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Isolation and mass spectrometry characterization of molecular species of lactosylceramides using liquid chromatography-electrospray ion trap mass spectrometry
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 337 (2005) 316–324 www.elsevier.com/locate/yabio
Isolation and mass spectrometry characterization of molecular species of lactosylceramides using liquid chromatography-electrospray ion trap mass spectrometry Naoko Kagaa, Saiko Kazunoa, Hikari Takaa, Kazuhisa Iwabuchib,c, Kimie Murayamaa,* a
b
Division of Proteomics and Biomolecular Science, BioMedical Research Center, Juntendo University Graduate School of Medicine, Tokyo 113-8421, Japan Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, Tokyo 113-8421, Japan c Laboratory of Biochemistry, Juntendo University School of Healthcare and Nursing, Chiba, Japan Received 27 September 2004 Available online 2 December 2004
Abstract Reverse-phase liquid chromatography/electrospray ion trap mass spectrometry (LC-ESI-MSn) was established for identification of the molecular species of lactosylceramides. Lactosylceramides derived from porcine blood cells were separated on a CapcellPak C8 column using a mixture of methanol and 1 mM ammonium formate from the C16 to C26 fatty acyl chains based on the length of total carbon chains and the nature of sphingoid bases (w00 ) and fatty acyl chains (Y00 w00 ) was identified by MS3 as their [M + H]+ ions. The same number of fatty acyl moieties appeared in the order of unsaturated, (2-)hydroxylated, and saturated components. The molecular species of lactosylceramides derived from porcine blood cells totaled more than 33 and included mainly C24:0-d18:1, Ch24:0-d18:1, Ch24:1-d18:1, C24:1-d18:1, and C22:0-d18:1 in addition to 28 minor species from C16:0 to C26:0 fatty acyl moieties. The molecular species of lactosylceramides in the membrane microdomain fraction of HL-60 cells (70% were differentiated into macrophagelineage cells) were identified as C24:0-d18:1, C24:1-d18:1, C22:0-d18:1, C16:0-d18:1, and more than 21 other minor species. Our results suggest that reverse-phase LC-ESI-MSn is a useful and simple method for identification of lactosylceramide molecular species. 2004 Elsevier Inc. All rights reserved. Keywords: Lactosylceramide; Molecular species; Fatty acyl chain moiety; Long-chain base moiety; LC-ESI-MSn
Studies on molecular membrane biology have focused on the dynamics of cellular proteins and the lipid microenvironment at the cell surface [1]. It is known that glycosphingolipids play important roles in cell–cell interaction, recognition, transmembrane signaling, and cellular growth and differentiation in animal cells [2]. These compounds self-aggregate easily based on their nature and cluster with sphingomyelin with or without cholesterol and various signal transducer molecules to form various types of microdomains called ‘‘lipid-raft’’ at plasma membrane [3–6]. Although the lipid composition of the *
Corresponding author. Fax: +81 3 3818 6330. E-mail address: [email protected] (K. Murayama).
0003-2697/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.11.003
rafts in different cells is not clear, lactosylceramides are known to localize with ‘‘sphingolipid-sterol rafts’’ and trigger raft-related signal transduction processes. In neutrophils, lactosylceramide-enriched microdomains on the cell surface coupled with tyrosine protein kinase LYN (Lyn)1 induce the activation of Lyn via lactosylceramides and lead to superoxide generation [3]. To understand the biological activity and physical behavior of lactosylceramides, it is important to deter1
Abbreviations used: Lyn, tyrosine protein kinase LYN; FAB, fast atom bombardment; CAD, collisional-activated dissociation; CID, collision-induced dissociation; ESI, electrospray ionization; APCI, atmospheric pressure chemical ionization; DFP, diisopropylphosphorofluoridate; SIM, single ion monitoring; 3D, three-dimensional.
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mine their structural heterogeneity. Lactosylceramides contain lactose and a ceramide moiety. Various classes of the sphingoid base have been identified, such as the dihydro type (sphinganine), the additional hydroxyl group type (phytosphingosine), and the 2-amino-4-ene1,3-diol type with 14–22 carbon chains (sphingenine). The commonest type in animal tissues is C18 sphingenine (-d18:1). In addition, significant variation can also occur in fatty acyl moiety with regard to chain length, unsaturation, and hydroxylation. Lactosylceramides containing saturated fatty acyl chains display relatively high thermotropic and pressure-induced transition, but the introduction of a single cis double bond into the fatty acyl chains markedly reduce these properties, especially when the double bonds are located near the middle of the fatty acyl chain [6]. Mass spectrometry (MS) is a powerful tool for lipid structure analysis. Several methods incorporating tandem MS for identification of intact ceramides and neutral sphingolipids such as glucosyl- and lactosylceramides have been published. In early 1990s, Costello and Vath [7] used fast atom bombardment tandem mass spectrometry (FAB-MS/MS) and reported valuable data about the structure of glycosphingolipids, such as the molecular weight of fatty acyl chain and sphingoid base, by analyzing the MS/MS spectra of [M + H]+ and [M H] ions. Ann and Adams [8,9] and Duarte et al. [10] also reported that, in the presence of alkaline metal, [M + metal]+ ions were formed with ceramides and glucosylceramides and these showed a much higher sensitivity than the [M + H]+ ions. In addition, they provided more informative MS/MS spectra for the location of double bonds at high-energy CAD, by using charge remote cleavage. At present, electrospray ionization tandem mass spectrometry (ESI-MS/MS) and atmospheric pressure chemical ionization tandem mass spectrometry (APCI-MS/MS) are used for determination of the structure of intact ceramides and lactosylceramides with low-energy CID [11–13] or high-energy CID [14]. Lee et al. [15] and Pettus et al. [16] reported analysis of the molecular species of ceramides by reversephase LC-ESI/MS/MS and normal-phase LC-APCI/ MS/MS, respectively. We report here the design of a simple and useful method for separation and MSn characterization of molecular species of lactosylceramides using reversephase HPLC-ESI ion trap mass spectrometry.
Materials and methods Materials Lactosylceramide derived from porcine blood cells was purchased from Matreya (Pleasant Gap, PA). The
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synthetic lactosylceramide for C24:1-d18:1 was provided by Professor Sandro Sonnino (Center of Excellence on Neurodegenerative Diseases, Department of Medical Chemistry, Biochemistry and Biotechnology, University of Milan, Italy). All reagents used in this study were of the highest grade, especially those used for HPLC, spectrometry, and amino acid analysis. Chloroform, acetone, sodium chloride, methanol, ethylenediaminetetraacetic acid (EDTA), Triton X-100, Tris–HCl, vitamin D3, and diisopropylphosphorofluoridate (DFP) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Lithium chloride anhydrous and ethylene glycol bis (b-aminoethyl ether)-N,N,N 0 ,N 0 -tetraacetic acid (EGTA) were obtained from Nacalai Tesque (Kyoto, Japan). Ammonium acetate, ammonium formate, and primulin were from Sigma–Aldrich (St. Louis, MO). Complete is a product of Roche (Switzerland). Ultrapure water was prepared using the Millipore Milli-Q purification system (Bedford, MA). Mass spectrometric analysis All analyses were performed in positive mode using a LCQ DECA XP ion trap mass spectrometer (Thermo Electron, San Jose, CA) with a conventional electrospray ionization source. Infusion-ESI-MSn analysis Lactosylceramide at a concentration of 5–10 ng/ll was dissolved in methanol with or without alkaline and ammonium salts (1 mM LiCl, 1 mM NaCl, and 10 mM ammonium acetate). Sample solution was directly infused at a rate of 3–5 ll/min into the ESI source without separation of the molecular species of lactosylceramides. The temperature of the ion transfer tube was set at 150–400 C. The spray voltage was set at 5 kV and the nitrogen sheath gas pressure used was 10 U. In MSn (n = 2–4) analysis, the collision energy was fixed at 100%. HPLC-ESI-MSn analysis. Reverse-phase HPLC was carried out with the Magic2002 system (Michrom BioResources, Auburn, CA). The column was Capcellpak C8 UG120, 1.0 mm ID · 150 mm (Shiseido Fine Chemicals, Tokyo). The mobile phases were as follows: solvent A was 1 mM ammonium formate in methanol:water (76:24 v/v) and solvent B was 1 mM ammonium formate in methanol:water (96:4 v/v). The elution program was isocratic elution with 80% B for 5 min, increase to 95% B, and then hold for 30 min. The flow rate was 50 ll/ min. The HPLC was joined to the mass spectrometer, LCQ DECA XP. The MS conditions were as follows: ion transfer tube temperature 250 C, spray voltage 5 kV, sheath gas pressure 20 U. MSn (n = 4) analysis was performed on data-dependent experiments (DDE) with collision energy 80%.
