraction (A, see lane f in Figure 1A), the crude lipid extract without PLC treatment (B, see lane a in Figure 1B), and after PLC treatment (C, see lane f of Figure 1B). Non-GSL contamination peaks derived from poly(ethylene glycol)s are marked by asterisks.

detectable in the untreated lipid extract (Figure 3B). The extremely low peak intensities did not allow for unequivocal assignment and/or proposal of GSL structures as indicated by question marks. Pretreatment of the lipid extract with PLC significantly improved the detection of Gb3Cer species as demonstrated by clear-cut signals corresponding to ions of the Gb3Cer (d18:1, C16:0) and Gb3Cer (d18:1, C18:0) lipoforms (Figure 3C). TLC-IR-MALDI MS1 analysis of upper Gb3Cer bands confirmed these results inasmuch as MS1 spectra obtained from the purified neutral GSL fraction and the PLC-pretreated lipid extract revealed the same major Gb3Cer species identified at m/z 1158.79/1158.76 (d18:1, C24:0) and m/z 1156.77/1156.75 (d18:1, C24:1), and minor signals at m/z 1130.75/1130.73, which correspond to Gb3Cer (d18:1, 1219



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extract as expected. Phospholipid signals disappeared after PLC treatment, and signals of Gb4Cer carrying long-chain fatty acids (as selectively shown in the mass scale expanded partial spectrum of Figure S-4C of the Supporting Information) appeared after PLC treatment accompanied by poly(ethylene glycol)-derived peaks with characteristic increments of 44 u throughout the whole mass range. The peaks in the lower m/z region are TLC-IR-MALDI MS-immanent glycerol- and/or glycerol/silica clusters, and most of them have been identified previously.56 nLc4Cer Lipoforms. As shown by single TLC overlay assays using purified neutral GSLs (Figure 1A) and the PLC-treated lipid extract (Figure 1B), lower Gb4Cer and upper nLc4Cer bands cochromatograph (see lanes d and e in Figure 1A, respectively, and lanes d and e in Figure 1B, respectively). This results in a triple band when using the antibody mixture for the fraction of purified neutral GSLs (see Figure 1A, lane f) and the PLC-treated lipid extract (see Figure 1B, lane f). In the MS1 spectrum of the lower one of the triple band of the purified neutral GSL preparation a peak at m/z 1249.74 points to nLc4Cer (d18:1, C16:0), also detectable but with lower intensity in the PLC-treated lipid extract, whereas only a very weak nLc4Cer-related signal was observed in the untreated lipid extract (Figure S-7 in the Supporting Information). The MS2 spectra attained from precursor ions at m/z 1249.75 and 1249.71 of the purified neutral GSL fraction and the PLCtreated lipid extract, respectively, gave some evidence for proposed nLc4Cer (d18:1, C16:0) structure (Figure S-8 in the Supporting Information).

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DISCUSSION Currently, accessory techniques for mass spectrometry like the TLC overlay assay are extremely helpful by providing essential structural information on the sugar epitopes of GSLs using antibodies or bacterial toxins with high carbohydrate binding specificity.57 TLC immunostaining of GSLs combined with direct UV-MALDI MS34,35 or direct IR-MALDI MS36,38 provides structural details of the oligosaccharide chain length as well of isomeric configuration of individual sugars, which cannot be accomplished by MS alone. This strategy has been successfully applied for determining tumor-associated GSLs in small surgical samples of various human malignancies using lipid extracts, which have been liberated from coextracted alkaline labile glycerophospholipids, triglycerides, etc. by mild saponification.13,58,59 Removal of lipid contamination by alkaline treatment is also required for high-performance liquid chromatography electrospray ionization (ESI) MS as described for neutral GSLs derived from human fibroblasts27 and human erythrocytes60 in order to avoid disturbances in analysis caused by coextracted glycerophospholipids and others. Lipids with quaternary ammonia groups like phosphatidylcholine and sphingomyelin are more sensitively detectable than other ones and are, therefore, candidates, which may prevent the detection of minor GSLs in crude lipid extracts.25 This might be at least one reason that often only selected lipid classes are detected if crude lipid mixtures are analyzed.26 The glycerophospholipid phosphatidylcholine and the sphingophospholipid sphingomyelin share the same phosphocholine headgroup, which is a target for PLC of mammalian and bacterial origin.61,62 Thus, their phosphocholine group can be cleaved off in crude lipid extracts by means of PLC enhancing efforts to study de-N-acetyl-gangliosides circumventing alkaline treatment.63 Prevention of degradation, for example, of alkaline

Figure 4. Direct TLC-IR-MALDI MS1 spectra of the lower band of antibody-detected Gb4Cer species in the purified neutral GSL fraction (A), the crude lipid extract (B), and the PLC-pretreated crude lipid extract (C) of THP-1 cells. MS1 spectra of Gb4Cer (arrowheads) were obtained from multiply immunostained TLC lanes of the purified neutral GSL fraction (A, see lane f in Figure 1A), the crude lipid extract without PLC treatment (B, see lane a in Figure 1B), and after PLC treatment (C, see lane f of Figure 1B).