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Identification of molecular species of lactosylceramides in the membrane microdomain fraction of HL-60 cells Fractionation of the membrane microdomain fraction of HL-60 cells. Cells of the myeloid leukemia cell line HL-60 were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum. HL-60 cells (2 · 108 cells) were treated with active form vitamin D3 (final concentration, 100 nM) for 5 days. Flow cytometric analysis using anti-CD11b antibody was performed as described previously [3]. Iwabuchi and Nagaoka [3] indicated that about 70% of the cells were differentiated into macrophage-lineage cells. Cells were resuspended in relaxation buffer (10 mM 1,4 piperazinediehanesulfonic acid, 100 mM KCl, 3 mM NaCl, 3.5 mM MgCl2, and 1 mM ATP, pH 7.4) containing a mixture of protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 0.4 lM aprotinin, 10 lM E-64, 20 lM leupeptin, 10 lM bestatin, 5 lM N-acetyl-Leu-Leu-Met-al, and 2 lM pepstatin) and disrupted by nitrogen cavitation (N2 for 20 min at 350 psi). Disrupted cells were dropped gradually into EGTA solution (final concentration, 1.25 mM). All manipulations were carried out at 4 C. The plasma membrane was fractionated after sequential centrifugation; 500g at 4 C for 10 min, 8500g for 20 min, and 200,000g for 1 h. This fraction was suspended in TNE-T buffer [1% Triton X-100, 10 mM Tris–HCl (pH 7.5), 150 mM Nacl, 1 mM DFP] containing 1/20 Complete. Extraction of crude lactosylceramides. The crude lipids in the membrane microdomain fraction were extracted with a chloroform:methanol mixture (2:1 v/v). A fifth of them (20 ll/100 ll crude lipids) were separated on a 10 · 10-cm high-performance thin-layer chromatography (HPTLC) plate (Merck, Rahway, NJ) with chloroform/methanol/water 65:25:4 (v/v/v). After development, lactosylceramides were visualized using primulin reagent (0.03% in 80% acetone) under ultraviolet light. Lactosylceramide-containing bands were scratched out from the plate and extracted with chloroform/methanol (2:1 v/v). After drying, lactosylceramides were dissolved in 20 ll of methanol and 4 ll of the solution was loaded on the column for LC-ESIMSn analysis. Finally, lactosylceramides from HL-60 (8 · 106 cells) were analyzed.
Results and discussion Effects of salts and temperature on ionization of synthetic lactosylceramide at C24:1-d18:1 To compare the effects of salts and ion transfer tube temperature on ionization of lactosylceramide by infusion analysis, synthetic lactosylceramide at C24:1-d18:1 was dissolved in methanol with/without 1 mM LiCl,
Fig. 1. Effects of salts and temperature on ionization of lactosylceramide by infusion analysis. Synthetic lactosylceramide of C24:1-d18:1 was dissolved in methanol containing each salt at 5 ng/ll concentration. The flow rate was 3 ll/min.
NaCl, or 10 mM ammonium acetate at 5 ng/ll and analyzed with the ion trap mass spectrometer. In salt-free condition, lactosylceramide at C24:1-d18:1 did not produce the [M + H]+ ion at 400 C, but the [M + Na]+ ion, which was produced from concomitant sodium in the sample or reagents, was stable. The addition of 1 mM NaCl to lactosylceramide markedly increased the amount of [M + Na]+ ion at 400 C (Fig. 1). On the other hand, the amount of [M + Li]+ ion was twice as much as that of [M + Na]+ ion at the same temperature. However, the addition of 10 mM ammonium acetate tended to produce the [M + H]+ ion at 200 C. The higher temperature (400 C) for the [M + H]+ ion was associated with the generation of dehydrated ion and resulted in a complex spectrum. On the other hand, the [M + Na]+ ion of lactosylceramide could not be produced at 200 C. The optimal temperature of the ion transfer tube was 400 C for the [M + metal]+ ion and 200 C for the [M + H]+ ion. MSn analysis of [M + Li]+ and [M + H]+ ion to characterize fatty acyl chain and long-chain base moiety We performed MSn analysis for [M + Li]+ and [M + H]+ ion of synthetic lactosylceramide (C24:1-d18:1) at optimal temperature (Fig. 2). In MSn analysis for [M + Li]+ ion, following the loss of one or two hexose(s) [Y1,Y0] and water [represented as 0 , 00 ], a HCHO was eliminated to form [a2] ion11 on the MS2/MS3 spectra (Fig. 2A). [Y0] and [a2] ions were extremely stable at 400 C and no further cleavage occurred on the MS4 spectrum. We could not obtain information on the fatty acyl chain and long-chain base, even though the dissociation energy increased up to 100%. However, lactosylceramides containing 2-hydroxy fatty acyl chain underwent marked cleavage of the N–CO bond on the MS3 spectrum and characterized the fatty acyl chain (data not shown). On the other hand, in MSn analysis of [M + H]+ ion, the loss of two hexoses and water [Y00 ] on the MS2 spec-
Characterization of molecular species of lactosylceramides / N. Kaga et al. / Anal. Biochem. 337 (2005) 316–324
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Fig. 2. MSn spectra of synthetic lactosylceramide of C24:1-d18:1 using ESI-ion trap mass spectrometer. (A) MSn spectra (n = 1–4) of the lithiated molecular ion [M + Li]+ with 1 mM LiCl in methanol at 400 C. (B) MSn spectra (n = 1–3) of the protonated molecular ion [M + H]+ with 10 mM ammonium acetate in methanol at 200 C. The collision energy was 100%. (C) Predicted cleavage scheme for lactosylceramide shows hexose (Hex), loss of hexoses (Y0), long-chain base (w series), and fatty acyl chain (COR-H).
trum was associated with cleavage of the immediate N–CO bond on the MS3 spectrum and identification of a long-chain base (w ion series) and a fatty acyl chain ([Y00 w0 ¼ COR H]) [7] (Fig. 2B). On the MS3 spectrum, m/z 264 was found as a fragment ion of Y00 regarded as w00 ion, which was assigned to d18:1, and the fatty acyl chain was identified as C24:1 [7]. The use of sequential MSn analysis allowed us to obtain the dissociation patterns of a single ion in multiple steps when that was compared with MS/MS analysis using FAB-MS or triple stage ESI/MS. The patterns were simple, thus allowing speculation on the structure of the molecular species. Molecular species of lactosylceramides derived from porcine blood cells using infusion analysis The molecular species of lactosylceramides derived from porcine blood cells were determined using infusion analysis. The MS spectrum for those ionized with 1 mM ammonium acetate is shown in Fig. 3. The molecular species of lactosylceramides comprised more than 14 different structures from C16:0-d18:1 to Ch26:1-d18:1. The main species were C22:0-d18:1, C24:1d18:1, C24:0-d18:1, Ch24:1-d18:1, and Ch24:0-d18:1, similar to the results of a previous study [11].
Fig. 3. ESI-MS spectrum of lactosylceramides derived from porcine blood cells ionized with ammonium salt using infusion analysis. Lactosylceramides were dissolved in 10 mM ammonium acetate in methanol at 10 ng/ll concentration, the ion transfer tube was set at 200 C. The molecules were detected as protonated molecular ions, [M + H]+. The parenthetic formulae of lactosylceramides were characterized using other methods. *Represent those revealing background ions from the reagents. Some ions at m/z 1001 and 1003 were similar to the [M + H]+ ions of C26:1-d18:1 and C26:0-d18:1 but they were distinguished from the background ions on MSn analysis.
The use of infusion analysis was associated with one problem; it was impossible to distinguish fatty acyl chains between Cn:x and Ch(n 1):(x + 1) by MSn
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analysis because of the same value of COR-H at m/z 364 for C25:0 and Ch24:1 fatty acyl chains. LC-ESI-MS for classification of molecular species of lactosylceramides by single ion monitoring (SIM) mode The molecular species of lactosylceramides were separated on a C8 reverse-phase column and identified using the LCQ ion trap mass spectrometer by characterizing their fatty acyl chain and long-chain base moieties by MSn analysis. Fig. 4A shows the three-dimensional (3D) HPLC mass chromatogram of 200 ng of total lactosylceramides derived from porcine blood cells. The abscissa, ordinate, and z axis indicate the retention time, relative abundance of ions, and m/z, respectively.
In the case of LC-MS, we used methanol containing 1 mM ammonium formate as a solvent because the formate produced stable and abundant [M + H]+ ions and [M + Na]+ ions were minimum as contaminants. It was assumed that the long chain base of lactosylceramides has d18:1 sphingenine, because this is the commonest type in animal tissues [17]. In Figs. 4B–E, [M + H]+ ions of lactosylceramide molecular species were monitored in the SIM mode for classification according to fatty acyl carbon chain into (B) straight saturated (n:0), (C) straight unsaturated (n:1), (D) saturated and hydroxylated (hn:0), and (E) unsaturated and hydroxylated (hn:1) fatty acyl chains. We also monitored d18:0 sphinganine, but no significant peak of lactosylceramides appeared in porcine blood cells (data not
Fig. 4. Three-dimensional HPLC mass chromatogram of lactosylceramides derived from porcine blood cells and selected ion monitoring (SIM) chromatograms. (A) 3D-HPLC-ESI/MS chromatogram of 200 ng of lactosylceramide. SIM chromatograms monitored the [M + H]+ ions of lactosylceramide molecular species classified according to fatty acyl carbon chain into (B) straight saturated (n:0), (C) straight unsaturated (n:1), (D) saturated and hydroxylated (hn:0), and (E) unsaturated and hydroxylated (hn:1).
Characterization of molecular species of lactosylceramides / N. Kaga et al. / Anal. Biochem. 337 (2005) 316–324
shown). In the straight saturated molecular species (Fig. 4B), the retention time of lactosylceramides correlated with the length of their fatty acyl chain from 10 min for C16:0 to 30 min for C26:0. Although the [M + H]+ ions of lactosylceramides at C25:0 and Ch24:1 had the same value at m/z 989 and could not be identified by infusion analysis, they were completely separated on the column. Their retention times were 17.3 min for Ch24:1 and 24.6 min for C25:0. The former had 2-hydroxylated fatty acyl chains and was more hydrophilic than the latter. Lactosylceramides with the same carbon number of fatty acyl chain of saturated (C24:0) at m/z 975, unsatu-
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rated (C24:1) at m/z 973, and 2-hydroxylated (Ch24:0) at m/z 991 fatty acyl chain moiety appeared at 22.2, 18.6, and 20.8 min on the chromatogram, respectively (Figs. 4B–D). These results suggest that lactosylceramides classified as unsaturated, 2-hydroxylated, and saturated fatty acyl chain were eluted in that order on the column. Determination of molecular species of porcine-blood cell-derived lactosylceramides We also identified more than 33 different structures as molecular species from porcine-blood-cell-derived lactosylceramides (Fig. 5A). The profile was constructed
Fig. 5. Distribution of molecular species of lactosylceramides derived from porcine blood cells. (A) Abscissa represents the carbon unit of fatty acyl chain and long-chain base (d18:1 is not shown). The intensity of lactosylceramides was estimated by the peak area on Figs. 3B–E. The ordinate represents the peak area. The z axis represents the class of fatty acyl chain such as saturated, unsaturated, and hydroxylated species. (B) The SIM chromatogram of the same [M + H]+ ions at m/z 1003 for C26:0-d18:1 and C24:0-d20:1 and (C) MSn analysis (n = 1–3) for m/z 1003.
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based on the peak area of SIM chromatogram for lactosylceramides. Some lactosylceramides with long-chain base moiety at d20:1 were confirmed by MS3 analysis and are shown in Fig. 5C. The retention time of lactosylceramide at m/z 1003 for C26:0-d18:1 or C24:0-d20:1 was 28 min in Fig. 5B, but the m/z 264 and m/z 292 ions were produced on the MS3 spectrum. This means that there were two other types of w00 ions associated with d18:1 and d20:1 sphingenines. Therefore, it was considered that separation of lactosylceramides was based on total carbon chain length and numbers of double bonds in the fatty acyl chain and long-chain base. Thus, lactosylceramides at Cn-dx and Cn2-dx+2 were not separated on the column in the system, however, we could at least confirm by MS3 analysis the presence or absence of the two types of lactosylceramides. The molecular species of porcine-blood-cell-derived lactosylceramides contained more than 33 different structures, which were mainly C24:0-d18:1, Ch24:0-d18:1, Ch24:1-d18:1, C24:1-d18:1, and C22:0-d18:1, with 28 other minor species. Minor species of long-carbon-chain fatty
acids such as C22 and C26 were also identified as saturated, unsaturated, and hydroxylated species. The number of molecular species of lactosylceramides determined by LC-ESI/MS was twice that confirmed by infusion analysis. The amount of lactosylceramides used was 200 ng for determination of the [M + H]+ ions and automatic MSn analysis with LC-ESI/MS. In the case of infusion analysis, lactosylceramides at 10 ng/ll were infused into the ion source for 10–15 min depending on the number of MSn analyses of [M + H]+ ions manually. The sample amounts used for both analyses were not different and LC-ESI/MS was convenient and precise for determination of low-intensity [M + H]+ ions. Since the standard compound of the molecular species of lactosylceramides could not be reported and no product could be identified, we were unable to perform quantification analysis of these species. However, the SIM chromatograms of 10–200 ng of porcine-bloodcell-derived lactosylceramides showed almost linear relationships between the ratio of the peak area and the total amount of lactosylceramides (data not shown).
Fig. 6. Determination of molecular species of lactosylceramides derived from membrane microdomain of HL-60 cells. (A) The pattern of lactosylceramides for membrane microdomain fractionated from HL-60 cells separated on HPTLC. (B) Molecular species of lactosylceramides in the upper band and (C) those in the lower band derived from HL-60 cells (8 · 106 cells). The abscissa, ordinate, and z axis are similar to those used in Fig. 5.