C16:0). With these experiments we could again demonstrate the reliability of MS1 GSL analysis. Subsequent fragmentation of selected corresponding precursor ions of the highly purified neutral GSL fraction and the PLC-treated lipid extract resulted in identical Gb4Cer fragmentation patterns (Figure S-5, parts A and B, respectively, in the Supporting Information). In addition to the partial spectra shown in this paper, full mass range spectra of the Gb4Cer upper band (see Figure S-4 of the Supporting Information) are exemplarily shown for a crude lipid extract and a PLC-treated crude lipid extract in Figure S-6, parts A and B, respectively, of the Supporting Information. No GSL-related peaks at all but a number of phospholipid-derived signals are detectable in the crude lipid 1220



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sensitive modifications like O-acetylation of neutral GSLs, which have been recently discovered in mammalian myelin,64,65 can be easily achieved by PLC treatment and recommends the use of this enzyme for the isolation of GSLs carrying alkaline sensible substituents. We employed this strategy for sensitive detection of neutral GSLs in PLC-treated crude lipid extracts of monocytic THP-1 cells, which have been recently reported to harbor phosphatidylcholine and sphingomyelin as major phospholipids.40 PLC treatment results in higher ionization efficiency of GSLs and allowed for structural assignment of neutral GSLs determined by means of MS1 and MS2, combined with oligosaccharide identification using a cocktail of various anti-GSL reactive antibodies. The antibody-mediated detection of GSLs allows for unequivocal discrimination between GSL lipoforms of immunopositive upper and lower bands, which preferentially harbor lipoforms carrying long- (C22 and C24) and short-chain fatty acids (C16 and C18), respectively. MS analysis enables further distinction of minor differences in fatty acid composition to differentiate, for example, between GSL species containing C24:1 or C24:0 fatty acids. Using MS1 analysis we observed changes in the mutual ratios of lipoform doublets of C24:1/C24:0 carrying Lc2Cer (Figure 2), Gb3Cer (Figure S-2 in the Supporting Information), and Gb4Cer species (Figure S4 in the Supporting Information). For example, low signal intensity of Lc2Cer (d18:1, C24:1) and high signal intensity of Lc2Cer (d18:1, C24:0) from the purified neutral GSL fraction (Figure 2A) switched to reversed relative distribution in the PLC-exposed crude lipid extract (Figure 2C). The same holds true for the doublet of Gb3Cer and Gb4Cer C24:1/C24:0 (see Figures S-2 and S-4 in the Supporting Information, respectively) recognized in the purified sample versus the PLC-treated crude lipid extracts. This finding suggests a possible selectivity of PLC treatment in the sample cleanup procedure, i.e., multifold downstream cleanup steps in preparing purified neutral GSLs compared with simple extraction of GSLs in providing crude lipid extracts. The explanation for this discrepancy is that the highly pure neutral GSLs were obtained from THP-1 cells produced on bioreactor scale,39 whereas the crude lipid extract was obtained from THP1 cells conventionally propagated in culture flasks.40 We suppose that low oxygen supply in surface aerated culture flasks, compared to optimal submerged aeration in bioreactors, might lead to a relative increase of GSL lipoforms with monounsaturated C24:1 fatty acid. This novel finding stimulates us for future in-depth investigation of cell culture-dependent changes in GSL expression, which have been previously described by us when analyzing ganglioside fractions from mouse lymphoma cells grown in serum-supplemented and serum-free cell culture medium.66

Article

ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +49-251-8355192. Fax: +49-251-8355341. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the International Graduate School “Molecular Interactions of Pathogens with Biotic and Abiotic Surfaces” (GRK 1409, collaboration between the projects 3.10 of J.M. and 3.6 of H.K.) funded by the “Deutsche Forschungsgemeinschaft” (DFG), the DFG project MU845/ 4-2 (J.M.), by the German Federal Ministry of Education and Research (BMBF) conducted under the umbrella of the German Research Platform for Zoonoses (01KI1106) and the German Center for Infection Research (DZIF, TTU 06.801). We thank Katja Neukirch and Nikola Skutta for excellent technical assistance.

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REFERENCES

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CONCLUSIONS So far only few data are available for GSLs from lipidomics and (glyco)sphingolipidomics approaches of total lipid extracts most likely due to their rather low abundance and low ionization in lipid mixtures containing coextracted phospholipids, cholesterol, and other lipids. The merged approach combining PLC pretreatment with multiple TLC immunostaining and direct IR-MALDI MS opens new doors by delivering data on the GSL composition and specific structural information of individual GSLs in complex mixtures with limited investment in sample preparation. 1221



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Progress in Detection and Structural Characterization of

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