Characterization of molecular species of lactosylceramides / N. Kaga et al. / Anal. Biochem. 337 (2005) 316–324
Analysis of molecular species of lactosylceramides in the membrane microdomain fraction of HL-60 cells Crude lipids were extracted from the membrane microdomain fraction of HL-60 cells using a mixture of chloroform and methanol (2:1 v/v). A fifth of the crude lipids was separated on HPTLC with a mixture solution of chloroform/methanol/water (65:25:4 v/v/v) and detected by primulin reagent. Lactosylceramides marked as upper and lower bands in Fig. 6A were removed and collected in each glass tube and crude lactosylceramides were extracted with a mixture of chloroform and methanol (2:1 v/v). A fifth of the crude lactosylceramides was loaded on a column and determined by LCESI-MSn analysis, respectively. Figs. 6B and C show the distribution of molecular species of lactosylceramides in the upper and the lower bands, respectively. A total of 25 species of lactosylceramides was detected in the upper and lower bands on the HPTLC plate. Lactosylceramides in the upper band consisted mainly of C24:0-d18:1, C24:1-d18:1, and C22:0-d18:1 and those in the lower band were mainly C16:0-d18:1 but higher carbon chain structures such as Ch24:0-d18:1 fatty acyl chain moiety at m/z 991 were also detected. The high molecular species in the lower band seemed to be insufficiently separated on the HPTLC plate and rather coexisted with those of the upper band. It is possible, however, that the hydrophilic property of Ch24:0 could have affected the separation of lactosylceramides on the HPTLC plate. On the other hand, lactosylceramide for C16:0-d18:0 also appeared in the upper band, suggesting that it might be more hydrophobic than lactosylceramide for C16:0-d18:1 on the HPTLC plate. Numerous minor lactosylceramides that contained odd numbers of fatty acyl chains and saturated long-chain base moieties were identified also, although we could not clarify whether these structures have any biological relevance in HL-60 cells, of which about 70% differentiate into macrophage-lineage cells. Further studies are necessary to identify key ions of various types of lactosylceramides such as branched, polyunsaturated fatty acyl moieties, and other types of sphingoids like phytosphingosine to characterize their structures and biological significance. There is also a need to determine the position of double bonds and hydroxylation in both fatty acyl chain and long-chain base moieties of lactosylceramides by LC-ESI ion trap mass spectrometry.
Conclusions Using mass spectrometry, lactosylceramide was ionized with alkaline metals or ammonium salts to produce molecular ions of [M + Li/Na]+ at 400 C or [M + H]+ at 200 C. The [M + Li]+ ion was stable and not frag-
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mented into fatty acyl chain and long-chain base moiety. However, MS3 analysis showed some dissociation of lactosylceramide [M + H]+ ion. The loss of two hexoses and water resulted in cleavage of the amide bond, which allowed information on the long chain base and fatty acyl moiety necessary to determine the molecular species of lactosylceramide to be obtained. In the present study, we developed a useful and simple method to identify the molecular species of lactosylceramides using reverse-phase LC-ESI ion trap mass spectrometry. Using sequential MSn analysis, we could obtain in multiple steps the dissociation patterns of a single ion. The patterns became simple and allowed us to speculate on the structure of the molecular species. The obtained molecular species were classified as [M + H]+ ions, which were produced with ammonium formate. In our analysis, 200 ng of lactosylceramides derived from porcine blood cells contained more than 33 molecular species. Lactosylceramides were eluted from a column depending on the length of their carbon chain and appeared in the order of unsaturated, 2-hydroxylated, and saturated fatty acyl chain with the same number of carbon atoms. We also used our method to determine the molecular species of lactosylceramides present in the membrane microdomain fraction of HL-60 cells (about 70% were differentiated cells). Five species of major lactosylceramides and about 20 species of minor ones were detected in the upper and lower bands on the HPTLC plate. The biological relevance of the minor lactosylceramides, which consisted of about 20 species from C16 to C26, is not clear yet.
Acknowledgments We thank Professor Sandro Sonnino and Dr. Alessandro Prinetti from the Department of Medical Chemistry, Biochemistry and Biotechnology, University of Milan, Italy for their generous gift of some synthetic lactosylceramides used in this study. This work was supported in part by Grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan 16017293 for K.I. This work was supported, in part, by Thermo Electron Corp.
References [1] K. Simons, D. Toomre, Lipid rafts and signal transduction, Nat. Rev. Mol. Cell Biol. 1 (2000) 31–39. [2] S. Hakomori, Cell adhesion/recognition and signal transduction through glycosphingolipid microdomain, Glycoconj. J. 17 (2000) 143–151. [3] K. Iwabuchi, I. Nagaoka, Lactosylceramide-enriched glycosphingolipid signaling domain mediates superoxide generation from human neutrophils, Blood 100 (2002) 1454–1464.
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[4] T.Y. Wang, J.R. Silvius, Sphingolipid partitioning into ordered domains in cholesterol-free and cholesterol-containing lipid bilayers, Biophys. J. 84 (2003) 367–378. [5] D. Hoekstra, O. Maier, J.M. Vander Wouden, T.A. Slimane, S.C.D. Van Ijzendoorn, Membrane dynamics and cell polarity: the role of sphingolipids, J. Lipid Res. 44 (2003) 869–877. [6] X.M. Li, M.M. Momsen, H.L. Brockman, R.E. Brown, Lactosylceramide: effect of acyl chain structure on phase behavior and molecular packing, Biophys. J. 83 (2002) 1535–1546. [7] C.E. Costello, J.E. Vath, Tandem mass spectrometry of glycolipids, Methods Enzymol. 193 (1990) 738–768. [8] Q. Ann, J. Adams, Structure determination of ceramides and neutral glycosphingolipids by collisional activation of [M + Li]+ ions, J. Am. Soc. Mass Spectrom. 3 (1992) 260–263. [9] Q. Ann, J. Adams, Structure-specific collision-induced fragmentations of ceramides cationized with alkali-metal ions, Anal. Chem. 65 (1993) 7–13. [10] R.S. Duarte, C.R. Polycarpo, R. Wait, R. Hartmann, E.B. Bergter, Structural characterization of neutral glycosphingolipids from Fusarium species, Biochim. Biophys. Acta 1390 (1998) 186– 196. [11] F.F. Hsu, J. Turk, Structural determination of glycosphingolipids as lithiated adducts by electrospray ionization mass spectrometry using low-energy collisional-activated dissociation on a triple stage quadrupole instrument, J. Am. Soc. Mass Spectrom. 12 (2001) 61–79.
[12] F.F. Hsu, J. Turk, Characterization of ceramides by low energy collisional-activated dissociation tandem mass spectrometry with negative-ion electrospray ionization, J. Am. Soc. Mass Spectrom. 13 (2002) 558–570. [13] X. Han, Characterization and direct quantitation of ceramide molecular species from lipid extracts of biological samples by electrospray ionization tandem mass spectrometry, Anal. Biochem. 302 (2002) 199–212. [14] G. Pocsfalvi, A. Malorni, I. Mancini, G. Guella, F. Pietra, Molecular characterization of a highly heterogeneous mixture of glucosylceramides from a deep-water Mediterranean scleractinian coral Dendrophyllia cornigera, Rapid Commun. Mass Spectrom. 14 (2000) 2247–2259. [15] M.H. Lee, G.H. Lee, J.S. Yoo, Analysis of ceramides in cosmetics by reversed-phase liquid chromatography/electrospray ionization mass spectrometry with collision-induced dissociation, Rapid Commun. Mass Spectrom. 17 (2003) 64–75. [16] B.J. Pettus, A. Bielawska, B.J. Kroesen, P.D.R. Moeller, Z.M. Szulc, Y.A. Hannun, M. Busman, Observation of different ceramide species from crude cellular extracts by normal-phase high-performance liquid chromatography coupled to atmospheric pressure chemical ionization mass spectrometry, Rapid Commun. Mass Spectrom. 17 (2003) 1203–1211. [17] F. Sako, S. Gasa, A. Makita, A. Hayashi, S. Nozawa, Human blood group glycosphinglipids of porcine erythrocytes, Arch. Biochem. Biophys. 278 (1990) 228–237.
Isolation and mass spectrometry characterization of molecular species of lactosylceramides using liquid chromatography-electrospray ion trap mass spectrometry Naoko Kagaa, Saiko Kazunoa, Hikari Takaa, Kazuhisa Iwabuchib,c, Kimie Murayamaa,* a
b
Division of Proteomics and Biomolecular Science, BioMedical Research Center, Juntendo University Graduate School of Medicine, Tokyo 113-8421, Japan Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, Tokyo 113-8421, Japan c Laboratory of Biochemistry, Juntendo University School of Healthcare and Nursing, Chiba, Japan Received 27 September 2004 Available online 2 December 2004
Abstract Reverse-phase liquid chromatography/electrospray ion trap mass spectrometry (LC-ESI-MSn) was established for identification of the molecular species of lactosylceramides. Lactosylceramides derived from porcine blood cells were separated on a CapcellPak C8 column using a mixture of methanol and 1 mM ammonium formate from the C16 to C26 fatty acyl chains based on the length of total carbon chains and the nature of sphingoid bases (w00 ) and fatty acyl chains (Y00 w00 ) was identified by MS3 as their [M + H]+ ions. The same number of fatty acyl moieties appeared in the order of unsaturated, (2-)hydroxylated, and saturated components. The molecular species of lactosylceramides derived from porcine blood cells totaled more than 33 and included mainly C24:0-d18:1, Ch24:0-d18:1, Ch24:1-d18:1, C24:1-d18:1, and C22:0-d18:1 in addition to 28 minor species from C16:0 to C26:0 fatty acyl moieties. The molecular species of lactosylceramides in the membrane microdomain fraction of HL-60 cells (70% were differentiated into macrophagelineage cells) were identified as C24:0-d18:1, C24:1-d18:1, C22:0-d18:1, C16:0-d18:1, and more than 21 other minor species. Our results suggest that reverse-phase LC-ESI-MSn is a useful and simple method for identification of lactosylceramide molecular species. 2004 Elsevier Inc. All rights reserved. Keywords: Lactosylceramide; Molecular species; Fatty acyl chain moiety; Long-chain base moiety; LC-ESI-MSn
Studies on molecular membrane biology have focused on the dynamics of cellular proteins and the lipid microenvironment at the cell surface [1]. It is known that glycosphingolipids play important roles in cell–cell interaction, recognition, transmembrane signaling, and cellular growth and differentiation in animal cells [2]. These compounds self-aggregate easily based on their nature and cluster with sphingomyelin with or without cholesterol and various signal transducer molecules to form various types of microdomains called ‘‘lipid-raft’’ at plasma membrane [3–6]. Although the lipid composition of the *
Corresponding author. Fax: +81 3 3818 6330. E-mail address: [email protected] (K. Murayama).
0003-2697/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.11.003
rafts in different cells is not clear, lactosylceramides are known to localize with ‘‘sphingolipid-sterol rafts’’ and trigger raft-related signal transduction processes. In neutrophils, lactosylceramide-enriched microdomains on the cell surface coupled with tyrosine protein kinase LYN (Lyn)1 induce the activation of Lyn via lactosylceramides and lead to superoxide generation [3]. To understand the biological activity and physical behavior of lactosylceramides, it is important to deter1
Abbreviations used: Lyn, tyrosine protein kinase LYN; FAB, fast atom bombardment; CAD, collisional-activated dissociation; CID, collision-induced dissociation; ESI, electrospray ionization; APCI, atmospheric pressure chemical ionization; DFP, diisopropylphosphorofluoridate; SIM, single ion monitoring; 3D, three-dimensional.
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mine their structural heterogeneity. Lactosylceramides contain lactose and a ceramide moiety. Various classes of the sphingoid base have been identified, such as the dihydro type (sphinganine), the additional hydroxyl group type (phytosphingosine), and the 2-amino-4-ene1,3-diol type with 14–22 carbon chains (sphingenine). The commonest type in animal tissues is C18 sphingenine (-d18:1). In addition, significant variation can also occur in fatty acyl moiety with regard to chain length, unsaturation, and hydroxylation. Lactosylceramides containing saturated fatty acyl chains display relatively high thermotropic and pressure-induced transition, but the introduction of a single cis double bond into the fatty acyl chains markedly reduce these properties, especially when the double bonds are located near the middle of the fatty acyl chain [6]. Mass spectrometry (MS) is a powerful tool for lipid structure analysis. Several methods incorporating tandem MS for identification of intact ceramides and neutral sphingolipids such as glucosyl- and lactosylceramides have been published. In early 1990s, Costello and Vath [7] used fast atom bombardment tandem mass spectrometry (FAB-MS/MS) and reported valuable data about the structure of glycosphingolipids, such as the molecular weight of fatty acyl chain and sphingoid base, by analyzing the MS/MS spectra of [M + H]+ and [M H] ions. Ann and Adams [8,9] and Duarte et al. [10] also reported that, in the presence of alkaline metal, [M + metal]+ ions were formed with ceramides and glucosylceramides and these showed a much higher sensitivity than the [M + H]+ ions. In addition, they provided more informative MS/MS spectra for the location of double bonds at high-energy CAD, by using charge remote cleavage. At present, electrospray ionization tandem mass spectrometry (ESI-MS/MS) and atmospheric pressure chemical ionization tandem mass spectrometry (APCI-MS/MS) are used for determination of the structure of intact ceramides and lactosylceramides with low-energy CID [11–13] or high-energy CID [14]. Lee et al. [15] and Pettus et al. [16] reported analysis of the molecular species of ceramides by reversephase LC-ESI/MS/MS and normal-phase LC-APCI/ MS/MS, respectively. We report here the design of a simple and useful method for separation and MSn characterization of molecular species of lactosylceramides using reversephase HPLC-ESI ion trap mass spectrometry.
Materials and methods Materials Lactosylceramide derived from porcine blood cells was purchased from Matreya (Pleasant Gap, PA). The
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synthetic lactosylceramide for C24:1-d18:1 was provided by Professor Sandro Sonnino (Center of Excellence on Neurodegenerative Diseases, Department of Medical Chemistry, Biochemistry and Biotechnology, University of Milan, Italy). All reagents used in this study were of the highest grade, especially those used for HPLC, spectrometry, and amino acid analysis. Chloroform, acetone, sodium chloride, methanol, ethylenediaminetetraacetic acid (EDTA), Triton X-100, Tris–HCl, vitamin D3, and diisopropylphosphorofluoridate (DFP) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Lithium chloride anhydrous and ethylene glycol bis (b-aminoethyl ether)-N,N,N 0 ,N 0 -tetraacetic acid (EGTA) were obtained from Nacalai Tesque (Kyoto, Japan). Ammonium acetate, ammonium formate, and primulin were from Sigma–Aldrich (St. Louis, MO). Complete is a product of Roche (Switzerland). Ultrapure water was prepared using the Millipore Milli-Q purification system (Bedford, MA). Mass spectrometric analysis All analyses were performed in positive mode using a LCQ DECA XP ion trap mass spectrometer (Thermo Electron, San Jose, CA) with a conventional electrospray ionization source. Infusion-ESI-MSn analysis Lactosylceramide at a concentration of 5–10 ng/ll was dissolved in methanol with or without alkaline and ammonium salts (1 mM LiCl, 1 mM NaCl, and 10 mM ammonium acetate). Sample solution was directly infused at a rate of 3–5 ll/min into the ESI source without separation of the molecular species of lactosylceramides. The temperature of the ion transfer tube was set at 150–400 C. The spray voltage was set at 5 kV and the nitrogen sheath gas pressure used was 10 U. In MSn (n = 2–4) analysis, the collision energy was fixed at 100%. HPLC-ESI-MSn analysis. Reverse-phase HPLC was carried out with the Magic2002 system (Michrom BioResources, Auburn, CA). The column was Capcellpak C8 UG120, 1.0 mm ID · 150 mm (Shiseido Fine Chemicals, Tokyo). The mobile phases were as follows: solvent A was 1 mM ammonium formate in methanol:water (76:24 v/v) and solvent B was 1 mM ammonium formate in methanol:water (96:4 v/v). The elution program was isocratic elution with 80% B for 5 min, increase to 95% B, and then hold for 30 min. The flow rate was 50 ll/ min. The HPLC was joined to the mass spectrometer, LCQ DECA XP. The MS conditions were as follows: ion transfer tube temperature 250 C, spray voltage 5 kV, sheath gas pressure 20 U. MSn (n = 4) analysis was performed on data-dependent experiments (DDE) with collision energy 80%.
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Identification of molecular species of lactosylceramides in the membrane microdomain fraction of HL-60 cells Fractionation of the membrane microdomain fraction of HL-60 cells. Cells of the myeloid leukemia cell line HL-60 were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum. HL-60 cells (2 · 108 cells) were treated with active form vitamin D3 (final concentration, 100 nM) for 5 days. Flow cytometric analysis using anti-CD11b antibody was performed as described previously [3]. Iwabuchi and Nagaoka [3] indicated that about 70% of the cells were differentiated into macrophage-lineage cells. Cells were resuspended in relaxation buffer (10 mM 1,4 piperazinediehanesulfonic acid, 100 mM KCl, 3 mM NaCl, 3.5 mM MgCl2, and 1 mM ATP, pH 7.4) containing a mixture of protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 0.4 lM aprotinin, 10 lM E-64, 20 lM leupeptin, 10 lM bestatin, 5 lM N-acetyl-Leu-Leu-Met-al, and 2 lM pepstatin) and disrupted by nitrogen cavitation (N2 for 20 min at 350 psi). Disrupted cells were dropped gradually into EGTA solution (final concentration, 1.25 mM). All manipulations were carried out at 4 C. The plasma membrane was fractionated after sequential centrifugation; 500g at 4 C for 10 min, 8500g for 20 min, and 200,000g for 1 h. This fraction was suspended in TNE-T buffer [1% Triton X-100, 10 mM Tris–HCl (pH 7.5), 150 mM Nacl, 1 mM DFP] containing 1/20 Complete. Extraction of crude lactosylceramides. The crude lipids in the membrane microdomain fraction were extracted with a chloroform:methanol mixture (2:1 v/v). A fifth of them (20 ll/100 ll crude lipids) were separated on a 10 · 10-cm high-performance thin-layer chromatography (HPTLC) plate (Merck, Rahway, NJ) with chloroform/methanol/water 65:25:4 (v/v/v). After development, lactosylceramides were visualized using primulin reagent (0.03% in 80% acetone) under ultraviolet light. Lactosylceramide-containing bands were scratched out from the plate and extracted with chloroform/methanol (2:1 v/v). After drying, lactosylceramides were dissolved in 20 ll of methanol and 4 ll of the solution was loaded on the column for LC-ESIMSn analysis. Finally, lactosylceramides from HL-60 (8 · 106 cells) were analyzed.
Results and discussion Effects of salts and temperature on ionization of synthetic lactosylceramide at C24:1-d18:1 To compare the effects of salts and ion transfer tube temperature on ionization of lactosylceramide by infusion analysis, synthetic lactosylceramide at C24:1-d18:1 was dissolved in methanol with/without 1 mM LiCl,
Fig. 1. Effects of salts and temperature on ionization of lactosylceramide by infusion analysis. Synthetic lactosylceramide of C24:1-d18:1 was dissolved in methanol containing each salt at 5 ng/ll concentration. The flow rate was 3 ll/min.
NaCl, or 10 mM ammonium acetate at 5 ng/ll and analyzed with the ion trap mass spectrometer. In salt-free condition, lactosylceramide at C24:1-d18:1 did not produce the [M + H]+ ion at 400 C, but the [M + Na]+ ion, which was produced from concomitant sodium in the sample or reagents, was stable. The addition of 1 mM NaCl to lactosylceramide markedly increased the amount of [M + Na]+ ion at 400 C (Fig. 1). On the other hand, the amount of [M + Li]+ ion was twice as much as that of [M + Na]+ ion at the same temperature. However, the addition of 10 mM ammonium acetate tended to produce the [M + H]+ ion at 200 C. The higher temperature (400 C) for the [M + H]+ ion was associated with the generation of dehydrated ion and resulted in a complex spectrum. On the other hand, the [M + Na]+ ion of lactosylceramide could not be produced at 200 C. The optimal temperature of the ion transfer tube was 400 C for the [M + metal]+ ion and 200 C for the [M + H]+ ion. MSn analysis of [M + Li]+ and [M + H]+ ion to characterize fatty acyl chain and long-chain base moiety We performed MSn analysis for [M + Li]+ and [M + H]+ ion of synthetic lactosylceramide (C24:1-d18:1) at optimal temperature (Fig. 2). In MSn analysis for [M + Li]+ ion, following the loss of one or two hexose(s) [Y1,Y0] and water [represented as 0 , 00 ], a HCHO was eliminated to form [a2] ion11 on the MS2/MS3 spectra (Fig. 2A). [Y0] and [a2] ions were extremely stable at 400 C and no further cleavage occurred on the MS4 spectrum. We could not obtain information on the fatty acyl chain and long-chain base, even though the dissociation energy increased up to 100%. However, lactosylceramides containing 2-hydroxy fatty acyl chain underwent marked cleavage of the N–CO bond on the MS3 spectrum and characterized the fatty acyl chain (data not shown). On the other hand, in MSn analysis of [M + H]+ ion, the loss of two hexoses and water [Y00 ] on the MS2 spec-
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Fig. 2. MSn spectra of synthetic lactosylceramide of C24:1-d18:1 using ESI-ion trap mass spectrometer. (A) MSn spectra (n = 1–4) of the lithiated molecular ion [M + Li]+ with 1 mM LiCl in methanol at 400 C. (B) MSn spectra (n = 1–3) of the protonated molecular ion [M + H]+ with 10 mM ammonium acetate in methanol at 200 C. The collision energy was 100%. (C) Predicted cleavage scheme for lactosylceramide shows hexose (Hex), loss of hexoses (Y0), long-chain base (w series), and fatty acyl chain (COR-H).
trum was associated with cleavage of the immediate N–CO bond on the MS3 spectrum and identification of a long-chain base (w ion series) and a fatty acyl chain ([Y00 w0 ¼ COR H]) [7] (Fig. 2B). On the MS3 spectrum, m/z 264 was found as a fragment ion of Y00 regarded as w00 ion, which was assigned to d18:1, and the fatty acyl chain was identified as C24:1 [7]. The use of sequential MSn analysis allowed us to obtain the dissociation patterns of a single ion in multiple steps when that was compared with MS/MS analysis using FAB-MS or triple stage ESI/MS. The patterns were simple, thus allowing speculation on the structure of the molecular species. Molecular species of lactosylceramides derived from porcine blood cells using infusion analysis The molecular species of lactosylceramides derived from porcine blood cells were determined using infusion analysis. The MS spectrum for those ionized with 1 mM ammonium acetate is shown in Fig. 3. The molecular species of lactosylceramides comprised more than 14 different structures from C16:0-d18:1 to Ch26:1-d18:1. The main species were C22:0-d18:1, C24:1d18:1, C24:0-d18:1, Ch24:1-d18:1, and Ch24:0-d18:1, similar to the results of a previous study [11].
Fig. 3. ESI-MS spectrum of lactosylceramides derived from porcine blood cells ionized with ammonium salt using infusion analysis. Lactosylceramides were dissolved in 10 mM ammonium acetate in methanol at 10 ng/ll concentration, the ion transfer tube was set at 200 C. The molecules were detected as protonated molecular ions, [M + H]+. The parenthetic formulae of lactosylceramides were characterized using other methods. *Represent those revealing background ions from the reagents. Some ions at m/z 1001 and 1003 were similar to the [M + H]+ ions of C26:1-d18:1 and C26:0-d18:1 but they were distinguished from the background ions on MSn analysis.
The use of infusion analysis was associated with one problem; it was impossible to distinguish fatty acyl chains between Cn:x and Ch(n 1):(x + 1) by MSn
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analysis because of the same value of COR-H at m/z 364 for C25:0 and Ch24:1 fatty acyl chains. LC-ESI-MS for classification of molecular species of lactosylceramides by single ion monitoring (SIM) mode The molecular species of lactosylceramides were separated on a C8 reverse-phase column and identified using the LCQ ion trap mass spectrometer by characterizing their fatty acyl chain and long-chain base moieties by MSn analysis. Fig. 4A shows the three-dimensional (3D) HPLC mass chromatogram of 200 ng of total lactosylceramides derived from porcine blood cells. The abscissa, ordinate, and z axis indicate the retention time, relative abundance of ions, and m/z, respectively.
In the case of LC-MS, we used methanol containing 1 mM ammonium formate as a solvent because the formate produced stable and abundant [M + H]+ ions and [M + Na]+ ions were minimum as contaminants. It was assumed that the long chain base of lactosylceramides has d18:1 sphingenine, because this is the commonest type in animal tissues [17]. In Figs. 4B–E, [M + H]+ ions of lactosylceramide molecular species were monitored in the SIM mode for classification according to fatty acyl carbon chain into (B) straight saturated (n:0), (C) straight unsaturated (n:1), (D) saturated and hydroxylated (hn:0), and (E) unsaturated and hydroxylated (hn:1) fatty acyl chains. We also monitored d18:0 sphinganine, but no significant peak of lactosylceramides appeared in porcine blood cells (data not
Fig. 4. Three-dimensional HPLC mass chromatogram of lactosylceramides derived from porcine blood cells and selected ion monitoring (SIM) chromatograms. (A) 3D-HPLC-ESI/MS chromatogram of 200 ng of lactosylceramide. SIM chromatograms monitored the [M + H]+ ions of lactosylceramide molecular species classified according to fatty acyl carbon chain into (B) straight saturated (n:0), (C) straight unsaturated (n:1), (D) saturated and hydroxylated (hn:0), and (E) unsaturated and hydroxylated (hn:1).
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shown). In the straight saturated molecular species (Fig. 4B), the retention time of lactosylceramides correlated with the length of their fatty acyl chain from 10 min for C16:0 to 30 min for C26:0. Although the [M + H]+ ions of lactosylceramides at C25:0 and Ch24:1 had the same value at m/z 989 and could not be identified by infusion analysis, they were completely separated on the column. Their retention times were 17.3 min for Ch24:1 and 24.6 min for C25:0. The former had 2-hydroxylated fatty acyl chains and was more hydrophilic than the latter. Lactosylceramides with the same carbon number of fatty acyl chain of saturated (C24:0) at m/z 975, unsatu-
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rated (C24:1) at m/z 973, and 2-hydroxylated (Ch24:0) at m/z 991 fatty acyl chain moiety appeared at 22.2, 18.6, and 20.8 min on the chromatogram, respectively (Figs. 4B–D). These results suggest that lactosylceramides classified as unsaturated, 2-hydroxylated, and saturated fatty acyl chain were eluted in that order on the column. Determination of molecular species of porcine-blood cell-derived lactosylceramides We also identified more than 33 different structures as molecular species from porcine-blood-cell-derived lactosylceramides (Fig. 5A). The profile was constructed
Fig. 5. Distribution of molecular species of lactosylceramides derived from porcine blood cells. (A) Abscissa represents the carbon unit of fatty acyl chain and long-chain base (d18:1 is not shown). The intensity of lactosylceramides was estimated by the peak area on Figs. 3B–E. The ordinate represents the peak area. The z axis represents the class of fatty acyl chain such as saturated, unsaturated, and hydroxylated species. (B) The SIM chromatogram of the same [M + H]+ ions at m/z 1003 for C26:0-d18:1 and C24:0-d20:1 and (C) MSn analysis (n = 1–3) for m/z 1003.
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based on the peak area of SIM chromatogram for lactosylceramides. Some lactosylceramides with long-chain base moiety at d20:1 were confirmed by MS3 analysis and are shown in Fig. 5C. The retention time of lactosylceramide at m/z 1003 for C26:0-d18:1 or C24:0-d20:1 was 28 min in Fig. 5B, but the m/z 264 and m/z 292 ions were produced on the MS3 spectrum. This means that there were two other types of w00 ions associated with d18:1 and d20:1 sphingenines. Therefore, it was considered that separation of lactosylceramides was based on total carbon chain length and numbers of double bonds in the fatty acyl chain and long-chain base. Thus, lactosylceramides at Cn-dx and Cn2-dx+2 were not separated on the column in the system, however, we could at least confirm by MS3 analysis the presence or absence of the two types of lactosylceramides. The molecular species of porcine-blood-cell-derived lactosylceramides contained more than 33 different structures, which were mainly C24:0-d18:1, Ch24:0-d18:1, Ch24:1-d18:1, C24:1-d18:1, and C22:0-d18:1, with 28 other minor species. Minor species of long-carbon-chain fatty
acids such as C22 and C26 were also identified as saturated, unsaturated, and hydroxylated species. The number of molecular species of lactosylceramides determined by LC-ESI/MS was twice that confirmed by infusion analysis. The amount of lactosylceramides used was 200 ng for determination of the [M + H]+ ions and automatic MSn analysis with LC-ESI/MS. In the case of infusion analysis, lactosylceramides at 10 ng/ll were infused into the ion source for 10–15 min depending on the number of MSn analyses of [M + H]+ ions manually. The sample amounts used for both analyses were not different and LC-ESI/MS was convenient and precise for determination of low-intensity [M + H]+ ions. Since the standard compound of the molecular species of lactosylceramides could not be reported and no product could be identified, we were unable to perform quantification analysis of these species. However, the SIM chromatograms of 10–200 ng of porcine-bloodcell-derived lactosylceramides showed almost linear relationships between the ratio of the peak area and the total amount of lactosylceramides (data not shown).
Fig. 6. Determination of molecular species of lactosylceramides derived from membrane microdomain of HL-60 cells. (A) The pattern of lactosylceramides for membrane microdomain fractionated from HL-60 cells separated on HPTLC. (B) Molecular species of lactosylceramides in the upper band and (C) those in the lower band derived from HL-60 cells (8 · 106 cells). The abscissa, ordinate, and z axis are similar to those used in Fig. 5.
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Analysis of molecular species of lactosylceramides in the membrane microdomain fraction of HL-60 cells Crude lipids were extracted from the membrane microdomain fraction of HL-60 cells using a mixture of chloroform and methanol (2:1 v/v). A fifth of the crude lipids was separated on HPTLC with a mixture solution of chloroform/methanol/water (65:25:4 v/v/v) and detected by primulin reagent. Lactosylceramides marked as upper and lower bands in Fig. 6A were removed and collected in each glass tube and crude lactosylceramides were extracted with a mixture of chloroform and methanol (2:1 v/v). A fifth of the crude lactosylceramides was loaded on a column and determined by LCESI-MSn analysis, respectively. Figs. 6B and C show the distribution of molecular species of lactosylceramides in the upper and the lower bands, respectively. A total of 25 species of lactosylceramides was detected in the upper and lower bands on the HPTLC plate. Lactosylceramides in the upper band consisted mainly of C24:0-d18:1, C24:1-d18:1, and C22:0-d18:1 and those in the lower band were mainly C16:0-d18:1 but higher carbon chain structures such as Ch24:0-d18:1 fatty acyl chain moiety at m/z 991 were also detected. The high molecular species in the lower band seemed to be insufficiently separated on the HPTLC plate and rather coexisted with those of the upper band. It is possible, however, that the hydrophilic property of Ch24:0 could have affected the separation of lactosylceramides on the HPTLC plate. On the other hand, lactosylceramide for C16:0-d18:0 also appeared in the upper band, suggesting that it might be more hydrophobic than lactosylceramide for C16:0-d18:1 on the HPTLC plate. Numerous minor lactosylceramides that contained odd numbers of fatty acyl chains and saturated long-chain base moieties were identified also, although we could not clarify whether these structures have any biological relevance in HL-60 cells, of which about 70% differentiate into macrophage-lineage cells. Further studies are necessary to identify key ions of various types of lactosylceramides such as branched, polyunsaturated fatty acyl moieties, and other types of sphingoids like phytosphingosine to characterize their structures and biological significance. There is also a need to determine the position of double bonds and hydroxylation in both fatty acyl chain and long-chain base moieties of lactosylceramides by LC-ESI ion trap mass spectrometry.
Conclusions Using mass spectrometry, lactosylceramide was ionized with alkaline metals or ammonium salts to produce molecular ions of [M + Li/Na]+ at 400 C or [M + H]+ at 200 C. The [M + Li]+ ion was stable and not frag-
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mented into fatty acyl chain and long-chain base moiety. However, MS3 analysis showed some dissociation of lactosylceramide [M + H]+ ion. The loss of two hexoses and water resulted in cleavage of the amide bond, which allowed information on the long chain base and fatty acyl moiety necessary to determine the molecular species of lactosylceramide to be obtained. In the present study, we developed a useful and simple method to identify the molecular species of lactosylceramides using reverse-phase LC-ESI ion trap mass spectrometry. Using sequential MSn analysis, we could obtain in multiple steps the dissociation patterns of a single ion. The patterns became simple and allowed us to speculate on the structure of the molecular species. The obtained molecular species were classified as [M + H]+ ions, which were produced with ammonium formate. In our analysis, 200 ng of lactosylceramides derived from porcine blood cells contained more than 33 molecular species. Lactosylceramides were eluted from a column depending on the length of their carbon chain and appeared in the order of unsaturated, 2-hydroxylated, and saturated fatty acyl chain with the same number of carbon atoms. We also used our method to determine the molecular species of lactosylceramides present in the membrane microdomain fraction of HL-60 cells (about 70% were differentiated cells). Five species of major lactosylceramides and about 20 species of minor ones were detected in the upper and lower bands on the HPTLC plate. The biological relevance of the minor lactosylceramides, which consisted of about 20 species from C16 to C26, is not clear yet.
Acknowledgments We thank Professor Sandro Sonnino and Dr. Alessandro Prinetti from the Department of Medical Chemistry, Biochemistry and Biotechnology, University of Milan, Italy for their generous gift of some synthetic lactosylceramides used in this study. This work was supported in part by Grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan 16017293 for K.I. This work was supported, in part, by Thermo Electron Corp.
References [1] K. Simons, D. Toomre, Lipid rafts and signal transduction, Nat. Rev. Mol. Cell Biol. 1 (2000) 31–39. [2] S. Hakomori, Cell adhesion/recognition and signal transduction through glycosphingolipid microdomain, Glycoconj. J. 17 (2000) 143–151. [3] K. Iwabuchi, I. Nagaoka, Lactosylceramide-enriched glycosphingolipid signaling domain mediates superoxide generation from human neutrophils, Blood 100 (2002) 1454–1464.
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[4] T.Y. Wang, J.R. Silvius, Sphingolipid partitioning into ordered domains in cholesterol-free and cholesterol-containing lipid bilayers, Biophys. J. 84 (2003) 367–378. [5] D. Hoekstra, O. Maier, J.M. Vander Wouden, T.A. Slimane, S.C.D. Van Ijzendoorn, Membrane dynamics and cell polarity: the role of sphingolipids, J. Lipid Res. 44 (2003) 869–877. [6] X.M. Li, M.M. Momsen, H.L. Brockman, R.E. Brown, Lactosylceramide: effect of acyl chain structure on phase behavior and molecular packing, Biophys. J. 83 (2002) 1535–1546. [7] C.E. Costello, J.E. Vath, Tandem mass spectrometry of glycolipids, Methods Enzymol. 193 (1990) 738–768. [8] Q. Ann, J. Adams, Structure determination of ceramides and neutral glycosphingolipids by collisional activation of [M + Li]+ ions, J. Am. Soc. Mass Spectrom. 3 (1992) 260–263. [9] Q. Ann, J. Adams, Structure-specific collision-induced fragmentations of ceramides cationized with alkali-metal ions, Anal. Chem. 65 (1993) 7–13. [10] R.S. Duarte, C.R. Polycarpo, R. Wait, R. Hartmann, E.B. Bergter, Structural characterization of neutral glycosphingolipids from Fusarium species, Biochim. Biophys. Acta 1390 (1998) 186– 196. [11] F.F. Hsu, J. Turk, Structural determination of glycosphingolipids as lithiated adducts by electrospray ionization mass spectrometry using low-energy collisional-activated dissociation on a triple stage quadrupole instrument, J. Am. Soc. Mass Spectrom. 12 (2001) 61–79.
[12] F.F. Hsu, J. Turk, Characterization of ceramides by low energy collisional-activated dissociation tandem mass spectrometry with negative-ion electrospray ionization, J. Am. Soc. Mass Spectrom. 13 (2002) 558–570. [13] X. Han, Characterization and direct quantitation of ceramide molecular species from lipid extracts of biological samples by electrospray ionization tandem mass spectrometry, Anal. Biochem. 302 (2002) 199–212. [14] G. Pocsfalvi, A. Malorni, I. Mancini, G. Guella, F. Pietra, Molecular characterization of a highly heterogeneous mixture of glucosylceramides from a deep-water Mediterranean scleractinian coral Dendrophyllia cornigera, Rapid Commun. Mass Spectrom. 14 (2000) 2247–2259. [15] M.H. Lee, G.H. Lee, J.S. Yoo, Analysis of ceramides in cosmetics by reversed-phase liquid chromatography/electrospray ionization mass spectrometry with collision-induced dissociation, Rapid Commun. Mass Spectrom. 17 (2003) 64–75. [16] B.J. Pettus, A. Bielawska, B.J. Kroesen, P.D.R. Moeller, Z.M. Szulc, Y.A. Hannun, M. Busman, Observation of different ceramide species from crude cellular extracts by normal-phase high-performance liquid chromatography coupled to atmospheric pressure chemical ionization mass spectrometry, Rapid Commun. Mass Spectrom. 17 (2003) 1203–1211. [17] F. Sako, S. Gasa, A. Makita, A. Hayashi, S. Nozawa, Human blood group glycosphinglipids of porcine erythrocytes, Arch. Biochem. Biophys. 278 (1990) 228–237.